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Acromial spur formation in patients with rotator cuff tears
Acromial spur formation in patients with rotator cuff tears A. F. W. Chambler, BSc, MS, FRCS(Orth),a A. A. Pitsillides, BSc(Hons), PhD,b and R. J. H. Emery, MS, FRCS(Ed),a London, United Kingdom
In this study we analyzed the acromial spurs of 15 patients with impingement syndrome undergoing open rotator cuff repair. Mineral apposition analysis and quantitative cytochemical techniques for glucose-6phosphate dehydrogenase (G6PD) activity (pentose phosphate pathway), alkaline phosphatase (ALP) activity (osteoblast activity), and tartrate-resistant acid phosphatase (TRAP) activity (osteoclast phenotype) were used to examine the distribution and level of activity of selected marker enzymes within the acromial spur insertion into the coracoacromial ligament in order to establish whether local behavior of bone cells is consistent with the proposed secondary development of the acromial spur. Our results indicate that G6PD and ALP activity was higher in osteoblasts on the inferior surface compared with the superior surface of the acromial spur in all patients (P ⬍ .001). This area correlated to the most intense area of mineral apposition shown by dual tetracycline labeling. TRAP activity revealed a heterogeneous distribution within the samples. A greater G6PD activity per cell (mean increase of 87%) was seen at the tip compared with that in post- and pre-tip zones within the coronal plane (P ⬍ .0002). The qualitative and quantitative enzyme analyses show that the acromial insertion of the coracoacromial ligament is actively involved in bone turnover. The spatial distribution patterns of metabolically active bone-forming osteoblastic cells compared with a heterogeneous distribution of TRAP-positive osteoclasts provide evidence of bone remodeling consistent with the morphologic contours of the acromial enthesis. The sites of oxytetracycline labeling appear to correlate with the sites of high ALP and G6PD activity, which From the Department of Orthopaedics,a St Mary’s Hospital, and Department of Veterinary Basic Sciences,b The Royal Veterinary College, London, United Kingdom. Reprint request: A. F. W. Chambler, BSc, MS, FRCS(Orth), 8 Woodford Mill, Mill Street, Witney, OX28 6DE. (E-mail: [email protected]). Copyright © 2003 by Journal of Shoulder and Elbow Surgery Board of Trustees. 1058-2746/2003/$35.00 ⫹ 0 doi:10.1016/S1058-2746(03)00030-2
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supports the concept of spur formation being a secondary phenomenon in the presence of established rotator cuff tears. (J Shoulder Elbow Surg 2003;12: 314-21.)
I
t is well established that both intrinsic factors within the rotator cuff tendon and extrinsic factors from the surrounding soft- and hard-tissue structures appear to be related to each other in a complex relationship and that each may play a role in rotator cuff disease.12 Enthesophytic bony spurs have been proposed to be associated with rotator cuff tears. However, there has been debate as to the etiology of such acromial spurs and their precise role in impingement and rotator cuff disease. Bigliani et al1 showed a correlation between acromial morphology and rotator cuff tears, and this association has been used to support the theory of Neer16 for extrinsic factors as a cause of rotator cuff tears. Examination of the undersurface of the acromion, however, has led to the suggestion that the spur may represent a secondary change induced by a primary bursal-side cuff tear.17 Moreover, Uhthoff et al20 and Sarker et al19 concluded from their ultrastructural analyses of the coracoacromial arch that the coracoacromial ligament does not appear to be the primary factor in the initiation of impingement and the subsequent tear process. Together, these later studies would appear to support the theory of Codman7 for the intrinsic etiology of cuff tears. It is clear, therefore, that the pathogenesis and pathophysiology of this syndrome remain enigmatic. To establish whether indices of the bone remodeling process and the behavior of bone cells are consistent with the proposed secondary development of the acromial spur, we used mineral apposition analysis and quantitative cytochemical techniques to examine the distribution and activity of selected marker enzymes within both sagittal and coronal planes of the acromial spur insertion site into the coracoacromial ligament in patients with rotator cuff tears. These quantitative cytochemical techniques were used to measure glucose-6-phosphate dehydrogenase (G6PD) activity (pentose phosphate pathway), alkaline phosphatase (ALP) activity (osteoblastic activity),
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and tartrate-resistant acid phosphatase (TRAP) activity (osteoclastic phenotype) in sections of the acromial spur insertion site. This allowed assessment of spatial distribution and the level of activity of these selected marker enzymes within individual cells at defined histologic locations within the acromial spur insertion into the coracoacromial ligament. An analysis of these patterns of activity, together with oxytetracycline-based assessment of local bone formation rates, would establish whether local behavior of bone cells is consistent with the proposed secondary development of the acromial spur. METHODS Acromial samples were taken from 15 consecutive patients undergoing modified open acromioplasty and rotator cuff repair. All had medium-sized cuff tears. The mean age was 62.2 ⫾ 1.75 years, with a male-to-female ratio of 11:4. Ethical committee approval was obtained before the study was undertaken. Informed consent was obtained with regard to the reason for this study and the collection of suitable acromial samples. Patients also gave their permission to be given oxytetracycline tablets preoperatively to allow bone apposition rates to be studied. None of the patients who were approached refused to participate in this study.
Bone apposition analysis So that differences in bone apposition rates could be examined in these patients, two separate doses of oral fluorochrome were given.11,15 Thirteen patients were given oral oxytetracycline. The dosing regimen consisted of 250 mg of oxytetracycline twice a day for 2 days. This resulted in a total dose of 1 g over the 48-hour period. An interval of 2 weeks was imposed before this regimen was repeated. The patients then underwent surgery 4 days after the second dosing regimen. After cryostat sectioning, the samples were reviewed under an ultraviolet light microscope. Although the prerequisite use of cryostat sections for cytochemical analysis precluded the possibility of accurate measures of bone turnover to be determined, a semiquantitative blind assessment was undertaken for the detection of labels. Assessment of the quantity and extent of doublelabel surfaces in each of the samples was undertaken without knowledge of the patient’s details.
