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The biomechanical, morphologic, and histochemical properties of the costal cartilages in children with pectus excavatum
The Biomechanical, Morphologic, and Histochemical Properties of the Costal Cartilages in Children With Pectus Excavatum By Jiexiong Feng, Tingze Hu, Wenying Liu, Shangfu Zhang, Yunman Tang, Rui Chen, Xiaoping Jiang, and Fukang Wei Chengdu, China
Background/Purpose: The cause of the pectus excavatum (PE) remains unclear, although some results of research have indicated that the disturbance of the sternum or costal cartilage might be responsible for this deformity. But no decisive evidence has been gained. The authors have analyzed the biomechanical, morphologic, and histochemical properties of the cartilage in PE and intend to support the belief that the disturbance of the cartilage might contribute to the development of PE. Methods: Thirty-eight specimens of the sixth cartilage were obtained at operation for the PE group (aged from 3 to 6 years; mean, 4.2 years). And 28 specimens of the control group (aged from 3 to 6 years; mean, 4.4 years) were gained from routine postmortem examinations in which the cause of death was unlikely to have affected the cartilage. The biomechanical test was carried out in a material testing machine (Shimadzu AG-10TA, Tokyo, Japan). The relation curve of load-deformation in tensile and compressive tests and the curve of load-time in the flexuous test were recorded automatically. The values of the ultimate strength and strain were calculated from this relation curve. The specimens also underwent H&E staining. The values of the area, circumference, mean diameter, maximal diameter, and morphologic factor of the cell and the nucleus of the cartilage in superficial and deep area were determined with the help of image analysis software (GT-2 model, China). The superficial zone (SZ) and deep zone (DZ) of the cartilage were examinated with electron microscopy (JEM-100SX, Japan). The distribution and intensity of type II collagen was shown by immunohistochemistry staining and analyzed with the image analysis software (GT-2 model, Huakang Co, Chengdu, China). The extent and distribution of proteoglycan were analyzed after Safranin-O and periodic acid shiff (PAS) staining.
P
ECTUS EXCAVATUM (PE) is the most commonly found anterior chest wall deformity and is characterized by posterior depression of the sternum and the lower costal cartilages. It occurs more frequently in boys
From the Departments of Pediatric Surgery, Pathology, and Biomedical Engineering, First University Hospital, West China University of Medical Sciences, Chengdu, China. Address reprint requests to Jiexiong Feng, MD, Department of Pediatric Surgery, First University Hospital, West China University of Medical Sciences. Chengdu 610041, China. Copyright © 2001 by W.B. Saunders Company 0022-3468/01/3612-0009$35.00/0 doi:10.1053/jpsu.2001.28820 1770
Results: The mean strength of the costal cartilage in the experimental group was less than that in the control group in terms of tension, compression, and flexure (P ⬍ .05). The shape of the stress-strain curve for tension and compression in the experimental group was different from the control group. The fracture load in the experimental group was less than in the control group in tension (1.5 MPa versus 2.8 MPa) and in compression (.2 MPa versus 8.3 MPa). The time of fracture in experimental group was 30 seconds compared with 38 seconds in control group. No denaturation or necrosis could be found in light microscopical examination. There was no manifestation of hyperplasia or hypoplasia in the costal cartilage of the PE group. In SZ and DZ areas, the pattern and the number of mitochondria, endoplasmic reticulum, and Golgi in the experimental group were the same as the control group in transmission electron microscopy. Furthermore, the distribution and the number of proteoglycan in the 2 groups did not show a significant difference both in SZ and DZ areas. Although the distribution of the collagen in SZ areas was normal, this pattern was disturbed in DZ areas in the experiment group. The results of type II collagen immunohistochemistry examination was concordant with that change. No significant difference between control and experimental group could be seen in Safranin-O and PAS staining for proteoglycan. Conclusions: The biomechanical stability of the cartilage was decreased in the PE group. This might be caused by the disorderly arrangement and distribution of the collagen in the cartilage of PE patients. J Pediatr Surg 36:1770-1776. Copyright © 2001 by W.B. Saunders Company. INDEX WORDS: Pectus excavatum, costal cartilage, biomechanics, morphology, histochemistry.
than in girls by a ratio of 4:1.1-3 The deformity can decrease the volume of the chest, restrict the pulmonary movement, and force the heart into a rotated position. Those pathologic changes may impair the cardiopulmonary function in children with PE.3-8 Moreover, the deformity of the chest wall may adversely affect the patient’s self-esteem and mental health. Therefore, many pediatric surgeons emphasize the necessity of surgical intervention. Different procedures are performed in different units. The results of the operation are excellent, and the operative complications can be controlled. Contrary to the excellent achievements in clinical research, the cause of PE has not been clarified. Proposed
Journal of Pediatric Surgery, Vol 36, No 12 (December), 2001: pp 1770-1776
COSTAL CARTILAGE AND PECTUS EXCAVATUM
theories include intrauterine pressure, rickets, respiratory obstruction, and abnormalities of the diaphragm that result in posterior traction on the sternum.3 But the evidence for this suggestion had not been found. Mullard9 (1956) mentioned that there were so many objections to the theory of primary diaphragmatic shortening that the theory should be discarded. Meanwhile, he advocated reverting to an earlier theory that suggested an intrinsic failure of osteogenesis and chondrongenesis; the deformity would thus result from the effect of the respiratory pressure gradient on ribs and cartilages insufficiently rigid to resist it. PE is associated with other musculoskeletal abnormalities, particularly scoliosis and Marfan’s syndrome.10 Results of biochemical studies have shown abnormalities in the costal cartilage, including decreased levels of zinc and increased levels of magnesium and calcium.3 A family history of chest wall deformity was identified in 37% of cases.10 The growth disturbance of the sternum in PE patients was pictured by image studies.11 Other factors involved in the pathogenesis include the immunologic abnormality, collagen synthesis disturbance in skin fibroblasts, abnormal structure of type II collagen of costal cartilage, and so on.12 All those manifestations of PE indicated that there was abnormality in the cartilage and bone matter of the ribs and the sternum. This abnormality might contribute to the development of PE. But up until now, no direct evidence from biomechanical, morphologic, and histochemical studies of cartilage in PE patients has been provided. In this article, we present the results of our biomechanical, morphologic, and histochemical studies of the properties of cartilage in PE patients. In this way, we intend to further support the theory of an intrinsic disturbance of osteogenesis and chondrongenesis as a cause of PE. MATERIALS AND METHODS
Biomechanics Test Thirty-eight specimens of the sixth cartilage were obtained at operation for the PE group (aged from 3 to 6 years; mean, 4.2 years), and 28 specimens of the control group (aged from 3 to 6 years; mean, 4.4 years) were gained from routine postmortem examinations in which the cause of death was unlikely to have affected the cartilage. Before use, the specimens were stored at ⫺20°C in sealed plastic bags, a temperature which does not affect the mechanical properties of the cartilage. The specimens were then trimmed to 3 cm ⫻ 0.8 cm and stored in Ringer’s solution at 4°C for 2 hours before testing. Ten specimens from the control group and 14 specimens from the experimental group were tested for tension and compression, respectively, and 8 specimens from control group and 10 specimens from experimental group were tested for flexure. The test was carried out on a material testing machine (Shimadzu Model AG-10TA, Tokyo, Japan) with the cross-head set to move at 5 mm/min. The specimens of cartilage were tested for uniaxial compression and tension in the direction parallel to the cartilage surface and for flexure in the direction perpendicular to the surface. During the
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test the specimen was kept in buffered saline, and the environmental temperature was maintained at 25°C. The relation curve of loaddeformation in the tensile and compressive test and the curve of load-time in the flexuous test were recorded automatically. Then, the relation curve of load-deformation was transformed into the curve of stress-strain. Likewise, the curve of the load-time was transformed into the curve of stress-time. The values of the ultimate strength and strain were calculated from this relation curve.
