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Biochemical composition and histologic structure of the forearm interosseous membrane
Biochemical Composition and Histologic Structure of the Forearm Interosseous Membrane Joseph C. McGinley, MS, Joshua E. Heller, BS, Andrzej Fertala, PhD, John P. Gaughan, PhD, Scott H. Kozin, MD, Philadelphia, PA
Purpose: The purpose of this study was to determine the structure and composition of the forearm interosseous membrane (IOM). Methods: The IOM of 12 cadaver forearms was fixed in formalin. After fixation 5 individual IOM fiber bundles per arm were separated by dissection, excised, and processed with hematoxylineosin, trichrome, and Verhoff-vanGeison stains. Nine additional fresh forearms were dissected and 5 IOM fiber bundles per arm were analyzed using the hydroxyproline assay. Bundles were evaluated at ulnar, central, and radial locations. Results: Histologic analysis of the IOM bundles obtained from the 12 fixed forearms showed an abundance of collagen in the main bundle central location (84% ⫾ 7.8%). A progressive increase in collagen was noted from distal to proximal bundles (r ⫽.72). The hydroxyproline assay of collagen content of the main IOM bundle’s central location from the 9 additional fresh forearms was 99.3% ⫾ 16.5%. There was no difference between bundles or location (power ⫽ 0.25 and 0.46). Conclusions: We found that the IOM possesses a large collagen content arranged in fibrillar structures surrounded by elastin. Collagen was abundant in the proximal bundles and decreased in the distal bundles. (J Hand Surg 2003;28A:503–510. Copyright © 2003 by the American Society for Surgery of the Hand.) Key words: Interosseous membrane, forearm, histology, biochemical.
The interosseous membrane (IOM) of the forearm is a unique anatomic structure that connects the radius and ulna. The IOM is described as a soft tissue From the School of Medicine, Department of Biostatistics, Temple University; Department of Dermatology and Cutaneous Biology, Thomas Jefferson University, Philadelphia; and Shriners Hospital for Children, Philadelphia, PA. Received for publication August 20, 2002; accepted in revised form January 13, 2003. No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article. Reprint requests: Scott H. Kozin, MD, Shriners Hospital for Children, Pediatric Hand & Upper Extremity Surgery, 3551 N Broad St, Philadelphia, PA 19140. Copyright © 2003 by the American Society for Surgery of the Hand 0363-5023/03/28A03-0023$30.00/0 doi:10.1053/jhsu.2003.50059
located within the interosseous space of the forearm that possesses a distinct oblique radioulnar direction of 20°.1,2 The IOM is the origin of several muscle groups within the forearm (flexor digitorum profundus, flexor pollicis longus, extensor pollicis brevis, abductor pollicis longus, extensor indicis, and extensor pollicis longus).3,4 There are several fibrous bundles directed from the radius to the ulna surrounded by a supporting matrix.5 Each fiber bundle represents a distinct grouping of collagen fibrils and shows a consistent organization with several large bundles located proximally within the central third of the IOM and a varying number of smaller bundles located distally within the forearm.5 The functional integration of the various fiber bundles and the supporting matrix provides the mechanics necessary for The Journal of Hand Surgery
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Figure 1. (A) IOM bundles were dissected as a distinct fiber group with a complete enclosing sheath. This figure shows the location of bundle excision. A varying number of larger distinct fiber bundles were found within the central third of the IOM. Smaller distal fibers were located in the membranous region of the IOM. (B) Each fiber bundle was excised from the forearm at a point 2 mm from the bony insertion thereby eliminating the contribution of sharpy fibers. Each fiber bundle was further dissected into a radial, central, and ulnar portion.
transferring forces in a graded fashion from the radius to the ulna. Several studies have investigated the various functions of the IOM. Rabinowitz et al6 described the role of the IOM in forearm stability and determined that considerable proximal migration of the radius (⬎7 mm) results from damage to its midportion and triangular fibrocartilage complex. Werner and Koebke7 identified the function of the IOM in relation to its origin for the extensor and flexor musculature and showed that muscle fibers formed specific angles with the membrane that result in the anatomic orientation of the fibers within the IOM. Shang et al8 studied the function of the IOM in coronal forearm stability. Tensile force between the radius and ulna tended to separate the 2 bones, which tightened the IOM and resisted separation. Pfaeffle et al9 recently described the IOM as ligamentous in function secondary to its bone-to-bone connection and mechanical stiffness. Previous researchers have described the anatomy and mechanics of the IOM. The purpose of our study was to characterize the microstructure and collagen organization of the IOM using histologic and biochemical analysis and relate its composition to forearm function.