Sample collection and preparation The coracoacromial ligament was identified on the anterior aspect and inferior surface of the acromion extending to the coracoid process. The ligament was released near its coracoid attachment, leaving the insertion into the acromion intact for later analysis. A Neer acromioplasty was performed with an oscillating saw. This sample contained the anterior part of the acromion, the spur, and the insertion of the remaining coracoacromial ligament. Suture material and markings, made by a sterile marker pen, were used to facilitate orientation of the sample. Unwanted soft tissue
Figure 1 A, G6PD activity (formazan salt) on inferior surface of acromial sample (original magnification ⫻20). The salt appears blue, with darker color representing higher activity. B, G6PD activity (formazan salt) on superior surface of acromial sample (original magnification ⫻20).
was removed from the sample, and immediately after the sample was harvested, it was immersed for 3 minutes in an aqueous 5% (wt/vol) solution of polyvinyl alcohol (grade GO4). This immersion aided the formation of a strong bond when a specimen was mounted into the cryostat chuck. The sample was then chilled by precipitate immersion in a chilling bath containing n-hexane (BDH, Poole, UK) (low in aromatic hydrocarbons; boiling range, 67°C-70°C) in a small (15-mL) beaker surrounded by a slurry of carbon dioxide (dry ice, Guildford, Surrey, UK) and industrial alcohol. When the hexane had reached the temperature of ⫺65°C or below, the specimen was dropped straight in without touching the sides of the beaker and left for at least 1 minute before it was removed by means of cold forceps and placed in precooled specimen containers at ⫺70°C. These were then stored in carbon dioxide ice in an insulated container for transport. The specimens were mounted by anchoring them firmly onto a metal chuck with 5% polyvinyl alcohol before being placed in the cryostat. Sections were cut with a heavy-duty microtome in a Bright’s cryostat with a cabinet temperature of ⫺35°C to ⫺25°C (Bright Instruments, Huntingdon, UK). A tungsten-tipped steel knife was used, and the thickness of the sections was set at 10 m. Fifteen specimens were mounted in an orientation so that the sectioning could take
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Figure 2 G6PD activity at superior and inferior surfaces of acromial spurs in patients with rotator cuff tears. MIE, Mean integrated extinction.
place laterally to medially in the sagittal plane. This allowed comparison between the superior and inferior surfaces of the bony sample. Sectioning in 5 cases, anterior to posterior, in the coronal plane allowed comparison between the tip of the bony sample and its descending surfaces on either side.
Quantitative cytochemistry Fresh, unfixed, undecalcified sections were then analyzed by quantitative cytochemistry (Chayen5). This technique allows the biochemical activity within individual cells in the section to be related to the histology of the sample. It is a nondisruptive form of biochemistry (retaining spatial relationships between cells and their matrix) and between the subcomponents of the cells. It is very sensitive, so it can be used to measure enzymatic activities in a small biopsy specimen or a small area within that biopsy specimen. Consequently, accommodation of heterogeneity of the tissue within a sample can also be made. In these studies tetrazolium reactions were used for demonstration of G6PD activity, whereas azo-dye– coupling reactions were used for the detection of the acid and alkaline phosphate activities within the specimens. The methods used were those validated and described by Chayen and Bitensky6 and by Pitsillides et al.18
G6PD activity assay For G6PD activity assay, fresh, unfixed, undecalcified sagittal and coronal 10-m sections of the acromial spur insertion site were incubated in medium containing 3.3 mmol/L glucose-6-phosphate (disodium salt), 3 mmol/L nicotinamide adenine dinucleotide phosphate in 40% Polypep
(Bovine protein digest 5115 Sigma, Poole, UK) in 0.05 mol/L glycylglycine buffer, and 3.0 mmol/L nitro blue tetrazolium, at pH 8.0 and warmed to 37°C. This reaction medium was deoxygenated by bubbling with humidified nitrogen, and the intermediate hydrogen acceptor phenazine methosulfate (at 0.7 mmol/L) was added just before use. Perspex rings were placed around the sections to hold the reaction medium, and the sections were incubated at 37°C for 20 minutes. After the reaction, sections were washed in water, allowed to dry, and mounted in Aquamount (Merck, Poole, UK).
ALP activity assay For ALP activity assay, the incubation medium consisted of 2% barbital sodium, 0.16 mmol/L ␣–naphthyl acid phosphate, and 10% magnesium chloride adjusted to pH 9.2 at 37°C. The coupler, 1.0 mg/mL Fast Blue RR (Sigma, Poole, UK), was stirred into the medium and filtered immediately into Coplin jars (BDH, Poole, UK) containing the slides. At the end of the reaction time (4 minutes), the slides were washed in distilled water and placed in 1% acetic acid for 1 minute, after which they were rinsed in distilled water and allowed to dry.
TRAP activity assay For TRAP activity assay, the medium consisted of 1.4 mg/mL naphthol AS-TR phosphate (Sigma, Poole, UK), 5 L/mL N,N-dimethylformamide, and 0.1 mol/L sodium acetate and sodium tartrate adjusted to pH 5.2 with acetic acid. Fast Red TR (at 1.4 mg/mL, Sigma, Poole, UK) was used as the coupler, and after mixing, the medium was filtered immediately into Coplin jars containing the slides.
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G6PD and 5 for ALP and TRAP). In practice, a ⫻40 objective was used and a scanning spot was 0.4 m in diameter in the plane of the section. The values were calculated as mean integrated extinction of the colored end product per cell. In any spatially defined area, the enzyme activity in at least 10 cells was measured per slide, and 5 slides were used to investigate each area. Thus, the corresponding values of enzyme activity per cell within each histologic location for each patient represented a mean enzyme activity of at least 50 cells.
RESULTS Differences in bone cell enzyme activities in the sagittal plane
Figure 3 ALP activity on superior and inferior surfaces of acromial sample (original magnification ⫻20). Note difference also on trabecular surface (arrows). ALP activity appears blue/black on the pale bone background.
At the end of the reaction time (5 minutes), the slides were washed in distilled water, allowed to dry, and mounted in Aquamount. The relative distribution and activity per cell of G6PD (pentose phosphate pathway),8 ALP (marker of osteoblastic phenotype),2 and TRAP (marker of osteoclast phenotype)10 were measured in individual cells in distinct histologically defined zones by scanning and integrating microdensitometry.