Light Microscopy Blocks were cut perpendicular to the long axis of the costal cartilage, about 1 cm apart from the costochondral junction. The slices then were fixed by immersion in 10% neutral formalin. After rinsing in water, the specimens were dehydrated in a series of graded ethanols. Embedding was done in paraffin. Six-micrometer sections were deparaffinized and stained with H & E. The values of the area, circumference, mean diameter, maximal diameter, and morphologic factor of cell and nucleus in superficial and deep areas were determined with the help of image analysis software (GT-2 model, Huakang Co, Chengdu, China).
Transmission Electron Microscopy Specimens for electron microscopy were collected from areas near the perichondrium (superficial zone, SZ) and from the deep zone (DZ) more than 3 mm apart from the perichondrium. Four blocks in each group were sampled at random. The tissue was fixed by immersion in 2.5% glutaraldehyde and 0.2 mol/L phosphate buffer for 2 hours. After rinsing in the respective buffer (2 hours), the specimens were postfixed in 1% osmium tetroxide. Dehydration was performed in a series of graded ethanols and finally in propylene oxide. Thin sections were cut after embedding, and then stained for 7 minutes with uranyl acetate and lead citrate. Samples were examined under an electron microscope (JEM-100SX, Japan).
Type II Collagen Immunohistochemistry The sections were dehydrated in graded ethanols after having been deparaffined and pretreated with 0.1% trysin (EC 3.4.21.4) in Trissaline. Later, they were immersed in phosphate-buffered saline (PBS) for 1 hour at 37°C. To eliminate nonspecific antibody binding, the sections were exposed to undiluted heat-inactive normal rabbit serum with the addition of 4% bovine serum for 30 minutes at 37°C. After rinsing, the sections were incubated with type II collagen monoclonal antibody (dilution, 1:50; NeoMarkers, Freemont, CA) and left overnight at 4°C. After washing, the sections were exposed to sheep anti-mouse antibody (dilution, 1:200, Vector Laboratories, Burlingame, CA). Then the sections were exposed to streptavidin-avidin-biotin complex (Vector Laboratories) for 30 minutes at 37°C and later to 3,3⬘-diaminobezidin tetrahydrochloride (Zymed Laboratories, South San Francisco, CA). Finally, the sections were washed and mounted in glycerin jelly. For negative controls, the specific antibody was omitted. The distribution and intensity of staining was analyzed by image analysis software (GT-2 model).
Table 1. The Mean Strength of Biomechanics of the Sixth Costal Cartilage Between Experimental Group and Control Group (MPa) Group
Tension
Compression
Flexure
Experimental Control
1.52 ⫾ 0.37 2.27 ⫾ 0.23
1.33 ⫾ 0.22 8.29 ⫾ 0.98
4.13 ⫾ 1.22 7.64 ⫾ 1.88
NOTE. P ⬍ .05.
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Fig 3.
The curve of stress-time of the costal cartilage in flexure.
RESULTS Fig 1. sion.
The curve of stress-strain of the costal cartilage in ten-
Safranin-O Stain for Proteoglycan The sections were deparaffinized in xylene and hydrated in a graded series of ethanol solutions to distilled water. The staining was carried out for 6 minutes with 0.1% (wt/vol) Safranin-O (Courtesy of Professor Guo Xiong). The sections then were dehydrated twice in 95% ethanol solution and once in absolute ethanol, cleared by xylene, and mounted. The distribution and intensity of staining were analyzed by image analysis software (GT-2 model, China).
Periodic Acid-Shiff Stain for Proteoglycan The standard staining procedure of periodic acid-shiff (PAS) staining was adopted. The sections were deparaffinized and dehydrated in a graded series of ethanol. Subsequently, the slices were rinsed in water and immersed in 1% periodic acid (Merck Co, Darmstadt, Hessen, Germany) for 6 minutes. After washing, the sections were exposed to Schiff’s reagent (Chemical Reagent Company, Shanghai, China) for 10 minutes. Finally, the sections were dehydrated and mounted. The distribution and intensity of staining were analyzed by image analysis software (GT-2 model, China).
Statistical Analysis Results are expressed as mean values ⫾ SEM. The students’s t test was used to look for statistically significant differences between control group and experimental group. A P value of less than .05 was considered statistically significant.
Fig 2. The curve of stress-strain of the costal cartilage in compression.
Biomechanical Test The results of biomechanical test are listed in Table 1, and representative curves of the stress-strain or stresstime are shown in Figs 1-3. Table 1 shows that the mean strength of the costal cartilage is less in the experimental group than in the control group in regard to tension, compression, and flexure (P ⬍ .05). The curves of tensile stress versus tensile strain for 2 groups are shown in Fig 1. The curve of the control group is similar to the characteristic curve of the hyaline cartilage, but the shape of the curve differs from that of the experimental group. The costal cartilage was damaged when loaded with 1.5 MPa in the experimental group, compared with 2.8 MPa in the control group. Figure 2 shows curves of the compressive stress versus compressive strain in these 2 groups. Similar to tensile stress-strain curve in Fig 1, the curve of experimental group again turns out to be significantly different from the curve of the control group. The damage load of the cartilage in the experimental group was 2 MPa, which is significantly less than the 8.3 MPa in the control group. Figure 3 shows curves of the flexuous stress-time. Although the character of the curves is similar in these 2 groups, the time of damage in the experimental group is 30 seconds, and shorter than 38 seconds measured in the control group.