Methods Dissection Twenty-one fresh-frozen forearms of cadavers aged 45 to 86 years were thawed in a warm isotonic saline solution. Each forearm was dissected, removing the overlying soft tissues and musculature and isolating
the radius, ulna, and IOM. Each arm was then photographed using a fluorescent light to show the gross organization of the IOM fiber bundles. For the histologic studies described later 12 of the dissected forearm specimens, aged 60 to 86 years (73.2 ⫾ 10.9 y), were fixed in a 10% formalin solution for 48 hours to ensure complete fixation. The remaining 9 freshly dissected forearm specimens (nonfixed), of cadavers aged 45 to 79 years (70.3 ⫾ 11.4 y), were used for the biochemical analysis described later. Using the fixed forearm specimens for histology and the fresh forearm specimens for biochemistry 5 discrete IOM fiber bundles were isolated from each arm at various locations (Fig. 1A). An IOM fiber bundle was defined as an independent dissectible grouping of fibers supported by an enclosing sheath. A total of 60 fixed IOM fiber bundles were obtained for histology (5 per arm) and 45 fresh IOM fiber bundles were obtained for biochemical analysis. Each bundle was dissected at a distance of 2 mm from the bony insertion thereby eliminating the contribution of Sharpey’s fibers. The location of the origin, center, and insertion of each bundle excised was measured relative to its position along the radius and reported as a percentage distance (actual distance divided by total radius length) from the styloid process of the radius. Each IOM fiber bundle was then sharply dissected into 3 samples based on location: radial, central, and ulnar (Fig. 1B).
Histologic Studies The fixed samples were embedded in paraffin and sectioned with a microtome into 7-m slices and
McGinley et al / Composition and Structure of Forearm IOM
placed on a slide. The slides were stained using a hematoxylin-eosin, trichrome, and Verhoff-vanGeison stain.10 Each slide was examined at various magnifications to qualitatively describe the IOM structure and organization including collagen and elastin distribution. The central regions of each trichromestained sample were photographed using a digital camera at a magnification of 40 to quantitatively analyze the collagen content. The trichrome images were analyzed and the staining intensity distribution was determined (SigmaScan Pro software, SPPS Inc, Chicago, IL). The number of pixels in a measured distance was determined by using a calibrated micrometer slide. By using the calibrated values of distance the area of each section was measured. Threelayer settings were used based on calibrated intensity values for collagen and elastin. Layer 1 included the collagenous tissue, layer 2 represented elastin and other noncollagenous tissue, and layer 3 included the entire membrane area. With the calibrated distance the percentage of collagen was determined by dividing layer 1 (collagen area) by layer 3 (total area). The results were plotted based on the percentage distance from the styloid process of the radius, and comparisons of collagen content were made relative to the location within the forearm.
Biochemical Analysis (Hydroxyproline Assay) Each fresh sample obtained from the dissection described earlier was placed into a cold porcelain mortar pan and filled with liquid nitrogen.11 The sample was crushed into powder form, dried in an oven at 80 °C, and weighed (dry weight). The sample was then hydrolyzed using 12 mol/L of hydrochloric acid. The hydrochloric acid was evaporated under a chemical hood, leaving a sample residue that was resuspended in deionized water. The samples were diluted for spectrophotometer readings of hydroxyproline content. Hydroxyproline standards (Biocolor Ltd., Newtownabbey, Northern Ireland) were prepared using 0, 0.5, 1, 3, 4, and 5 g of collagen (at higher concentrations the relation between absorbance and collagen concentration becomes nonlinear).12 Collagen concentration was calculated based on a 10% hydroxyproline content. Chloramine T, perchloric acid, and Ehrlich’s reagent were added to each standard and sample vial. After a short incubation period the absorbance of each sample was recorded at a wavelength of 557 nm. The data were analyzed based on the section origin (radial, central, ulnar) and location with regard to the radial styloid.
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Figure 2. Trichrome stain of the central location of the main IOM bundle (⫻40). The solid arrows indicate the individual collagen fascicles and the dashed arrow shows the supporting elastin sheath.
Statistical Methods The dependent variables (ie, collagen content [trichrome staining] and collagen content measured by hydroxyproline) were treated as continuous variables for all analyses. Means, standard deviations, standard errors, and p values are presented for the different analyses by percentage distance from the styloid process and by bundle and location. The experimental design was a 2-factor (bundle and location) mixed design with repeated measures on both factors. The null hypothesis was that there was no difference in the measured parameters among the different bundles and locations. Before analysis all data were tested for normality using the ShapiroWilk test.13 The data for the dependent variables were significantly nonnormal. To apply ANOVA methods, a normalized-rank transformation was applied to the data.14,15 The rank-transformed data were analyzed by using a mixed-model ANOVA for repeated measures followed by multiple comparisons to detect significant individual mean differences between bundles and locations. Multiple pair-wise comparisons used the Dunn-Bonferroni adjustment to maintain an experiment-wise type I error of .05 or less.16 Differences between group means (rejection of the null hypothesis) were considered significant when the probability of chance occurrence was .05 or less using 2-tailed tests. Collagen content was plotted versus percentage distance to examine the relationship between distance from the styloid process and collagen.
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Figure 3. Trichrome stain of the central location of the main IOM bundle (⫻400). The solid arrow indicates the substance of a single collagen fascicle with the arrowhead showing the surrounding elastin sheath. The dashed arrow highlights the surrounding elastin fibers grouping the collagen fascicles into a single collagen bundle.