Microdensitometry The amount of reaction product per cell was measured by a Vickers M85A scanning and integrating microdensitometer (Vickers Instruments, York, UK). Microdensitometry is a means by which to measure the mass of a chromophore (or the optical density of a colored end product) in a defined projected area of material, such as an individual cell. It differs from spectrophotometry in that microdensitometry is a microscopic technique, able to dissect specific regions of sections of a tissue optically and to measure the amount of chromophore within cells. Monochromatic light (585 nm for G6PD and ALP and 550 nm for TRAP) is used to scan the area enclosed within the masked field (mask size 4 for
G6PD activity. The mean G6PD activity per cell in osteoblasts on the inferior aspect of the tip of the acromial enthesis (0.92 ⫾ 0.04 [mean ⫾ SEM]) (Figure 1, A) was statistically significantly higher (P ⬍ .001, paired t test) than on the superior aspect (0.80 ⫾ 0.03) (Figure 1, B). This increased level of G6PD activity on the acromion’s inferior aspect was also evident, within any single patient, when the activity of these two sites were compared (Figure 2), and this was also reflected in a significant direct correlation (P ⬍ .001, r2 ⫽ 0.863) between resident cells’ G6PD activity on the inferior and superior surfaces in different patients. ALP activity. In all 15 acromial enthesophytes examined, the ALP activity in bone cells on the inferior aspect was significantly higher (P ⬍ .001) than on the superior aspect of the acromion (Figure 3), with mean ALP activity per cell of 0.89 ⫾ 0.04 and 1.02 ⫾ 0.03 on the inferior and superior aspects, respectively (Figure 4). In addition, further analysis of these results showed that intersite variability within specimens exhibited a direct and statistically significant correlation (P ⬍ .0001, r2 ⫽ 0.856). TRAP activity. The mean level of TRAP activity per cell in different sections of the same sample, each reacted for different reaction times (2, 5, 10, 15, and 20 minutes), failed to show a consistent trend. An assessment of the distribution of TRAP-positive bone cells failed to show any consistent spatial pattern of osteoclast distribution, exhibiting marked heterogeneity throughout sagittal-tissue sections. The surfaces of the bone trabeculae and not external aspects of the enthesophyte most commonly contained the largest numbers of TRAP-positive cells (Figure 5). Differences in bone cell enzyme activities in the coronal plane
G6PD activity in individual bone cells was assessed within the spur at sites defined in the coronal plane in 5 patients. In 4 of 5 patients (Figure 6), G6PD activity was consistently higher in the cells at the tip of the spur than at either side of the spur’s tip. These samples showed a greater G6PD activity per
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Figure 4 ALP activity on superior and inferior surfaces of acromial spur in patients with rotator cuff tears. MIE, Mean integrated extinction.
showed no significant difference in the level of activity per cell. Nevertheless, it was apparent that the variation in TRAP activity was considerable between different samples (range at tip, 1.82-0.63). Fluorochrome labeling in acromial spurs in patients with rotator cuff tears
Figure 5 TRAP-positive (osteoclastic) cells within acromial spur (original magnification ⫻20). Activity appears red, with darker color representing higher activity. CA, Coracoacromial.
cell (mean increase of 87%) at the tip compared with that in the post- and pre-tip zones within the coronal plane (P ⬍ .0002). In contrast to the spatially selected increase in G6PD activity evident in cells at the various sites of the coronal plane, in all but 1 (of 4) of the samples examined, bone cell ALP activity showed no variation between the sites at or on either side of the enthesophyte’s tip. TRAP activity per cell at different sites in each individual sample was compared in sections reacted for 5 minutes. In each of the 3 sites assessed in the coronal plane, osteoclasts in the enthesophytes
A semiquantitative assessment for the detection of labels, their quantity, and the extent of double-labeled surfaces was undertaken. No tetracycline labeling was seen in the control sample (an acromial sample from a patient who did not undergo oxytetracycline labeling) or in 3 samples from the patient group. This was assumed to be the result of a lack of compliance rather than failure of mineralization. Of the remaining 9 patients, 5 showed pronounced evidence of active bone apposition as defined by the scoring system. Tetracycline labeling yielded different patterns of distribution throughout the samples. Figure 7 reveals a double label at the bone surface, where the coracoacromial ligament has its attachment. Figure 8 reveals a double label only on one side of the trabeculae, which may represent a directional drift. This pattern was frequently seen and not thought to be cutting artifact. DISCUSSION This study concentrated on the coracoacromial ligament’s insertion onto the acromion, namely the
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Figure 6 Mean G6PD activity at pre-tip, tip, and post-tip sites in trabecular bone in acromial spur in patients with rotator cuff tears. MIE, Mean integrated extinction.
Figure 7 Double labeling seen at bone-ligament insertion site (original magnification ⫻40). Oxytetracycline appears yellow under ultraviolet light, and bone appears green.
Figure 8 Double labeling seen on one aspect of a trabecula: Directional effect (original magnification ⫻40). Oxytetracycline appears yellow under ultraviolet light, and bone appears green.