Fig 4. Cartilages in PE are immerged in each other in macroscopic examination.
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Table 2. The Index of the Cell of the Sixth Costal Cartilage Between Experimental Group and Control Group Group
A
C
D
MD
MF
Control Experimental
247.572 ⫾ 51.635 235.956 ⫾ 46.860
66.475 ⫾ 9.440 53.334 ⫾ 8.901
17.050 ⫾ 3.631 15.902 ⫾ 2.996
19.552 ⫾ 3.412 18.665 ⫾ 3.167
1.317 ⫾ 0.471 1.359 ⫾ 0.436
NOTE. P ⬎ .05. Abbreviations: A, area; C, circumference; D, diameter; MD, maximal diameter; MF, morphologic factor.
Light Microscopy Although some cartilages appeared to be abnormal in the macroscopic examination (Fig 4), no denaturation or necrosis could be found in the light microscopic examination. There was no hyperplasia or hypoplasia in the costal cartilage from PE patients. The number of vessels in the cartilage from the experimental group was the same as in the control group. The results of the image analysis are shown in Tables 2 and 3. The density, shape, and areas of the cell and nucleus of cartilage in the experimental group are all similar to the values of the control group. That indicates that the morphologic structure of the costal cartilage was intact in PE patients. Transmission Electron Microscopy In SZ areas, the pattern and the number of mitochondria, endoplasmic reticulum, and Golgi in experimental group were the same as in the control group. Similar results were found in DZ areas. The distribution and the number of the proteoglycans in the 2 groups showed no significant difference both in SZ and DZ areas. The collagen fibril diameter seemed to show a slight variability, but there was no difference in fibril diameters between the SZ and the DZ in both groups. The collagen fibrils were distributed equally and evenly in control group (Fig 5). In the experimental group, the distribution of the collagen fibril was similar to that in the control group in SZ areas, but the fibrils were distributed unequally and arranged irregularly in DZ areas. In several cases, some areas in DZ showed a significant scarcity of collagen fibril (Fig 6). Type II Collagen Immunohistochemistry The matrix of the cartilage was stained with yellow color, whereas the negative control specimens were stainless. In SZ areas, the intensity and distribution of staining were similar in both groups. But the difference was noted in DZ areas. In the control group, the intensity and distribution of staining was equal both in SZ and DZ
areas. However, the intensity of staining in experimental group in these areas was unequal and patchy. There was deep staining in some areas and light staining in the others. Some areas even appeared stainless (Figs 7 and 8). The results of the image analysis are outlined in Tables 4 and 5. Safranin-O and PAS Stain for Proteoglycans Large amounts of proteoglycan were seen in DZ areas and were slightly less in the region of SZ in both groups in Safranin-O staining. The staining was distributed equally and even in cartilage matrix in both groups. No significant difference in the density of the staining for proteoglycan between the control group and the experimental group were found by the image analysis (Table 6). The intensity and the distribution of PAS staining for proteoglycan were the same as in the Safranin-O staining (Table 7). DISCUSSION
Many theories have been established to explain the pathogenesis of PE, such as maldevelopment of the anterior and anterolateral muscular fibers of the diaphragm, the poststernal bands, and respiratory tract obstruction.13,14 Unfortunately, no reliable supportive anatomic or radiologic evidence has been found. On the contrary, it was suggested that intrinsic abnormalities of the sternum and costal cartilage might contribute to the development of PE. Genetic factors seem to play a part, and PE is associated with other anomalies of skeletomuscular system.3 Abnormal levels of certain chemical elements as well as abnormaltities in the collagen of the costal cartilage have been reported in patients with PE. The development of the sternum, seen by radiologic imaging, is delayed in patients with PE.11 All of these manifestations suggest that there is a relationship between abnormal rib and sternum cartilage and bone matter as well as PE formation. To date, however, no systematic research on the biomechanics, morphology,
Table 3. The Index of the Nucleus of the Sixth Costal Cartilage Between Experimental Group and Control Group Group
A
C
D
MD
MF
Control Experimental
25.775 ⫾ 6.712 24.890 ⫾ 5.906
19.806 ⫾ 3.563 19.516 ⫾ 2.675
5.867 ⫾ 0.715 5.275 ⫾ 0.613
6.517 ⫾ 0.915 5.948 ⫾ 1.025
1.330 ⫾ 0.150 1.322 ⫾ 0.142
NOTE. P ⬎ .05. Abbreviations: A, area; C, circumference; D, diameter; MD, maximal diameter; MF, morphologic factor.
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Fig 5. The collagen fibrils are distributed equally and evenfully in control group (TEM ⴛ 10,000).
and histochemistry of costal cartilage or sternum in children with PE has been reported. Furthermore, there have not been reports on the biomechanical properties of costal cartilage both in healthy individuals and patients with PE. We adopted the indentation method that had been used previously to examine the biomechanical properties of articular cartilage because both articular and costal cartilage belong to the hyaline cartilage.15 The character of the stress-strain curves both in tensile and compressive test in costal cartilage in the control group is similar to the curve of the articular cartilage known from the current study. The shape of this curve in the experimental group, however, is strikingly changed. In the control group, the curve of the yielding change can be found between the curve of the elastic change and the fracture. In the experimental group, however, the curve of the yielding change is absent. This indicates that in the experimental group the costal cartilage is fragile rather than elastic, as it is in the control group. The ultimate strength of the costal carti-
Fig 6. The collagen fibrils are distributed unequally and arranged irregularly in DZ areas in experimental group. Extremely, some areas in DZ are scanty of collagen fibril in experimental group (TEM ⴛ 10,000).
FENG ET AL
Fig 7. In the control group, the intensity and the distribution of type II collagen in DZ area are equal (SP ⴛ 175).
lage both in tension and compression is significantly less in the experimental group compared with the control group. Although the similar biomechanical properties can be found in the flexuous test for both group, the ultimate strength of the cartilage in the experimental group is less than that in the control group. Moreover, the time of fracture of the cartilage also is shorter in the experimental group. All these results show that the biomechanical stability of costal cartilage in children with PE is impaired in terms of tension, compression, and flexure. The abnormal costal cartilages in children with PE might be insufficiently rigid to resist the effect of the respiratory gradient, and so the funnel chest is formed. The movement of diaphragm, one of the main forms of fetal breathing activities, might retract the impaired costal cartilages and make them angled posteriorly. Therefore, the PE would be present at birth.16 Some unusual inspiratory efforts, such as crying spells, respiratory obstruction, or hiccupping may aggravate the effect of inspiratory retraction of the anterior sixth ribs, cartilages,
Fig 8. The intensity and the distribution of staining in experimental group in DZ areas are unequal (SP ⴛ 175).