Results Histology: 12 Arms The staining of the histologic sections revealed an abundance of collagen fibers with small amounts of elastin fibers. An orderly array of collagen fibers perpendicular to the cutting plane was observed. The collagen fibers were arranged in parallel into fascicles (Figs. 2, 3) with blood vessels and nerves located between fascicles. The elastin organization of the IOM was visualized as the areas of reduced
Figure 5. Collagen content distribution (percent of total area) from the radial styloid process using image intensity analysis of trichrome-stained histologic sections.
trichrome staining of the histologic specimens. The elastin provided a supporting structure surrounding each individual collagen fascicle and as an outer sheath grouping the fascicles into a single IOM bundle. The results of the image intensity analysis are summarized in Figure 4. The average data are based on the numbered IOM fiber bundle (1 ⫽ distal, 5 ⫽ proximal). The percentage distance from the styloid process of the radius of each fiber bundle was found to equal 37.2% ⫾ 5.2%, 45.7% ⫾ 3.8%, 51.7% ⫾
Figure 4. Collagen content of bundles 1–5 using digital analysis of trichrome-stained sections and hydroxy-proline assay. For histologic sections collagen content was calculated as a percentage of the total area and for biochemical analysis collagen content was determined based on the percentage of dry weight.
McGinley et al / Composition and Structure of Forearm IOM
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Figure 6. There were no significant differences in collagen content based on location within the forearm using hydroxy-proline analysis.
4.4%, 57.3% ⫾ 3.4%, and 63.5% ⫾ 3.6% for bundles 1 to 5, respectively. Image analysis of the trichrome-stained sections showed an abundance of collagen with an overall average value of 73.1% ⫾ 14.9%. The main fiber bundle (bundle 4) was composed of 84.1% ⫾ 7.8% collagen. The relative amount of collagen increased from the distal to proximal bundles (Fig. 5). Analysis of differences between bundles showed that bundles 1 and 2 contained
Table 1. Bundle Collagen Content Within Each Location (Hydroxy-Proline Assay)
Location Radial
Central
Ulnar
Bundle Number
Distance From Styloid Process (%)
Collagen Content (%)
1 2 3 4 5 1 2 3 4 5 1 2 3 4 5
43.0 ⫾ 4.4 47.3 ⫾ 6.1 51.8 ⫾ 5.3 57.7 ⫾ 5.2 62.6 ⫾ 6.4 38.7 ⫾ 4.0 43.1 ⫾ 6.2 48.2 ⫾ 5.5 54.4 ⫾ 5.8 59.9 ⫾ 5.3 34.4 ⫾ 4.0 38.9 ⫾ 6.9 44.6 ⫾ 5.2 51.0 ⫾ 6.5 57.2 ⫾ 4.7
87.6 ⫾ 18.2 90.7 ⫾ 12.9 88.5 ⫾ 12.3 85.8 ⫾ 10.8 94.4 ⫾ 18.7 84.5 ⫾ 15.8 94.9 ⫾ 6.8 92.0 ⫾ 8.5 92.8 ⫾ 5.1 99.3 ⫾ 16.5 96.3 ⫾ 16.7 82.6 ⫾ 11.9 86.6 ⫾ 8.5 86.5 ⫾ 9.8 86.0 ⫾ 8.1
significantly less collagen than bundles 4 and 5 (p ⬍ .001).
Collagen Content: 9 Arms The results of the hydroxyproline assay are shown in Figures 4 and 6 and Table 1. The percentage distance from the styloid process of the radius of each fiber bundle was found to equal 38.7% ⫾ 4.0%, 43.1% ⫾ 6.2%, 48.2% ⫾ 5.5%, 54.4% ⫾ 5.8%, and 59.9% ⫾ 5.3% for bundles 1 to 5, respectively. Figure 4 shows the collagen content based on fiber bundle (bundle 1 ⫽ distal, bundle 5 ⫽ proximal). Figure 6 shows the collagen percentages based on section location (radial, central, ulnar). Table 1 further classifies the data based on bundle number in each location. Column 1 lists the section location, column 2 lists the bundle number, column 3 lists bundle distance and column 4 summarizes the average collagen content. The average collagen content of the main fiber bundle (bundle 5) of the IOM was 93.2% ⫾ 15.5% (n ⫽ 24). Using the hydroxyproline assay there was no significant difference in collagen content between bundles (p ⬍ .67, power ⫽ 0.25). Comparisons of average collagen content of each section location (radial, central, ulnar) also showed no significant differences (89.4% ⫾ 14.5%, 92.7% ⫾ 12.0%, and 87.6% ⫾ 11.8%, respectively; p ⬍ .52; power ⫽ 0.46). Based on the hydroxyproline assay there was no relationship between collagen content and posi-
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Figure 7. Analysis of the IOM’s radial origin collagen content using the hydroxy-proline assay. Collagen content (percent dry weight) is plotted versus the percent distance from the radial styloid process.
tion within the forearm in the radial and central locations and a minimal negative relationship at the ulnar location (Figs. 7, 8, 9).