enthesis, and thus bone growth at the ligament insertion is described as an enthesophyte.4 Therefore, the commonly used term spur in this text refers to an acromial enthesophyte at the coracoacromial ligament’s insertion. G6PD activity is a rate-limiting step of the pentose phosphate shunt, which is used during energy metabolism for the production of reducing equivalents that contribute to many synthetic pathways, including those involved in mineralization, but specifically for the production of pentose nucleic acid synthesis.8 Our findings indicate that in all of the patients studied, G6PD activity was higher in the cells on the inferior surface of the acromial sample than on the superior
surface. These location-selective elevations in periosteal cell G6PD activity may, therefore, reflect persistent increases in matrix synthesis on the acromion’s inferior aspect. Similar persistent increases in G6PD activity have previously been described to be a component of the response of osteoblasts to increases in applied load bearing in vivo,8 and it is thus possible that they represent either enhanced rates of local matrix synthesis or a component of the local response to changes in dynamic mechanical loading. Furthermore, previous studies have also shown increases in bone cell G6PD activity at the site of subsequent new bone formation associated with fracture healing.9 The results observed suggest that the predominance of the
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inferior-aspect G6PD activity reflects elevated rates of bone formation, which may be promoted at this site by recent episodes of increased local mechanical loading. ALP activity has also been linked to bone formation by several authors.2,10 Indeed, measurements of mineral apposition rate and ALP activity in iliac crest biopsy specimens have shown correlation between fluctuations in mineral apposition rate and osteoblastic ALP activity, suggesting that bone formation appears to be at least partly dependent upon ALP activity and osteoblastic energy balance.2 Therefore, the activity of this enzyme was used in this study as a quantitative cytochemical indicator of local boneforming capacity of the acromial insertion of the coracoacromial ligament. The comparison of acromial osteoblasts on the superior and inferior surfaces (in the sagittal plane) did show differences, with the activity in the acromial osteoblasts on the inferior surface being significantly greater than on the superior surface in all 15 patients studied. As well as being consistent with the proposed increased rate of bone formation at these sites, these observations support a persistent loading-related elevation of local osteogenesis. The qualitative and quantitative enzyme analyses show that the acromial insertion of the coracoacromial ligament is likely to be actively involved in bone turnover. The spatial distribution patterns of metabolically active bone-forming osteoblastic cells with high G6PD and ALP activity showing a predominance on its anteroinferior aspect compared with a heterogeneous distribution of TRAP-positive osteoclasts provides evidence of bone remodeling consistent with a morphologic contour of the acromial sample. The sites of oxytetracycline labeling appear to correlate with the sites of high ALP and G6PD activity, which supports the notion for the presence of an osteogenic potential within the coracoacromial ligament’s insertion. Because of the lack of specimens, this study cannot describe the relative activities of these enzymes in bone cells in acromions in appropriate control groups, such as patients with impingement without rotator cuff tears or patients without impingement or indeed normal shoulders. Consequently, it is evident that our study does not rule out the possibility that such patterns of activity and the high rates of bone formation are not necessarily a characteristic that is always evident at the inferior aspect of the acromial insertion site. For this reason, it also remains possible that these high apparent rates of turnover represent a normal pattern of cellular behavior at all ligament insertions exposed to increased loading. However, given the quantitative nature of the increases in G6PD and ALP activities, our findings nevertheless support the con-
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clusion that higher rates of bone turnover are likely to be evident at the acromion’s inferior aspect. The mechanism by which subacromial impingement may contribute to producing an acromial enthesophyte cannot be determined from this study. It has been suggested, however, that the coracoacromial ligament is under a constant static tension.13 Furthermore, Burns and Whipple3 have demonstrated, in cadaveric studies, that the ligament is forced upward during impinging movements of the shoulder, adding a dynamic component to the tension experienced by the insertion of the ligament. Investigations by Lanyon and Rubin14 in an avian model system showed that dynamic loading of bone could lead to enhanced bone formation. Applying this theory to the human scenario, it is hypothesized that dynamic loading from the ligament during certain shoulder movements stimulates an osteogenic response within the acromial insertion site, thus encouraging enthesophyte formation. This, in turn, leads to further impingement and the production of Codman’s vicious cycle, which may lead to eventual cuff tear.7 These results reveal spatially defined differences in bone cell enzymatic activity with concomitant appositional bone growth within the enthesis of the coracoacromial ligament in patients with rotator cuff tears. This study demonstrates active bone formation in the presence of established rotator cuff tears, supporting the concept of spur formation being a secondary phenomenon. REFERENCES
1. Bigliani LU, Morrison DS, April EW. The morphology of the acromion and its relationship to rotator cuff tears. Orthop Trans 1986;10:216. 2. Bradbeer JN, Lindsay PC, Reeve J. Fluctuation of mineral apposition rate at individual bone remodeling sites in human iliac cancellous bone: independent correlations with osteoid width and osteoblastic alkaline phosphatase activity. J Bone Miner Res 1994;9:1679-86. 3. Burns WC, Whipple TL. Anatomic relationships in the shoulder impingement syndrome. Clin Orthop 1993;294:96-102. 4. Chambler AFW, Emery RJH. Acromial morphology: the enigma of terminology. Knee Surg Sports Traumatol Arthrosc 1997;5:268-72. 5. Chayen J. Quantitative cytochemistry: a precise form of cellular biochemistry. Biochem Soc Trans 1984;12:887-98. 6. Chayen J, Bitensky L. Practical histochemistry. New York: Wiley; 1991. 7. Codman E. Rare lesions of the shoulder. In: The shoulder. Boston: Thomas Todd; 1934. 468-509. 8. Dodds RA, Ali N, Pead MJ, Lanyon LE. Early loading-related changes in the activity of glucose 6-phosphate dehydrogenase in osteocytes and periosteal osteoblasts in rat fibulae in vivo. J Bone Miner Res 1993;8:261-7. 9. Dodds RA, Catterall A, Bitensky L, Chayen J. Effects on fracture healing of an antagonist of the vitamin K cycle. Calcif Tissue Int 1984;36:233-8. 10. Dodds RA, Gowen M, Bradbeer JN. Microcytophotometric analysis of human osteoclast metabolism: lack of activity in certain oxidative pathways indicates inability to sustain biosynthesis during resorption. J Histochem Cytochem 1994;42:599-606. 11. Frost H. Choice of marking agents and labelling schedule. In:
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12. 13. 14. 15. 16.
Recker RR, editor. Bone histomorphometry: techniques and interpretation. Boca Raton (FL): CRC Press; 1983. 37-52. Fu F, Klein AH. Shoulder impingement syndrome. Clin Orthop 1991;269:162-73. Harris JE, Blackney MC. The anatomy and function of the coracoacromial ligament. J Shoulder Elbow Surg 1993;2(Suppl):S6 [abstract]. Lanyon LE, Rubin CT. Static vs dynamic loads as an influence on bone remodelling. J Biomech 1984;17:897-905. Lee YS, Schlotzhauer T, Ott SM, et al. Skeletal status of men with early and late ankylosing spondylitis. Am J Med 1997;103:233-41. Neer CS. Anterior acromioplasty for the chronic impingement in the shoulder. A preliminary report. J Bone Joint Surg Am 1972;
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54:41-50. 17. Ozaki J, Fujimoto S, Nakagawa Y, Masuhara K, Tamai S. Tears of the rotator cuff of the shoulder associated with pathological changes in the acromion. J Bone Joint Surg Am 1988;70:1224-30. 18. Pitsillides AA, Rawlinson SCF, Mosley JR, Lanyon LE. Genetic selection for enhances growth at the expense of a skeletal adaptability to loading. J Bone Miner Res 1999;14:980-7. 19. Sarkar K, Taine W, Uhthoff HK. The ultrastructure of the coracoacromial ligament in patients with chronic impingement syndrome. Clin Orthop 1990;254:49-54. 20. Uhthoff HK, Hammond DI, Sarkar K, Hooper GJ, Papoff WJ. The role of the coracoacromial ligament in the impingement syndrome. Int Orthop 1988;12:97-104.