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Table 4. The Intensity and Distribution of the Type II Collagen in SZ of the Sixth Costal Cartilages Between Control and Experimental Group Group
ARLS
ARDS
GRLS
GRDS
MGR
Experimental Control
21.405 ⫾ 4.108 22.755 ⫾ 7.891
16.375 ⫾ 3.403 11.306 ⫾ 3.182
167.142 ⫾ 29.192 164.935 ⫾ 15.570
160.750 ⫾ 43.476 159.611 ⫾ 32.305
165.198 ⫾ 30.452 163.120 ⫾ 19.201
NOTE. P ⬎ .05. Abbreviations: RALS, area ratio of light staining; ARDS, area ratio of deep staining; GRLS, gray scale of light staining; GRDS, gray scale of deep staining; MGR, mean gray scale.
Table 5. The Intensity and Distribution of the Type II Collagen in DZ of the Sixth Costal Cartilages Between Control and Experimental Group Group
ARLS
ARDS
GRLS
GRDS
MGR
Experimental Control
34.037 ⫾ 7.525 19.738 ⫾ 4.245*
23.010 ⫾ 2.935 10.002 ⫾ 1.821*
166.842 ⫾ 31.110 165.935 ⫾ 24.833
155.667 ⫾ 42.675 161.435 ⫾ 26.402
163.973 ⫾ 34.145 164.371 ⫾ 27.334
Abbreviations: RALS, area ratio of light staining; ARDS, area ratio of deep staining; GRLS, gray scale of light staining; GRDS, gray scale of deep staining; MGR, mean gray scale. *P ⬍ .05.
and adjacent interspace because of the stronger spastic contraction of the diaphragm, and may induce or aggravate the deformity. This could be supported by the fact that the PE is intended to happen in children with respiratory obstruction.17 Mullard9 held that the impaired biomechanics of the costal cartilages in PE might be secondary to the failure of osteogenesis or chondrogenesis. Unfortunately, there is no evidence to support this belief from morphologic investigations in this study. The structure of the cell and the nucleus of the costal cartilage is intact, and no hyperplasia or hypoplasia of the cartilage can be found either in light microscopy or in transmission electron microscopy investigation. The number of the vessel in cartilage in PE, as found by Rupprecht et al18 in their research in cartilage of funnel chest, also remained the same as in the control group. Unlike the report in a previous study,18 stating that the number of the chondrocytes strongly increased within the single chondrons with rising age, we found that the number of the cartilage cells remains constant. This difference might find its explanation in the different mean age of the patients in our research and the previous study. The collagen in DZ areas, however, has shown a significant difference between control and experimental groups by transmission electron microscopy and immunohistochemistry. The disturbance of the distribution and the arrangement of the collagen can be seen by transmis-
sion electron microscopy. Moreover, this abnormality mainly results from the disturbance of the distribution and the arrangement of the type II collagen shown by immunohistochemistry. The different properties of collagen meshwork, found by other investigators, may well affect the results of the tensile or compressive test.19 For example, the size and number of the individual fibrils might be related to the strength of the cartilage. Meanwhile, tensile stiffness might reflect interactions between collagen and the noncollageous matrix. The degree of binding between macromolecules can be expected to have a bearing on how fast the network can rearrange itself when subjected to stress. Kempson et al20 regarded that the tensile stiffness of the cartilage at low values of applied stress was largely dependent on 2 factors. First, it depended on the extent to which the collagen fibers were aligned initially in the direction of the applied stress and second, it depended on the resistance that fibers experience when they aligned in the direction of the stress. The properties of the collagen also influences the biomechanical property in compression.19-23 If the collagen meshwork has been dysarranged, the collagen fibers would fail to restrain the imbibition of the water by the hydrophilic proteoglycans, and the total water content of the cartilage could be expected to increase, whereas the compressive modulus might decrease. The disturbance of the distribution and arrangement of the collagen in cartilage therefore is responsible for the damage of the
Table 6. The Intensity and Distribution of the Proteoglycan of the Sixth Costal Cartilages Between Control and Experimental Group (Safranin-O Staining) Group
ARLS
ARDS
GRLS
GRDS
MGR
Experimental Control
12.520 ⫾ 2.005 13.617 ⫾ 2.640
20.647 ⫾ 7.145 22.203 ⫾ 5.015
169.729 ⫾ 30.070 165.942 ⫾ 20.086
160.474 ⫾ 33.521 154.997 ⫾ 29.568
164.137 ⫾ 32.134 158.807 ⫾ 30.247
NOTE. P ⬎ .05. Abbreviations: RALS, area ratio of light staining; ARDS, area ratio of deep staining; GRLS, gray scale of light staining; GRDS, gray scale of deep staining; MGR, mean gray scale.
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Table 7. The Intensity and Distribution of the Proteoglycan of the Sixth Costal Cartilages Between Control and Experimental Group (PAS Staining) Group
ARLS
ARDS
GRLS
GRDS
MGR
Experimental Control
18.805 ⫾ 3.451 17.765 ⫾ 4.394
13.305 ⫾ 2.947 11.205 ⫾ 3.175
142.246 ⫾ 28.360 151.675 ⫾ 19.528
128.347 ⫾ 23.980 130.714 ⫾ 30.505
137.388 ⫾ 40.136 142.474 ⫾ 35.919
NOTE. P ⬎ .05. Abbreviations: RALS, area ratio of light staining; ARDS, area ratio of deep staining; GRLS, gray scale of light staining; GRDS, gray scale of deep staining; MGR, mean gray scale.
biomechanical properties of the cartilage from the children with PE. Proteoglycan, the other important element of the matrix of the cartilage, also contributes to the cartilage biomechanics. When a large proportion of the total proteoglycan content of the articular cartilage is degraded and released from the matrix by the action of either cathepsin D or cathpsin B1, the compressive stiffness of the tissue is reduced significantly.20 Similar reduction in the tensile stiffness of cartilage at low stresses can be found using the same way to deal with the cartilage.20 But the distribution and the content of the proteoglycans of the cartilage from the experimental group, shown by Safranin-O and PAS staining, are the same as in the control group. These results may indicate that the biomechanical properties of the costal
cartilage in PE have been damaged by disturbance of the distribution and arrangement of the collagen rather than disturbance of the proteoglycan. Many factors might influence the distribution and arrangement of the collagen in cartilage. Further investigations will determine in which way a particular factor may contribute to the disturbance of the collagen in PE in the future research. ACKNOWLEDGMENTS The authors wish thank Professor Song ZM for his help in the biomechanical tests, Professor GUO X and Zhang ZT for their assistance with Safranin-O staining, Professor Yang XY for her help in immunohistochemistry and PAS staining, Dr Zhang J for his help in transmission electron microscopy, and Dr Neubauer H for his help in the revised form of the manuscript.