Discussion The IOM has been described as a membrane, tendon, or ligament. Mechanical studies imply the IOM has a ligamentous function.9 Complete understanding of the IOM requires investigation of its anatomic arrangement, histology, composition, and function. Tendons are defined as cord-like structures that connect muscle to bone and possess an orderly arrangement of collagen fibrils that assemble into parallel fiber bundles.17 The collagen content of tendons is approximately 90% by dry weight with a minimal elastin contribution (5% to 7%).18 Because of the large portion of collagen relative to elastin, tendons possess superior tensile strength and are able to resist large tensile forces. Tendons also have a large elastic modulus and are mechanically stiff structures.18 In contrast ligaments connect bone to bone and their structure is arranged in a less orderly manner than tendons. The bone-to-bone connection requires a more elastic mechanical structure to permit some displacement when loaded in tension. The increased elasticity is a result of a lower collagen content (25% to 85%) and a less regularly arranged microstructure resulting in a decreased elastic modulus and tensile strength.18 Ligaments cannot withstand the large tensile forces maintained by tendons. To understand IOM physiology and analyze the function of the IOM within the forearm we compared published chemical
and mechanical studies of tendons and ligaments with our structural and biochemical findings. Grossly the IOM is a fibrous structure located within the interosseous space of the forearm. Its fiber bundles originate from the radius and are directed obliquely to the ulna (bone-bone connection). Numerous flexor and extensor muscle groups have origins on the dorsal and ventral surfaces of the IOM (muscle-bone connection). The bundles are arranged in distinct visible groups (main and accessory fiber bundles) and are surrounded by an overlying matrix. The trichrome stains of the histologic sections of the IOM showed an abundance of collagen arranged in an orderly fashion (Figs. 2, 3). The cross-sections of the IOM showed that the fibers are arranged in a parallel fashion perpendicular to the cutting plane. The collagen fibrils are grouped into fibers surrounded by an enclosing sheath. Furthermore the collagen fibers are subdivided into fascicles by an extension of the sheath. The hematoxylin-eosin and Verhoeff-vanGieson stains of the sections showed that the enclosing structures are composed largely of elastin. The elastin content in the membrane structure is minimal, functioning as a sheath around the collagen groups. There are several different types of collagen ␣ chains that are used to classify collagen into the categories of type I to XVI. Loose and dense connective tissue are composed mostly of type I collagen, which belongs to a group of fibrillar collagens and consists of 2 ␣ 1 chains and one ␣ 2 chain. The fibrillar collagen molecule is composed of 3 polypep-
Figure 8. Analysis of the IOM’s central location collagen content using the hydroxy-proline assay. Collagen content (percent dry weight) is plotted versus the percent distance from the radial styloid process.
McGinley et al / Composition and Structure of Forearm IOM
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Figure 9. Analysis of the IOM’s ulnar insertion collagen content using the hydroxy-proline assay. Collagen content (percent dry weight) is plotted versus the percent distance from the radial styloid process.
tide chains (␣ chains) interwoven into a right-handed helix.19 The triple helix shape is a result of the orderly arrangement of glycine-proline-hydroxyproline structure. The amino acid hydroxyproline makes up approximately 10% of the total amino acid content of collagen. Hydroxyproline is unique to collagen and is used as a marker to analyze the collagen content of various tissues. The determination of the collagen percentage was based on the hydroxyproline assay, which measures the amount of hydroxyproline present within the collagen triple helix. This assay provides an accurate determination of the collagen content. Collagen represented 99.3% ⫾ 16.5% of the central portion of the central fiber bundle. Our results showed an abundance of collagen across the IOM. The percentage of collagen for the radial, central, and ulnar portion of each fiber bundle was 89.4% ⫾ 14.5%, 92.7% ⫾ 12.0%, and 87.6% ⫾ 11.8%, respectively. There was no between-group difference or between–fiber bundle difference in collagen content. The large collagen content along with the orderly arrangement of the fiber bundles results in a structure with a large tensile strength that provides the necessary mechanical properties for transferring large forces from the radius to the ulna. Furthermore the angular direction of the fibers provides an origin for the muscular groups in line with its collagen structure. The histologic organization of the IOM shows a dense connective tissue with an abundance of colla-
gen arranged in parallel surrounded by an elastin sheath. Biochemical analysis confirms the large collagen composition. The collagen content and distinct fiber organization of the IOM result in superior mechanical properties that are able to withstand large tensile loads.
References 1. Schneiderman G, Meldrum RD, Bloebaum RD, Tarr R, Sarmiento A. The interosseous membrane of the forearm: structure and its role in Galeazzi fractures. J Trauma 1993; 35:879 – 885. 2. Skahen JR III, Palmer AK, Werner FW, Fortino MD. The interosseous membrane of the forearm: anatomy and function. J Hand Surg 1997;22A:981–985. 3. Hollister AM, Gellman H, Waters RL. The relationship of the interosseous membrane to the axis of rotation of the forearm. Clin Orthop 1994;298:272–276. 4. Netter FH. Atlas of human anatomy. East Hanover, NJ: Novartis, 1989:424 – 425. 5. McGinley JC, Kozin SH. Interosseous membrane anatomy and functional mechanics. Clin Orthop 2001;383:108 –122. 6. Rabinowitz RS, Light TR, Havey RM, Gourineni P, Patwardhan AG, Sartori MJ, et al. The role of the interosseous membrane and triangular fibrocartilage complex in forearm stability. J Hand Surg 1994;19A:385–393. 7. Werner JA, Koebke J. The function of the antebrachial interosseous membrane. Anat Embryol 1987;176:127–131. 8. Shang T-Y, Gu Y-W, Dong F-H. Treatment of forearm bone fractures by an integrated method of traditional Chinese and western medicine. Clin Orthop 1987;215:56 – 64. 9. Pfaeffle HJ, Tomaino MM, Grewal R, Xu J, Boardman ND, Woo SL-Y, et al. Tensile properties of the interosseous
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The Journal of Hand Surgery / Vol. 28A No. 3 May 2003 membrane of the human forearm. J Orthop Res 1996;14: 842– 845. Bancroft JD, Cook HC. Manual of histological techniques. New York: Churchill Livingstone, 1984:39 – 62. Prockop DJ, Udenfriend S. A specific method for the analysis of hydroxyproline in tissues and urine. Anal Biochem 1960;1:228 –239. Woessner JF. The determination of hydroxyproline in tissue and protein samples containing small proportions of this amino acid. Arch Biochem Biophys 1961;93:440 – 447. Armitage P, Berry G. Statistical methods in medical research. 3rd ed. Cambridge, MA: Blackwell Science, 1994: 395–398. Conover WJ, Iman RL. Rank transformations as a bridge
15. 16.