In this study we analyzed the acromial spurs of 15 patients with impingement syndrome undergoing open rotator cuff repair. Mineral apposition analysis and quantitative cytochemical techniques for glucose-6phosphate dehydrogenase (G6PD) activity (pentose phosphate pathway), alkaline phosphatase (ALP) activity (osteoblast activity), and tartrate-resistant acid phosphatase (TRAP) activity (osteoclast phenotype) were used to examine the distribution and level of activity of selected marker enzymes within the acromial spur insertion into the coracoacromial ligament in order to establish whether local behavior of bone cells is consistent with the proposed secondary development of the acromial spur. Our results indicate that G6PD and ALP activity was higher in osteoblasts on the inferior surface compared with the superior surface of the acromial spur in all patients (P ⬍ .001). This area correlated to the most intense area of mineral apposition shown by dual tetracycline labeling. TRAP activity revealed a heterogeneous distribution within the samples. A greater G6PD activity per cell (mean increase of 87%) was seen at the tip compared with that in post- and pre-tip zones within the coronal plane (P ⬍ .0002). The qualitative and quantitative enzyme analyses show that the acromial insertion of the coracoacromial ligament is actively involved in bone turnover. The spatial distribution patterns of metabolically active bone-forming osteoblastic cells compared with a heterogeneous distribution of TRAP-positive osteoclasts provide evidence of bone remodeling consistent with the morphologic contours of the acromial enthesis. The sites of oxytetracycline labeling appear to correlate with the sites of high ALP and G6PD activity, which From the Department of Orthopaedics,a St Mary’s Hospital, and Department of Veterinary Basic Sciences,b The Royal Veterinary College, London, United Kingdom. Reprint request: A. F. W. Chambler, BSc, MS, FRCS(Orth), 8 Woodford Mill, Mill Street, Witney, OX28 6DE. (E-mail: [email protected]). Copyright © 2003 by Journal of Shoulder and Elbow Surgery Board of Trustees. 1058-2746/2003/$35.00 ⫹ 0 doi:10.1016/S1058-2746(03)00030-2
314
supports the concept of spur formation being a secondary phenomenon in the presence of established rotator cuff tears. (J Shoulder Elbow Surg 2003;12: 314-21.)
I
t is well established that both intrinsic factors within the rotator cuff tendon and extrinsic factors from the surrounding soft- and hard-tissue structures appear to be related to each other in a complex relationship and that each may play a role in rotator cuff disease.12 Enthesophytic bony spurs have been proposed to be associated with rotator cuff tears. However, there has been debate as to the etiology of such acromial spurs and their precise role in impingement and rotator cuff disease. Bigliani et al1 showed a correlation between acromial morphology and rotator cuff tears, and this association has been used to support the theory of Neer16 for extrinsic factors as a cause of rotator cuff tears. Examination of the undersurface of the acromion, however, has led to the suggestion that the spur may represent a secondary change induced by a primary bursal-side cuff tear.17 Moreover, Uhthoff et al20 and Sarker et al19 concluded from their ultrastructural analyses of the coracoacromial arch that the coracoacromial ligament does not appear to be the primary factor in the initiation of impingement and the subsequent tear process. Together, these later studies would appear to support the theory of Codman7 for the intrinsic etiology of cuff tears. It is clear, therefore, that the pathogenesis and pathophysiology of this syndrome remain enigmatic. To establish whether indices of the bone remodeling process and the behavior of bone cells are consistent with the proposed secondary development of the acromial spur, we used mineral apposition analysis and quantitative cytochemical techniques to examine the distribution and activity of selected marker enzymes within both sagittal and coronal planes of the acromial spur insertion site into the coracoacromial ligament in patients with rotator cuff tears. These quantitative cytochemical techniques were used to measure glucose-6-phosphate dehydrogenase (G6PD) activity (pentose phosphate pathway), alkaline phosphatase (ALP) activity (osteoblastic activity),
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and tartrate-resistant acid phosphatase (TRAP) activity (osteoclastic phenotype) in sections of the acromial spur insertion site. This allowed assessment of spatial distribution and the level of activity of these selected marker enzymes within individual cells at defined histologic locations within the acromial spur insertion into the coracoacromial ligament. An analysis of these patterns of activity, together with oxytetracycline-based assessment of local bone formation rates, would establish whether local behavior of bone cells is consistent with the proposed secondary development of the acromial spur. METHODS Acromial samples were taken from 15 consecutive patients undergoing modified open acromioplasty and rotator cuff repair. All had medium-sized cuff tears. The mean age was 62.2 ⫾ 1.75 years, with a male-to-female ratio of 11:4. Ethical committee approval was obtained before the study was undertaken. Informed consent was obtained with regard to the reason for this study and the collection of suitable acromial samples. Patients also gave their permission to be given oxytetracycline tablets preoperatively to allow bone apposition rates to be studied. None of the patients who were approached refused to participate in this study.
Bone apposition analysis So that differences in bone apposition rates could be examined in these patients, two separate doses of oral fluorochrome were given.11,15 Thirteen patients were given oral oxytetracycline. The dosing regimen consisted of 250 mg of oxytetracycline twice a day for 2 days. This resulted in a total dose of 1 g over the 48-hour period. An interval of 2 weeks was imposed before this regimen was repeated. The patients then underwent surgery 4 days after the second dosing regimen. After cryostat sectioning, the samples were reviewed under an ultraviolet light microscope. Although the prerequisite use of cryostat sections for cytochemical analysis precluded the possibility of accurate measures of bone turnover to be determined, a semiquantitative blind assessment was undertaken for the detection of labels. Assessment of the quantity and extent of doublelabel surfaces in each of the samples was undertaken without knowledge of the patient’s details.