REFERENCES 1. Hu TZ, Feng JX, Liu WY, et al: Modified sternal elevation for children with pectus excavatum. CMJ 113:451-454, 2000 2. Hu TZ, Feng JX, Liu WY, et al: The long-term results of modified sternal elevation procedure for correction pectus excavatum. Chin J Thoracic Cardiovasc Surg 16:85-86, 2000 3. Shamberger RC: Congenital chest wall deformities. Curr Probl Surg 33:469-542, 1996 4. Feng JX, Hu TZ, Liu WY, et al: Assessment of cardiac function in children with pectus excavatum before and after operation. Chin J Thorac Cardiovasc Surg 6:242-244, 1999 5. Feng JX, Hu TZ, Liu WY, et al: The long-term follow-up of cardiac function after sternal elevation. Chin J Pediatr Surg 20:198200, 1999 6. Liu WY, Yuan YR, Hu TZ, et al: Assessment of the pulmonary function in patients with pectus excavatum. Chin J Pediatr Surg 13:198-200, 1992 7. Quigley PM, Haller JA, Jelus KL, et al: Cardiorespiratory function before and after corrective surgery in pectus excavatum. J Pediatr 128:638-643, 1996 8. Peterson RJ, Young WG Jr, Godwin JD, et al: Noninvasive assessment of exercise cardiac function before and after pectus excavatum repair. J Thorac Cardiovasc Surg 90:251-260, 1985 9. Mullard K: Observations on the etiology of pectus excavatum and other chest deformities, and a method of recording them. Br J Surg 54:115-120, 1967 10. Shamberger RC, Welch KJ: Surgical repair of pectus excavatum. J Pediatr Surg 23:615-622, 1988 11. Haje SA, Harcke HT, Bowen JR. Growth disturbance of the sternum and pectus deformities: imageing studies and clinical correlation. Pediatr Radiol 29:334-341, 1999. 12. Tsvetkova TA, Gorokhova TA, Delvig AA: The effect of ascorbic acid on the collagen synthesis in skin fibroblast of children with funnel chest. Vopr Med Khim 36:53-55, 1990
13. Chin EF: Surgery of funnel chest and congenital sternal prominence. Br J Surg 44:360-364, 1957 14. Brodkin HA: Congenital anterior chest wall deformities of diaphragmatic origin. Dis Chest 24:259-277, 1953 15. Silver FH, Glasgold AI: Cartilage wound healing—an overview. Otolaryngologic Clin Nor Am 28:847-864, 1995 16. Stark RI, Damel SS, Kin YI, et al: Patterns of development in fetal breathing activity in the latter third of gestation of the boon. Early Hum Dev 32:31-47, 1993 17. Van Klaveren RJ, Morshuis WJ, Lacquet LK, et al: Congenital bronchial atresia with regional emphysema associated with pectus excavatum. Thorax 47:1082-1083, 1992 18. Rupprecht H, Freiberger N: Light microscopic studies of the cartilage in funnel chest, a new view of the pathogenesis. Z Exp Chir Transplant Kunstliche Organe 22:314-318, 1989 19. Roberts S, Weightman B, Urban J, et al: Mechanical and biochemical properties of human articular cartilage in osteoarthritic femoral heads and in autopsy specimens. J Bone Joint Surg 68B:278288, 1986 20. Kempson GE, Tuke MA, Dingle JT, et al: The effect of proteolytic enzymes on the mechanical properties of adult human articular cartilage. Biochim Biophy Acta 428:741-760, 1976 21. Kempson GE, Muir H, Swanson SAV, et al: Correlations between stiffness and the chemical constituents of cartilage on the human femoral head. Biochim Biophy Acta 215:70-77, 1970 22. Kempson GE, Muir H, Pollard C, et al: The tensile properties of the cartilage of human femoral condyles related to the content of collagen and glycosaminoglycans. Biochim Biophy Acta 297:456-472, 1973 23. Bader DL, Kempson GE, Barrett AJ, et al: The effect of leucocyte elastase on the mechanical properties of adult human articular cartilage in tension. Biochim Biophy Acta 677:103-108, 1981
Background/Purpose: The cause of the pectus excavatum (PE) remains unclear, although some results of research have indicated that the disturbance of the sternum or costal cartilage might be responsible for this deformity. But no decisive evidence has been gained. The authors have analyzed the biomechanical, morphologic, and histochemical properties of the cartilage in PE and intend to support the belief that the disturbance of the cartilage might contribute to the development of PE. Methods: Thirty-eight specimens of the sixth cartilage were obtained at operation for the PE group (aged from 3 to 6 years; mean, 4.2 years). And 28 specimens of the control group (aged from 3 to 6 years; mean, 4.4 years) were gained from routine postmortem examinations in which the cause of death was unlikely to have affected the cartilage. The biomechanical test was carried out in a material testing machine (Shimadzu AG-10TA, Tokyo, Japan). The relation curve of load-deformation in tensile and compressive tests and the curve of load-time in the flexuous test were recorded automatically. The values of the ultimate strength and strain were calculated from this relation curve. The specimens also underwent H&E staining. The values of the area, circumference, mean diameter, maximal diameter, and morphologic factor of the cell and the nucleus of the cartilage in superficial and deep area were determined with the help of image analysis software (GT-2 model, China). The superficial zone (SZ) and deep zone (DZ) of the cartilage were examinated with electron microscopy (JEM-100SX, Japan). The distribution and intensity of type II collagen was shown by immunohistochemistry staining and analyzed with the image analysis software (GT-2 model, Huakang Co, Chengdu, China). The extent and distribution of proteoglycan were analyzed after Safranin-O and periodic acid shiff (PAS) staining.