17. 18. 19.
between parametric and nonparametric statistics. Am Statistician 1981;35:124 –129. Harter HL. Expected values of normal order statistics. Biometrika 1981;48:151–165. Winer BJ, Brown DR, Michels KM. Statistical principles in experimental design. 3rd ed. New York: McGraw-Hill, 1991:158 –166. Ross MH, Romrell LJ, Kaye GI. Histology. A text and atlas. 3rd ed. Baltimore: Williams & Wilkins, 1995:94 –125. Vincent JFV, ed. Biomechanics. Materials. A practical approach. New York: Oxford University Press, 1992:75–98. Marks DB, Marks AD, Smith CM. Basic medical biochemistry. A clinical approach. Baltimore: Williams & Wilkins, 1996:94 –95.
Purpose: The purpose of this study was to determine the structure and composition of the forearm interosseous membrane (IOM). Methods: The IOM of 12 cadaver forearms was fixed in formalin. After fixation 5 individual IOM fiber bundles per arm were separated by dissection, excised, and processed with hematoxylineosin, trichrome, and Verhoff-vanGeison stains. Nine additional fresh forearms were dissected and 5 IOM fiber bundles per arm were analyzed using the hydroxyproline assay. Bundles were evaluated at ulnar, central, and radial locations. Results: Histologic analysis of the IOM bundles obtained from the 12 fixed forearms showed an abundance of collagen in the main bundle central location (84% ⫾ 7.8%). A progressive increase in collagen was noted from distal to proximal bundles (r ⫽.72). The hydroxyproline assay of collagen content of the main IOM bundle’s central location from the 9 additional fresh forearms was 99.3% ⫾ 16.5%. There was no difference between bundles or location (power ⫽ 0.25 and 0.46). Conclusions: We found that the IOM possesses a large collagen content arranged in fibrillar structures surrounded by elastin. Collagen was abundant in the proximal bundles and decreased in the distal bundles. (J Hand Surg 2003;28A:503–510. Copyright © 2003 by the American Society for Surgery of the Hand.) Key words: Interosseous membrane, forearm, histology, biochemical.
The interosseous membrane (IOM) of the forearm is a unique anatomic structure that connects the radius and ulna. The IOM is described as a soft tissue From the School of Medicine, Department of Biostatistics, Temple University; Department of Dermatology and Cutaneous Biology, Thomas Jefferson University, Philadelphia; and Shriners Hospital for Children, Philadelphia, PA. Received for publication August 20, 2002; accepted in revised form January 13, 2003. No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article. Reprint requests: Scott H. Kozin, MD, Shriners Hospital for Children, Pediatric Hand & Upper Extremity Surgery, 3551 N Broad St, Philadelphia, PA 19140. Copyright © 2003 by the American Society for Surgery of the Hand 0363-5023/03/28A03-0023$30.00/0 doi:10.1053/jhsu.2003.50059
located within the interosseous space of the forearm that possesses a distinct oblique radioulnar direction of 20°.1,2 The IOM is the origin of several muscle groups within the forearm (flexor digitorum profundus, flexor pollicis longus, extensor pollicis brevis, abductor pollicis longus, extensor indicis, and extensor pollicis longus).3,4 There are several fibrous bundles directed from the radius to the ulna surrounded by a supporting matrix.5 Each fiber bundle represents a distinct grouping of collagen fibrils and shows a consistent organization with several large bundles located proximally within the central third of the IOM and a varying number of smaller bundles located distally within the forearm.5 The functional integration of the various fiber bundles and the supporting matrix provides the mechanics necessary for The Journal of Hand Surgery
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Figure 1. (A) IOM bundles were dissected as a distinct fiber group with a complete enclosing sheath. This figure shows the location of bundle excision. A varying number of larger distinct fiber bundles were found within the central third of the IOM. Smaller distal fibers were located in the membranous region of the IOM. (B) Each fiber bundle was excised from the forearm at a point 2 mm from the bony insertion thereby eliminating the contribution of sharpy fibers. Each fiber bundle was further dissected into a radial, central, and ulnar portion.