Sample collection and preparation The coracoacromial ligament was identified on the anterior aspect and inferior surface of the acromion extending to the coracoid process. The ligament was released near its coracoid attachment, leaving the insertion into the acromion intact for later analysis. A Neer acromioplasty was performed with an oscillating saw. This sample contained the anterior part of the acromion, the spur, and the insertion of the remaining coracoacromial ligament. Suture material and markings, made by a sterile marker pen, were used to facilitate orientation of the sample. Unwanted soft tissue
Figure 1 A, G6PD activity (formazan salt) on inferior surface of acromial sample (original magnification ⫻20). The salt appears blue, with darker color representing higher activity. B, G6PD activity (formazan salt) on superior surface of acromial sample (original magnification ⫻20).
was removed from the sample, and immediately after the sample was harvested, it was immersed for 3 minutes in an aqueous 5% (wt/vol) solution of polyvinyl alcohol (grade GO4). This immersion aided the formation of a strong bond when a specimen was mounted into the cryostat chuck. The sample was then chilled by precipitate immersion in a chilling bath containing n-hexane (BDH, Poole, UK) (low in aromatic hydrocarbons; boiling range, 67°C-70°C) in a small (15-mL) beaker surrounded by a slurry of carbon dioxide (dry ice, Guildford, Surrey, UK) and industrial alcohol. When the hexane had reached the temperature of ⫺65°C or below, the specimen was dropped straight in without touching the sides of the beaker and left for at least 1 minute before it was removed by means of cold forceps and placed in precooled specimen containers at ⫺70°C. These were then stored in carbon dioxide ice in an insulated container for transport. The specimens were mounted by anchoring them firmly onto a metal chuck with 5% polyvinyl alcohol before being placed in the cryostat. Sections were cut with a heavy-duty microtome in a Bright’s cryostat with a cabinet temperature of ⫺35°C to ⫺25°C (Bright Instruments, Huntingdon, UK). A tungsten-tipped steel knife was used, and the thickness of the sections was set at 10 m. Fifteen specimens were mounted in an orientation so that the sectioning could take
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Figure 2 G6PD activity at superior and inferior surfaces of acromial spurs in patients with rotator cuff tears. MIE, Mean integrated extinction.
place laterally to medially in the sagittal plane. This allowed comparison between the superior and inferior surfaces of the bony sample. Sectioning in 5 cases, anterior to posterior, in the coronal plane allowed comparison between the tip of the bony sample and its descending surfaces on either side.
Quantitative cytochemistry Fresh, unfixed, undecalcified sections were then analyzed by quantitative cytochemistry (Chayen5). This technique allows the biochemical activity within individual cells in the section to be related to the histology of the sample. It is a nondisruptive form of biochemistry (retaining spatial relationships between cells and their matrix) and between the subcomponents of the cells. It is very sensitive, so it can be used to measure enzymatic activities in a small biopsy specimen or a small area within that biopsy specimen. Consequently, accommodation of heterogeneity of the tissue within a sample can also be made. In these studies tetrazolium reactions were used for demonstration of G6PD activity, whereas azo-dye– coupling reactions were used for the detection of the acid and alkaline phosphate activities within the specimens. The methods used were those validated and described by Chayen and Bitensky6 and by Pitsillides et al.18
G6PD activity assay For G6PD activity assay, fresh, unfixed, undecalcified sagittal and coronal 10-m sections of the acromial spur insertion site were incubated in medium containing 3.3 mmol/L glucose-6-phosphate (disodium salt), 3 mmol/L nicotinamide adenine dinucleotide phosphate in 40% Polypep
(Bovine protein digest 5115 Sigma, Poole, UK) in 0.05 mol/L glycylglycine buffer, and 3.0 mmol/L nitro blue tetrazolium, at pH 8.0 and warmed to 37°C. This reaction medium was deoxygenated by bubbling with humidified nitrogen, and the intermediate hydrogen acceptor phenazine methosulfate (at 0.7 mmol/L) was added just before use. Perspex rings were placed around the sections to hold the reaction medium, and the sections were incubated at 37°C for 20 minutes. After the reaction, sections were washed in water, allowed to dry, and mounted in Aquamount (Merck, Poole, UK).
ALP activity assay For ALP activity assay, the incubation medium consisted of 2% barbital sodium, 0.16 mmol/L ␣–naphthyl acid phosphate, and 10% magnesium chloride adjusted to pH 9.2 at 37°C. The coupler, 1.0 mg/mL Fast Blue RR (Sigma, Poole, UK), was stirred into the medium and filtered immediately into Coplin jars (BDH, Poole, UK) containing the slides. At the end of the reaction time (4 minutes), the slides were washed in distilled water and placed in 1% acetic acid for 1 minute, after which they were rinsed in distilled water and allowed to dry.
TRAP activity assay For TRAP activity assay, the medium consisted of 1.4 mg/mL naphthol AS-TR phosphate (Sigma, Poole, UK), 5 L/mL N,N-dimethylformamide, and 0.1 mol/L sodium acetate and sodium tartrate adjusted to pH 5.2 with acetic acid. Fast Red TR (at 1.4 mg/mL, Sigma, Poole, UK) was used as the coupler, and after mixing, the medium was filtered immediately into Coplin jars containing the slides.
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G6PD and 5 for ALP and TRAP). In practice, a ⫻40 objective was used and a scanning spot was 0.4 m in diameter in the plane of the section. The values were calculated as mean integrated extinction of the colored end product per cell. In any spatially defined area, the enzyme activity in at least 10 cells was measured per slide, and 5 slides were used to investigate each area. Thus, the corresponding values of enzyme activity per cell within each histologic location for each patient represented a mean enzyme activity of at least 50 cells.
RESULTS Differences in bone cell enzyme activities in the sagittal plane
Figure 3 ALP activity on superior and inferior surfaces of acromial sample (original magnification ⫻20). Note difference also on trabecular surface (arrows). ALP activity appears blue/black on the pale bone background.
At the end of the reaction time (5 minutes), the slides were washed in distilled water, allowed to dry, and mounted in Aquamount. The relative distribution and activity per cell of G6PD (pentose phosphate pathway),8 ALP (marker of osteoblastic phenotype),2 and TRAP (marker of osteoclast phenotype)10 were measured in individual cells in distinct histologically defined zones by scanning and integrating microdensitometry.