P
ECTUS EXCAVATUM (PE) is the most commonly found anterior chest wall deformity and is characterized by posterior depression of the sternum and the lower costal cartilages. It occurs more frequently in boys
From the Departments of Pediatric Surgery, Pathology, and Biomedical Engineering, First University Hospital, West China University of Medical Sciences, Chengdu, China. Address reprint requests to Jiexiong Feng, MD, Department of Pediatric Surgery, First University Hospital, West China University of Medical Sciences. Chengdu 610041, China. Copyright © 2001 by W.B. Saunders Company 0022-3468/01/3612-0009$35.00/0 doi:10.1053/jpsu.2001.28820 1770
Results: The mean strength of the costal cartilage in the experimental group was less than that in the control group in terms of tension, compression, and flexure (P ⬍ .05). The shape of the stress-strain curve for tension and compression in the experimental group was different from the control group. The fracture load in the experimental group was less than in the control group in tension (1.5 MPa versus 2.8 MPa) and in compression (.2 MPa versus 8.3 MPa). The time of fracture in experimental group was 30 seconds compared with 38 seconds in control group. No denaturation or necrosis could be found in light microscopical examination. There was no manifestation of hyperplasia or hypoplasia in the costal cartilage of the PE group. In SZ and DZ areas, the pattern and the number of mitochondria, endoplasmic reticulum, and Golgi in the experimental group were the same as the control group in transmission electron microscopy. Furthermore, the distribution and the number of proteoglycan in the 2 groups did not show a significant difference both in SZ and DZ areas. Although the distribution of the collagen in SZ areas was normal, this pattern was disturbed in DZ areas in the experiment group. The results of type II collagen immunohistochemistry examination was concordant with that change. No significant difference between control and experimental group could be seen in Safranin-O and PAS staining for proteoglycan. Conclusions: The biomechanical stability of the cartilage was decreased in the PE group. This might be caused by the disorderly arrangement and distribution of the collagen in the cartilage of PE patients. J Pediatr Surg 36:1770-1776. Copyright © 2001 by W.B. Saunders Company. INDEX WORDS: Pectus excavatum, costal cartilage, biomechanics, morphology, histochemistry.
than in girls by a ratio of 4:1.1-3 The deformity can decrease the volume of the chest, restrict the pulmonary movement, and force the heart into a rotated position. Those pathologic changes may impair the cardiopulmonary function in children with PE.3-8 Moreover, the deformity of the chest wall may adversely affect the patient’s self-esteem and mental health. Therefore, many pediatric surgeons emphasize the necessity of surgical intervention. Different procedures are performed in different units. The results of the operation are excellent, and the operative complications can be controlled. Contrary to the excellent achievements in clinical research, the cause of PE has not been clarified. Proposed
Journal of Pediatric Surgery, Vol 36, No 12 (December), 2001: pp 1770-1776
COSTAL CARTILAGE AND PECTUS EXCAVATUM
theories include intrauterine pressure, rickets, respiratory obstruction, and abnormalities of the diaphragm that result in posterior traction on the sternum.3 But the evidence for this suggestion had not been found. Mullard9 (1956) mentioned that there were so many objections to the theory of primary diaphragmatic shortening that the theory should be discarded. Meanwhile, he advocated reverting to an earlier theory that suggested an intrinsic failure of osteogenesis and chondrongenesis; the deformity would thus result from the effect of the respiratory pressure gradient on ribs and cartilages insufficiently rigid to resist it. PE is associated with other musculoskeletal abnormalities, particularly scoliosis and Marfan’s syndrome.10 Results of biochemical studies have shown abnormalities in the costal cartilage, including decreased levels of zinc and increased levels of magnesium and calcium.3 A family history of chest wall deformity was identified in 37% of cases.10 The growth disturbance of the sternum in PE patients was pictured by image studies.11 Other factors involved in the pathogenesis include the immunologic abnormality, collagen synthesis disturbance in skin fibroblasts, abnormal structure of type II collagen of costal cartilage, and so on.12 All those manifestations of PE indicated that there was abnormality in the cartilage and bone matter of the ribs and the sternum. This abnormality might contribute to the development of PE. But up until now, no direct evidence from biomechanical, morphologic, and histochemical studies of cartilage in PE patients has been provided. In this article, we present the results of our biomechanical, morphologic, and histochemical studies of the properties of cartilage in PE patients. In this way, we intend to further support the theory of an intrinsic disturbance of osteogenesis and chondrongenesis as a cause of PE. MATERIALS AND METHODS
Biomechanics Test Thirty-eight specimens of the sixth cartilage were obtained at operation for the PE group (aged from 3 to 6 years; mean, 4.2 years), and 28 specimens of the control group (aged from 3 to 6 years; mean, 4.4 years) were gained from routine postmortem examinations in which the cause of death was unlikely to have affected the cartilage. Before use, the specimens were stored at ⫺20°C in sealed plastic bags, a temperature which does not affect the mechanical properties of the cartilage. The specimens were then trimmed to 3 cm ⫻ 0.8 cm and stored in Ringer’s solution at 4°C for 2 hours before testing. Ten specimens from the control group and 14 specimens from the experimental group were tested for tension and compression, respectively, and 8 specimens from control group and 10 specimens from experimental group were tested for flexure. The test was carried out on a material testing machine (Shimadzu Model AG-10TA, Tokyo, Japan) with the cross-head set to move at 5 mm/min. The specimens of cartilage were tested for uniaxial compression and tension in the direction parallel to the cartilage surface and for flexure in the direction perpendicular to the surface. During the
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test the specimen was kept in buffered saline, and the environmental temperature was maintained at 25°C. The relation curve of loaddeformation in the tensile and compressive test and the curve of load-time in the flexuous test were recorded automatically. Then, the relation curve of load-deformation was transformed into the curve of stress-strain. Likewise, the curve of the load-time was transformed into the curve of stress-time. The values of the ultimate strength and strain were calculated from this relation curve.
Light Microscopy Blocks were cut perpendicular to the long axis of the costal cartilage, about 1 cm apart from the costochondral junction. The slices then were fixed by immersion in 10% neutral formalin. After rinsing in water, the specimens were dehydrated in a series of graded ethanols. Embedding was done in paraffin. Six-micrometer sections were deparaffinized and stained with H & E. The values of the area, circumference, mean diameter, maximal diameter, and morphologic factor of cell and nucleus in superficial and deep areas were determined with the help of image analysis software (GT-2 model, Huakang Co, Chengdu, China).
Transmission Electron Microscopy Specimens for electron microscopy were collected from areas near the perichondrium (superficial zone, SZ) and from the deep zone (DZ) more than 3 mm apart from the perichondrium. Four blocks in each group were sampled at random. The tissue was fixed by immersion in 2.5% glutaraldehyde and 0.2 mol/L phosphate buffer for 2 hours. After rinsing in the respective buffer (2 hours), the specimens were postfixed in 1% osmium tetroxide. Dehydration was performed in a series of graded ethanols and finally in propylene oxide. Thin sections were cut after embedding, and then stained for 7 minutes with uranyl acetate and lead citrate. Samples were examined under an electron microscope (JEM-100SX, Japan).