transferring forces in a graded fashion from the radius to the ulna. Several studies have investigated the various functions of the IOM. Rabinowitz et al6 described the role of the IOM in forearm stability and determined that considerable proximal migration of the radius (⬎7 mm) results from damage to its midportion and triangular fibrocartilage complex. Werner and Koebke7 identified the function of the IOM in relation to its origin for the extensor and flexor musculature and showed that muscle fibers formed specific angles with the membrane that result in the anatomic orientation of the fibers within the IOM. Shang et al8 studied the function of the IOM in coronal forearm stability. Tensile force between the radius and ulna tended to separate the 2 bones, which tightened the IOM and resisted separation. Pfaeffle et al9 recently described the IOM as ligamentous in function secondary to its bone-to-bone connection and mechanical stiffness. Previous researchers have described the anatomy and mechanics of the IOM. The purpose of our study was to characterize the microstructure and collagen organization of the IOM using histologic and biochemical analysis and relate its composition to forearm function.
Methods Dissection Twenty-one fresh-frozen forearms of cadavers aged 45 to 86 years were thawed in a warm isotonic saline solution. Each forearm was dissected, removing the overlying soft tissues and musculature and isolating
the radius, ulna, and IOM. Each arm was then photographed using a fluorescent light to show the gross organization of the IOM fiber bundles. For the histologic studies described later 12 of the dissected forearm specimens, aged 60 to 86 years (73.2 ⫾ 10.9 y), were fixed in a 10% formalin solution for 48 hours to ensure complete fixation. The remaining 9 freshly dissected forearm specimens (nonfixed), of cadavers aged 45 to 79 years (70.3 ⫾ 11.4 y), were used for the biochemical analysis described later. Using the fixed forearm specimens for histology and the fresh forearm specimens for biochemistry 5 discrete IOM fiber bundles were isolated from each arm at various locations (Fig. 1A). An IOM fiber bundle was defined as an independent dissectible grouping of fibers supported by an enclosing sheath. A total of 60 fixed IOM fiber bundles were obtained for histology (5 per arm) and 45 fresh IOM fiber bundles were obtained for biochemical analysis. Each bundle was dissected at a distance of 2 mm from the bony insertion thereby eliminating the contribution of Sharpey’s fibers. The location of the origin, center, and insertion of each bundle excised was measured relative to its position along the radius and reported as a percentage distance (actual distance divided by total radius length) from the styloid process of the radius. Each IOM fiber bundle was then sharply dissected into 3 samples based on location: radial, central, and ulnar (Fig. 1B).
Histologic Studies The fixed samples were embedded in paraffin and sectioned with a microtome into 7-m slices and
McGinley et al / Composition and Structure of Forearm IOM
placed on a slide. The slides were stained using a hematoxylin-eosin, trichrome, and Verhoff-vanGeison stain.10 Each slide was examined at various magnifications to qualitatively describe the IOM structure and organization including collagen and elastin distribution. The central regions of each trichromestained sample were photographed using a digital camera at a magnification of 40 to quantitatively analyze the collagen content. The trichrome images were analyzed and the staining intensity distribution was determined (SigmaScan Pro software, SPPS Inc, Chicago, IL). The number of pixels in a measured distance was determined by using a calibrated micrometer slide. By using the calibrated values of distance the area of each section was measured. Threelayer settings were used based on calibrated intensity values for collagen and elastin. Layer 1 included the collagenous tissue, layer 2 represented elastin and other noncollagenous tissue, and layer 3 included the entire membrane area. With the calibrated distance the percentage of collagen was determined by dividing layer 1 (collagen area) by layer 3 (total area). The results were plotted based on the percentage distance from the styloid process of the radius, and comparisons of collagen content were made relative to the location within the forearm.
Biochemical Analysis (Hydroxyproline Assay) Each fresh sample obtained from the dissection described earlier was placed into a cold porcelain mortar pan and filled with liquid nitrogen.11 The sample was crushed into powder form, dried in an oven at 80 °C, and weighed (dry weight). The sample was then hydrolyzed using 12 mol/L of hydrochloric acid. The hydrochloric acid was evaporated under a chemical hood, leaving a sample residue that was resuspended in deionized water. The samples were diluted for spectrophotometer readings of hydroxyproline content. Hydroxyproline standards (Biocolor Ltd., Newtownabbey, Northern Ireland) were prepared using 0, 0.5, 1, 3, 4, and 5 g of collagen (at higher concentrations the relation between absorbance and collagen concentration becomes nonlinear).12 Collagen concentration was calculated based on a 10% hydroxyproline content. Chloramine T, perchloric acid, and Ehrlich’s reagent were added to each standard and sample vial. After a short incubation period the absorbance of each sample was recorded at a wavelength of 557 nm. The data were analyzed based on the section origin (radial, central, ulnar) and location with regard to the radial styloid.
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Figure 2. Trichrome stain of the central location of the main IOM bundle (⫻40). The solid arrows indicate the individual collagen fascicles and the dashed arrow shows the supporting elastin sheath.