Microdensitometry The amount of reaction product per cell was measured by a Vickers M85A scanning and integrating microdensitometer (Vickers Instruments, York, UK). Microdensitometry is a means by which to measure the mass of a chromophore (or the optical density of a colored end product) in a defined projected area of material, such as an individual cell. It differs from spectrophotometry in that microdensitometry is a microscopic technique, able to dissect specific regions of sections of a tissue optically and to measure the amount of chromophore within cells. Monochromatic light (585 nm for G6PD and ALP and 550 nm for TRAP) is used to scan the area enclosed within the masked field (mask size 4 for
G6PD activity. The mean G6PD activity per cell in osteoblasts on the inferior aspect of the tip of the acromial enthesis (0.92 ⫾ 0.04 [mean ⫾ SEM]) (Figure 1, A) was statistically significantly higher (P ⬍ .001, paired t test) than on the superior aspect (0.80 ⫾ 0.03) (Figure 1, B). This increased level of G6PD activity on the acromion’s inferior aspect was also evident, within any single patient, when the activity of these two sites were compared (Figure 2), and this was also reflected in a significant direct correlation (P ⬍ .001, r2 ⫽ 0.863) between resident cells’ G6PD activity on the inferior and superior surfaces in different patients. ALP activity. In all 15 acromial enthesophytes examined, the ALP activity in bone cells on the inferior aspect was significantly higher (P ⬍ .001) than on the superior aspect of the acromion (Figure 3), with mean ALP activity per cell of 0.89 ⫾ 0.04 and 1.02 ⫾ 0.03 on the inferior and superior aspects, respectively (Figure 4). In addition, further analysis of these results showed that intersite variability within specimens exhibited a direct and statistically significant correlation (P ⬍ .0001, r2 ⫽ 0.856). TRAP activity. The mean level of TRAP activity per cell in different sections of the same sample, each reacted for different reaction times (2, 5, 10, 15, and 20 minutes), failed to show a consistent trend. An assessment of the distribution of TRAP-positive bone cells failed to show any consistent spatial pattern of osteoclast distribution, exhibiting marked heterogeneity throughout sagittal-tissue sections. The surfaces of the bone trabeculae and not external aspects of the enthesophyte most commonly contained the largest numbers of TRAP-positive cells (Figure 5). Differences in bone cell enzyme activities in the coronal plane
G6PD activity in individual bone cells was assessed within the spur at sites defined in the coronal plane in 5 patients. In 4 of 5 patients (Figure 6), G6PD activity was consistently higher in the cells at the tip of the spur than at either side of the spur’s tip. These samples showed a greater G6PD activity per
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Figure 4 ALP activity on superior and inferior surfaces of acromial spur in patients with rotator cuff tears. MIE, Mean integrated extinction.
showed no significant difference in the level of activity per cell. Nevertheless, it was apparent that the variation in TRAP activity was considerable between different samples (range at tip, 1.82-0.63). Fluorochrome labeling in acromial spurs in patients with rotator cuff tears
Figure 5 TRAP-positive (osteoclastic) cells within acromial spur (original magnification ⫻20). Activity appears red, with darker color representing higher activity. CA, Coracoacromial.
cell (mean increase of 87%) at the tip compared with that in the post- and pre-tip zones within the coronal plane (P ⬍ .0002). In contrast to the spatially selected increase in G6PD activity evident in cells at the various sites of the coronal plane, in all but 1 (of 4) of the samples examined, bone cell ALP activity showed no variation between the sites at or on either side of the enthesophyte’s tip. TRAP activity per cell at different sites in each individual sample was compared in sections reacted for 5 minutes. In each of the 3 sites assessed in the coronal plane, osteoclasts in the enthesophytes
A semiquantitative assessment for the detection of labels, their quantity, and the extent of double-labeled surfaces was undertaken. No tetracycline labeling was seen in the control sample (an acromial sample from a patient who did not undergo oxytetracycline labeling) or in 3 samples from the patient group. This was assumed to be the result of a lack of compliance rather than failure of mineralization. Of the remaining 9 patients, 5 showed pronounced evidence of active bone apposition as defined by the scoring system. Tetracycline labeling yielded different patterns of distribution throughout the samples. Figure 7 reveals a double label at the bone surface, where the coracoacromial ligament has its attachment. Figure 8 reveals a double label only on one side of the trabeculae, which may represent a directional drift. This pattern was frequently seen and not thought to be cutting artifact. DISCUSSION This study concentrated on the coracoacromial ligament’s insertion onto the acromion, namely the
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Figure 6 Mean G6PD activity at pre-tip, tip, and post-tip sites in trabecular bone in acromial spur in patients with rotator cuff tears. MIE, Mean integrated extinction.
Figure 7 Double labeling seen at bone-ligament insertion site (original magnification ⫻40). Oxytetracycline appears yellow under ultraviolet light, and bone appears green.
Figure 8 Double labeling seen on one aspect of a trabecula: Directional effect (original magnification ⫻40). Oxytetracycline appears yellow under ultraviolet light, and bone appears green.