Type II Collagen Immunohistochemistry The sections were dehydrated in graded ethanols after having been deparaffined and pretreated with 0.1% trysin (EC 3.4.21.4) in Trissaline. Later, they were immersed in phosphate-buffered saline (PBS) for 1 hour at 37°C. To eliminate nonspecific antibody binding, the sections were exposed to undiluted heat-inactive normal rabbit serum with the addition of 4% bovine serum for 30 minutes at 37°C. After rinsing, the sections were incubated with type II collagen monoclonal antibody (dilution, 1:50; NeoMarkers, Freemont, CA) and left overnight at 4°C. After washing, the sections were exposed to sheep anti-mouse antibody (dilution, 1:200, Vector Laboratories, Burlingame, CA). Then the sections were exposed to streptavidin-avidin-biotin complex (Vector Laboratories) for 30 minutes at 37°C and later to 3,3⬘-diaminobezidin tetrahydrochloride (Zymed Laboratories, South San Francisco, CA). Finally, the sections were washed and mounted in glycerin jelly. For negative controls, the specific antibody was omitted. The distribution and intensity of staining was analyzed by image analysis software (GT-2 model).
Table 1. The Mean Strength of Biomechanics of the Sixth Costal Cartilage Between Experimental Group and Control Group (MPa) Group
Tension
Compression
Flexure
Experimental Control
1.52 ⫾ 0.37 2.27 ⫾ 0.23
1.33 ⫾ 0.22 8.29 ⫾ 0.98
4.13 ⫾ 1.22 7.64 ⫾ 1.88
NOTE. P ⬍ .05.
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Fig 3.
The curve of stress-time of the costal cartilage in flexure.
RESULTS Fig 1. sion.
The curve of stress-strain of the costal cartilage in ten-
Safranin-O Stain for Proteoglycan The sections were deparaffinized in xylene and hydrated in a graded series of ethanol solutions to distilled water. The staining was carried out for 6 minutes with 0.1% (wt/vol) Safranin-O (Courtesy of Professor Guo Xiong). The sections then were dehydrated twice in 95% ethanol solution and once in absolute ethanol, cleared by xylene, and mounted. The distribution and intensity of staining were analyzed by image analysis software (GT-2 model, China).
Periodic Acid-Shiff Stain for Proteoglycan The standard staining procedure of periodic acid-shiff (PAS) staining was adopted. The sections were deparaffinized and dehydrated in a graded series of ethanol. Subsequently, the slices were rinsed in water and immersed in 1% periodic acid (Merck Co, Darmstadt, Hessen, Germany) for 6 minutes. After washing, the sections were exposed to Schiff’s reagent (Chemical Reagent Company, Shanghai, China) for 10 minutes. Finally, the sections were dehydrated and mounted. The distribution and intensity of staining were analyzed by image analysis software (GT-2 model, China).
Statistical Analysis Results are expressed as mean values ⫾ SEM. The students’s t test was used to look for statistically significant differences between control group and experimental group. A P value of less than .05 was considered statistically significant.
Fig 2. The curve of stress-strain of the costal cartilage in compression.
Biomechanical Test The results of biomechanical test are listed in Table 1, and representative curves of the stress-strain or stresstime are shown in Figs 1-3. Table 1 shows that the mean strength of the costal cartilage is less in the experimental group than in the control group in regard to tension, compression, and flexure (P ⬍ .05). The curves of tensile stress versus tensile strain for 2 groups are shown in Fig 1. The curve of the control group is similar to the characteristic curve of the hyaline cartilage, but the shape of the curve differs from that of the experimental group. The costal cartilage was damaged when loaded with 1.5 MPa in the experimental group, compared with 2.8 MPa in the control group. Figure 2 shows curves of the compressive stress versus compressive strain in these 2 groups. Similar to tensile stress-strain curve in Fig 1, the curve of experimental group again turns out to be significantly different from the curve of the control group. The damage load of the cartilage in the experimental group was 2 MPa, which is significantly less than the 8.3 MPa in the control group. Figure 3 shows curves of the flexuous stress-time. Although the character of the curves is similar in these 2 groups, the time of damage in the experimental group is 30 seconds, and shorter than 38 seconds measured in the control group.
Fig 4. Cartilages in PE are immerged in each other in macroscopic examination.
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Table 2. The Index of the Cell of the Sixth Costal Cartilage Between Experimental Group and Control Group Group
A
C
D
MD
MF
Control Experimental
247.572 ⫾ 51.635 235.956 ⫾ 46.860
66.475 ⫾ 9.440 53.334 ⫾ 8.901
17.050 ⫾ 3.631 15.902 ⫾ 2.996
19.552 ⫾ 3.412 18.665 ⫾ 3.167
1.317 ⫾ 0.471 1.359 ⫾ 0.436
NOTE. P ⬎ .05. Abbreviations: A, area; C, circumference; D, diameter; MD, maximal diameter; MF, morphologic factor.