Statistical Methods The dependent variables (ie, collagen content [trichrome staining] and collagen content measured by hydroxyproline) were treated as continuous variables for all analyses. Means, standard deviations, standard errors, and p values are presented for the different analyses by percentage distance from the styloid process and by bundle and location. The experimental design was a 2-factor (bundle and location) mixed design with repeated measures on both factors. The null hypothesis was that there was no difference in the measured parameters among the different bundles and locations. Before analysis all data were tested for normality using the ShapiroWilk test.13 The data for the dependent variables were significantly nonnormal. To apply ANOVA methods, a normalized-rank transformation was applied to the data.14,15 The rank-transformed data were analyzed by using a mixed-model ANOVA for repeated measures followed by multiple comparisons to detect significant individual mean differences between bundles and locations. Multiple pair-wise comparisons used the Dunn-Bonferroni adjustment to maintain an experiment-wise type I error of .05 or less.16 Differences between group means (rejection of the null hypothesis) were considered significant when the probability of chance occurrence was .05 or less using 2-tailed tests. Collagen content was plotted versus percentage distance to examine the relationship between distance from the styloid process and collagen.
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Figure 3. Trichrome stain of the central location of the main IOM bundle (⫻400). The solid arrow indicates the substance of a single collagen fascicle with the arrowhead showing the surrounding elastin sheath. The dashed arrow highlights the surrounding elastin fibers grouping the collagen fascicles into a single collagen bundle.
Results Histology: 12 Arms The staining of the histologic sections revealed an abundance of collagen fibers with small amounts of elastin fibers. An orderly array of collagen fibers perpendicular to the cutting plane was observed. The collagen fibers were arranged in parallel into fascicles (Figs. 2, 3) with blood vessels and nerves located between fascicles. The elastin organization of the IOM was visualized as the areas of reduced
Figure 5. Collagen content distribution (percent of total area) from the radial styloid process using image intensity analysis of trichrome-stained histologic sections.
trichrome staining of the histologic specimens. The elastin provided a supporting structure surrounding each individual collagen fascicle and as an outer sheath grouping the fascicles into a single IOM bundle. The results of the image intensity analysis are summarized in Figure 4. The average data are based on the numbered IOM fiber bundle (1 ⫽ distal, 5 ⫽ proximal). The percentage distance from the styloid process of the radius of each fiber bundle was found to equal 37.2% ⫾ 5.2%, 45.7% ⫾ 3.8%, 51.7% ⫾
Figure 4. Collagen content of bundles 1–5 using digital analysis of trichrome-stained sections and hydroxy-proline assay. For histologic sections collagen content was calculated as a percentage of the total area and for biochemical analysis collagen content was determined based on the percentage of dry weight.
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Figure 6. There were no significant differences in collagen content based on location within the forearm using hydroxy-proline analysis.
4.4%, 57.3% ⫾ 3.4%, and 63.5% ⫾ 3.6% for bundles 1 to 5, respectively. Image analysis of the trichrome-stained sections showed an abundance of collagen with an overall average value of 73.1% ⫾ 14.9%. The main fiber bundle (bundle 4) was composed of 84.1% ⫾ 7.8% collagen. The relative amount of collagen increased from the distal to proximal bundles (Fig. 5). Analysis of differences between bundles showed that bundles 1 and 2 contained
Table 1. Bundle Collagen Content Within Each Location (Hydroxy-Proline Assay)
Location Radial
Central
Ulnar
Bundle Number
Distance From Styloid Process (%)
Collagen Content (%)
1 2 3 4 5 1 2 3 4 5 1 2 3 4 5
43.0 ⫾ 4.4 47.3 ⫾ 6.1 51.8 ⫾ 5.3 57.7 ⫾ 5.2 62.6 ⫾ 6.4 38.7 ⫾ 4.0 43.1 ⫾ 6.2 48.2 ⫾ 5.5 54.4 ⫾ 5.8 59.9 ⫾ 5.3 34.4 ⫾ 4.0 38.9 ⫾ 6.9 44.6 ⫾ 5.2 51.0 ⫾ 6.5 57.2 ⫾ 4.7
87.6 ⫾ 18.2 90.7 ⫾ 12.9 88.5 ⫾ 12.3 85.8 ⫾ 10.8 94.4 ⫾ 18.7 84.5 ⫾ 15.8 94.9 ⫾ 6.8 92.0 ⫾ 8.5 92.8 ⫾ 5.1 99.3 ⫾ 16.5 96.3 ⫾ 16.7 82.6 ⫾ 11.9 86.6 ⫾ 8.5 86.5 ⫾ 9.8 86.0 ⫾ 8.1
significantly less collagen than bundles 4 and 5 (p ⬍ .001).
Collagen Content: 9 Arms The results of the hydroxyproline assay are shown in Figures 4 and 6 and Table 1. The percentage distance from the styloid process of the radius of each fiber bundle was found to equal 38.7% ⫾ 4.0%, 43.1% ⫾ 6.2%, 48.2% ⫾ 5.5%, 54.4% ⫾ 5.8%, and 59.9% ⫾ 5.3% for bundles 1 to 5, respectively. Figure 4 shows the collagen content based on fiber bundle (bundle 1 ⫽ distal, bundle 5 ⫽ proximal). Figure 6 shows the collagen percentages based on section location (radial, central, ulnar). Table 1 further classifies the data based on bundle number in each location. Column 1 lists the section location, column 2 lists the bundle number, column 3 lists bundle distance and column 4 summarizes the average collagen content. The average collagen content of the main fiber bundle (bundle 5) of the IOM was 93.2% ⫾ 15.5% (n ⫽ 24). Using the hydroxyproline assay there was no significant difference in collagen content between bundles (p ⬍ .67, power ⫽ 0.25). Comparisons of average collagen content of each section location (radial, central, ulnar) also showed no significant differences (89.4% ⫾ 14.5%, 92.7% ⫾ 12.0%, and 87.6% ⫾ 11.8%, respectively; p ⬍ .52; power ⫽ 0.46). Based on the hydroxyproline assay there was no relationship between collagen content and posi-
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Figure 7. Analysis of the IOM’s radial origin collagen content using the hydroxy-proline assay. Collagen content (percent dry weight) is plotted versus the percent distance from the radial styloid process.