enthesis, and thus bone growth at the ligament insertion is described as an enthesophyte.4 Therefore, the commonly used term spur in this text refers to an acromial enthesophyte at the coracoacromial ligament’s insertion. G6PD activity is a rate-limiting step of the pentose phosphate shunt, which is used during energy metabolism for the production of reducing equivalents that contribute to many synthetic pathways, including those involved in mineralization, but specifically for the production of pentose nucleic acid synthesis.8 Our findings indicate that in all of the patients studied, G6PD activity was higher in the cells on the inferior surface of the acromial sample than on the superior
surface. These location-selective elevations in periosteal cell G6PD activity may, therefore, reflect persistent increases in matrix synthesis on the acromion’s inferior aspect. Similar persistent increases in G6PD activity have previously been described to be a component of the response of osteoblasts to increases in applied load bearing in vivo,8 and it is thus possible that they represent either enhanced rates of local matrix synthesis or a component of the local response to changes in dynamic mechanical loading. Furthermore, previous studies have also shown increases in bone cell G6PD activity at the site of subsequent new bone formation associated with fracture healing.9 The results observed suggest that the predominance of the
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inferior-aspect G6PD activity reflects elevated rates of bone formation, which may be promoted at this site by recent episodes of increased local mechanical loading. ALP activity has also been linked to bone formation by several authors.2,10 Indeed, measurements of mineral apposition rate and ALP activity in iliac crest biopsy specimens have shown correlation between fluctuations in mineral apposition rate and osteoblastic ALP activity, suggesting that bone formation appears to be at least partly dependent upon ALP activity and osteoblastic energy balance.2 Therefore, the activity of this enzyme was used in this study as a quantitative cytochemical indicator of local boneforming capacity of the acromial insertion of the coracoacromial ligament. The comparison of acromial osteoblasts on the superior and inferior surfaces (in the sagittal plane) did show differences, with the activity in the acromial osteoblasts on the inferior surface being significantly greater than on the superior surface in all 15 patients studied. As well as being consistent with the proposed increased rate of bone formation at these sites, these observations support a persistent loading-related elevation of local osteogenesis. The qualitative and quantitative enzyme analyses show that the acromial insertion of the coracoacromial ligament is likely to be actively involved in bone turnover. The spatial distribution patterns of metabolically active bone-forming osteoblastic cells with high G6PD and ALP activity showing a predominance on its anteroinferior aspect compared with a heterogeneous distribution of TRAP-positive osteoclasts provides evidence of bone remodeling consistent with a morphologic contour of the acromial sample. The sites of oxytetracycline labeling appear to correlate with the sites of high ALP and G6PD activity, which supports the notion for the presence of an osteogenic potential within the coracoacromial ligament’s insertion. Because of the lack of specimens, this study cannot describe the relative activities of these enzymes in bone cells in acromions in appropriate control groups, such as patients with impingement without rotator cuff tears or patients without impingement or indeed normal shoulders. Consequently, it is evident that our study does not rule out the possibility that such patterns of activity and the high rates of bone formation are not necessarily a characteristic that is always evident at the inferior aspect of the acromial insertion site. For this reason, it also remains possible that these high apparent rates of turnover represent a normal pattern of cellular behavior at all ligament insertions exposed to increased loading. However, given the quantitative nature of the increases in G6PD and ALP activities, our findings nevertheless support the con-
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clusion that higher rates of bone turnover are likely to be evident at the acromion’s inferior aspect. The mechanism by which subacromial impingement may contribute to producing an acromial enthesophyte cannot be determined from this study. It has been suggested, however, that the coracoacromial ligament is under a constant static tension.13 Furthermore, Burns and Whipple3 have demonstrated, in cadaveric studies, that the ligament is forced upward during impinging movements of the shoulder, adding a dynamic component to the tension experienced by the insertion of the ligament. Investigations by Lanyon and Rubin14 in an avian model system showed that dynamic loading of bone could lead to enhanced bone formation. Applying this theory to the human scenario, it is hypothesized that dynamic loading from the ligament during certain shoulder movements stimulates an osteogenic response within the acromial insertion site, thus encouraging enthesophyte formation. This, in turn, leads to further impingement and the production of Codman’s vicious cycle, which may lead to eventual cuff tear.7 These results reveal spatially defined differences in bone cell enzymatic activity with concomitant appositional bone growth within the enthesis of the coracoacromial ligament in patients with rotator cuff tears. This study demonstrates active bone formation in the presence of established rotator cuff tears, supporting the concept of spur formation being a secondary phenomenon. REFERENCES
1. Bigliani LU, Morrison DS, April EW. The morphology of the acromion and its relationship to rotator cuff tears. Orthop Trans 1986;10:216. 2. Bradbeer JN, Lindsay PC, Reeve J. Fluctuation of mineral apposition rate at individual bone remodeling sites in human iliac cancellous bone: independent correlations with osteoid width and osteoblastic alkaline phosphatase activity. J Bone Miner Res 1994;9:1679-86. 3. Burns WC, Whipple TL. Anatomic relationships in the shoulder impingement syndrome. Clin Orthop 1993;294:96-102. 4. Chambler AFW, Emery RJH. Acromial morphology: the enigma of terminology. Knee Surg Sports Traumatol Arthrosc 1997;5:268-72. 5. Chayen J. Quantitative cytochemistry: a precise form of cellular biochemistry. Biochem Soc Trans 1984;12:887-98. 6. Chayen J, Bitensky L. Practical histochemistry. New York: Wiley; 1991. 7. Codman E. Rare lesions of the shoulder. In: The shoulder. Boston: Thomas Todd; 1934. 468-509. 8. Dodds RA, Ali N, Pead MJ, Lanyon LE. Early loading-related changes in the activity of glucose 6-phosphate dehydrogenase in osteocytes and periosteal osteoblasts in rat fibulae in vivo. J Bone Miner Res 1993;8:261-7. 9. Dodds RA, Catterall A, Bitensky L, Chayen J. Effects on fracture healing of an antagonist of the vitamin K cycle. Calcif Tissue Int 1984;36:233-8. 10. Dodds RA, Gowen M, Bradbeer JN. Microcytophotometric analysis of human osteoclast metabolism: lack of activity in certain oxidative pathways indicates inability to sustain biosynthesis during resorption. J Histochem Cytochem 1994;42:599-606. 11. Frost H. Choice of marking agents and labelling schedule. In:
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Recker RR, editor. Bone histomorphometry: techniques and interpretation. Boca Raton (FL): CRC Press; 1983. 37-52. Fu F, Klein AH. Shoulder impingement syndrome. Clin Orthop 1991;269:162-73. Harris JE, Blackney MC. The anatomy and function of the coracoacromial ligament. J Shoulder Elbow Surg 1993;2(Suppl):S6 [abstract]. Lanyon LE, Rubin CT. Static vs dynamic loads as an influence on bone remodelling. J Biomech 1984;17:897-905. Lee YS, Schlotzhauer T, Ott SM, et al. Skeletal status of men with early and late ankylosing spondylitis. Am J Med 1997;103:233-41. Neer CS. Anterior acromioplasty for the chronic impingement in the shoulder. A preliminary report. J Bone Joint Surg Am 1972;
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54:41-50. 17. Ozaki J, Fujimoto S, Nakagawa Y, Masuhara K, Tamai S. Tears of the rotator cuff of the shoulder associated with pathological changes in the acromion. J Bone Joint Surg Am 1988;70:1224-30. 18. Pitsillides AA, Rawlinson SCF, Mosley JR, Lanyon LE. Genetic selection for enhances growth at the expense of a skeletal adaptability to loading. J Bone Miner Res 1999;14:980-7. 19. Sarkar K, Taine W, Uhthoff HK. The ultrastructure of the coracoacromial ligament in patients with chronic impingement syndrome. Clin Orthop 1990;254:49-54. 20. Uhthoff HK, Hammond DI, Sarkar K, Hooper GJ, Papoff WJ. The role of the coracoacromial ligament in the impingement syndrome. Int Orthop 1988;12:97-104.