tion within the forearm in the radial and central locations and a minimal negative relationship at the ulnar location (Figs. 7, 8, 9).
Discussion The IOM has been described as a membrane, tendon, or ligament. Mechanical studies imply the IOM has a ligamentous function.9 Complete understanding of the IOM requires investigation of its anatomic arrangement, histology, composition, and function. Tendons are defined as cord-like structures that connect muscle to bone and possess an orderly arrangement of collagen fibrils that assemble into parallel fiber bundles.17 The collagen content of tendons is approximately 90% by dry weight with a minimal elastin contribution (5% to 7%).18 Because of the large portion of collagen relative to elastin, tendons possess superior tensile strength and are able to resist large tensile forces. Tendons also have a large elastic modulus and are mechanically stiff structures.18 In contrast ligaments connect bone to bone and their structure is arranged in a less orderly manner than tendons. The bone-to-bone connection requires a more elastic mechanical structure to permit some displacement when loaded in tension. The increased elasticity is a result of a lower collagen content (25% to 85%) and a less regularly arranged microstructure resulting in a decreased elastic modulus and tensile strength.18 Ligaments cannot withstand the large tensile forces maintained by tendons. To understand IOM physiology and analyze the function of the IOM within the forearm we compared published chemical
and mechanical studies of tendons and ligaments with our structural and biochemical findings. Grossly the IOM is a fibrous structure located within the interosseous space of the forearm. Its fiber bundles originate from the radius and are directed obliquely to the ulna (bone-bone connection). Numerous flexor and extensor muscle groups have origins on the dorsal and ventral surfaces of the IOM (muscle-bone connection). The bundles are arranged in distinct visible groups (main and accessory fiber bundles) and are surrounded by an overlying matrix. The trichrome stains of the histologic sections of the IOM showed an abundance of collagen arranged in an orderly fashion (Figs. 2, 3). The cross-sections of the IOM showed that the fibers are arranged in a parallel fashion perpendicular to the cutting plane. The collagen fibrils are grouped into fibers surrounded by an enclosing sheath. Furthermore the collagen fibers are subdivided into fascicles by an extension of the sheath. The hematoxylin-eosin and Verhoeff-vanGieson stains of the sections showed that the enclosing structures are composed largely of elastin. The elastin content in the membrane structure is minimal, functioning as a sheath around the collagen groups. There are several different types of collagen ␣ chains that are used to classify collagen into the categories of type I to XVI. Loose and dense connective tissue are composed mostly of type I collagen, which belongs to a group of fibrillar collagens and consists of 2 ␣ 1 chains and one ␣ 2 chain. The fibrillar collagen molecule is composed of 3 polypep-
Figure 8. Analysis of the IOM’s central location collagen content using the hydroxy-proline assay. Collagen content (percent dry weight) is plotted versus the percent distance from the radial styloid process.
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Figure 9. Analysis of the IOM’s ulnar insertion collagen content using the hydroxy-proline assay. Collagen content (percent dry weight) is plotted versus the percent distance from the radial styloid process.
tide chains (␣ chains) interwoven into a right-handed helix.19 The triple helix shape is a result of the orderly arrangement of glycine-proline-hydroxyproline structure. The amino acid hydroxyproline makes up approximately 10% of the total amino acid content of collagen. Hydroxyproline is unique to collagen and is used as a marker to analyze the collagen content of various tissues. The determination of the collagen percentage was based on the hydroxyproline assay, which measures the amount of hydroxyproline present within the collagen triple helix. This assay provides an accurate determination of the collagen content. Collagen represented 99.3% ⫾ 16.5% of the central portion of the central fiber bundle. Our results showed an abundance of collagen across the IOM. The percentage of collagen for the radial, central, and ulnar portion of each fiber bundle was 89.4% ⫾ 14.5%, 92.7% ⫾ 12.0%, and 87.6% ⫾ 11.8%, respectively. There was no between-group difference or between–fiber bundle difference in collagen content. The large collagen content along with the orderly arrangement of the fiber bundles results in a structure with a large tensile strength that provides the necessary mechanical properties for transferring large forces from the radius to the ulna. Furthermore the angular direction of the fibers provides an origin for the muscular groups in line with its collagen structure. The histologic organization of the IOM shows a dense connective tissue with an abundance of colla-
gen arranged in parallel surrounded by an elastin sheath. Biochemical analysis confirms the large collagen composition. The collagen content and distinct fiber organization of the IOM result in superior mechanical properties that are able to withstand large tensile loads.
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