Light Microscopy Although some cartilages appeared to be abnormal in the macroscopic examination (Fig 4), no denaturation or necrosis could be found in the light microscopic examination. There was no hyperplasia or hypoplasia in the costal cartilage from PE patients. The number of vessels in the cartilage from the experimental group was the same as in the control group. The results of the image analysis are shown in Tables 2 and 3. The density, shape, and areas of the cell and nucleus of cartilage in the experimental group are all similar to the values of the control group. That indicates that the morphologic structure of the costal cartilage was intact in PE patients. Transmission Electron Microscopy In SZ areas, the pattern and the number of mitochondria, endoplasmic reticulum, and Golgi in experimental group were the same as in the control group. Similar results were found in DZ areas. The distribution and the number of the proteoglycans in the 2 groups showed no significant difference both in SZ and DZ areas. The collagen fibril diameter seemed to show a slight variability, but there was no difference in fibril diameters between the SZ and the DZ in both groups. The collagen fibrils were distributed equally and evenly in control group (Fig 5). In the experimental group, the distribution of the collagen fibril was similar to that in the control group in SZ areas, but the fibrils were distributed unequally and arranged irregularly in DZ areas. In several cases, some areas in DZ showed a significant scarcity of collagen fibril (Fig 6). Type II Collagen Immunohistochemistry The matrix of the cartilage was stained with yellow color, whereas the negative control specimens were stainless. In SZ areas, the intensity and distribution of staining were similar in both groups. But the difference was noted in DZ areas. In the control group, the intensity and distribution of staining was equal both in SZ and DZ
areas. However, the intensity of staining in experimental group in these areas was unequal and patchy. There was deep staining in some areas and light staining in the others. Some areas even appeared stainless (Figs 7 and 8). The results of the image analysis are outlined in Tables 4 and 5. Safranin-O and PAS Stain for Proteoglycans Large amounts of proteoglycan were seen in DZ areas and were slightly less in the region of SZ in both groups in Safranin-O staining. The staining was distributed equally and even in cartilage matrix in both groups. No significant difference in the density of the staining for proteoglycan between the control group and the experimental group were found by the image analysis (Table 6). The intensity and the distribution of PAS staining for proteoglycan were the same as in the Safranin-O staining (Table 7). DISCUSSION
Many theories have been established to explain the pathogenesis of PE, such as maldevelopment of the anterior and anterolateral muscular fibers of the diaphragm, the poststernal bands, and respiratory tract obstruction.13,14 Unfortunately, no reliable supportive anatomic or radiologic evidence has been found. On the contrary, it was suggested that intrinsic abnormalities of the sternum and costal cartilage might contribute to the development of PE. Genetic factors seem to play a part, and PE is associated with other anomalies of skeletomuscular system.3 Abnormal levels of certain chemical elements as well as abnormaltities in the collagen of the costal cartilage have been reported in patients with PE. The development of the sternum, seen by radiologic imaging, is delayed in patients with PE.11 All of these manifestations suggest that there is a relationship between abnormal rib and sternum cartilage and bone matter as well as PE formation. To date, however, no systematic research on the biomechanics, morphology,
Table 3. The Index of the Nucleus of the Sixth Costal Cartilage Between Experimental Group and Control Group Group
A
C
D
MD
MF
Control Experimental
25.775 ⫾ 6.712 24.890 ⫾ 5.906
19.806 ⫾ 3.563 19.516 ⫾ 2.675
5.867 ⫾ 0.715 5.275 ⫾ 0.613
6.517 ⫾ 0.915 5.948 ⫾ 1.025
1.330 ⫾ 0.150 1.322 ⫾ 0.142
NOTE. P ⬎ .05. Abbreviations: A, area; C, circumference; D, diameter; MD, maximal diameter; MF, morphologic factor.
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Fig 5. The collagen fibrils are distributed equally and evenfully in control group (TEM ⴛ 10,000).
and histochemistry of costal cartilage or sternum in children with PE has been reported. Furthermore, there have not been reports on the biomechanical properties of costal cartilage both in healthy individuals and patients with PE. We adopted the indentation method that had been used previously to examine the biomechanical properties of articular cartilage because both articular and costal cartilage belong to the hyaline cartilage.15 The character of the stress-strain curves both in tensile and compressive test in costal cartilage in the control group is similar to the curve of the articular cartilage known from the current study. The shape of this curve in the experimental group, however, is strikingly changed. In the control group, the curve of the yielding change can be found between the curve of the elastic change and the fracture. In the experimental group, however, the curve of the yielding change is absent. This indicates that in the experimental group the costal cartilage is fragile rather than elastic, as it is in the control group. The ultimate strength of the costal carti-
Fig 6. The collagen fibrils are distributed unequally and arranged irregularly in DZ areas in experimental group. Extremely, some areas in DZ are scanty of collagen fibril in experimental group (TEM ⴛ 10,000).
FENG ET AL
Fig 7. In the control group, the intensity and the distribution of type II collagen in DZ area are equal (SP ⴛ 175).
lage both in tension and compression is significantly less in the experimental group compared with the control group. Although the similar biomechanical properties can be found in the flexuous test for both group, the ultimate strength of the cartilage in the experimental group is less than that in the control group. Moreover, the time of fracture of the cartilage also is shorter in the experimental group. All these results show that the biomechanical stability of costal cartilage in children with PE is impaired in terms of tension, compression, and flexure. The abnormal costal cartilages in children with PE might be insufficiently rigid to resist the effect of the respiratory gradient, and so the funnel chest is formed. The movement of diaphragm, one of the main forms of fetal breathing activities, might retract the impaired costal cartilages and make them angled posteriorly. Therefore, the PE would be present at birth.16 Some unusual inspiratory efforts, such as crying spells, respiratory obstruction, or hiccupping may aggravate the effect of inspiratory retraction of the anterior sixth ribs, cartilages,
Fig 8. The intensity and the distribution of staining in experimental group in DZ areas are unequal (SP ⴛ 175).
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Table 4. The Intensity and Distribution of the Type II Collagen in SZ of the Sixth Costal Cartilages Between Control and Experimental Group Group
ARLS
ARDS
GRLS
GRDS
MGR
Experimental Control
21.405 ⫾ 4.108 22.755 ⫾ 7.891
16.375 ⫾ 3.403 11.306 ⫾ 3.182
167.142 ⫾ 29.192 164.935 ⫾ 15.570
160.750 ⫾ 43.476 159.611 ⫾ 32.305
165.198 ⫾ 30.452 163.120 ⫾ 19.201
NOTE. P ⬎ .05. Abbreviations: RALS, area ratio of light staining; ARDS, area ratio of deep staining; GRLS, gray scale of light staining; GRDS, gray scale of deep staining; MGR, mean gray scale.
Table 5. The Intensity and Distribution of the Type II Collagen in DZ of the Sixth Costal Cartilages Between Control and Experimental Group Group
ARLS
ARDS
GRLS
GRDS
MGR
Experimental Control
34.037 ⫾ 7.525 19.738 ⫾ 4.245*
23.010 ⫾ 2.935 10.002 ⫾ 1.821*
166.842 ⫾ 31.110 165.935 ⫾ 24.833
155.667 ⫾ 42.675 161.435 ⫾ 26.402
163.973 ⫾ 34.145 164.371 ⫾ 27.334
Abbreviations: RALS, area ratio of light staining; ARDS, area ratio of deep staining; GRLS, gray scale of light staining; GRDS, gray scale of deep staining; MGR, mean gray scale. *P ⬍ .05.
and adjacent interspace because of the stronger spastic contraction of the diaphragm, and may induce or aggravate the deformity. This could be supported by the fact that the PE is intended to happen in children with respiratory obstruction.17 Mullard9 held that the impaired biomechanics of the costal cartilages in PE might be secondary to the failure of osteogenesis or chondrogenesis. Unfortunately, there is no evidence to support this belief from morphologic investigations in this study. The structure of the cell and the nucleus of the costal cartilage is intact, and no hyperplasia or hypoplasia of the cartilage can be found either in light microscopy or in transmission electron microscopy investigation. The number of the vessel in cartilage in PE, as found by Rupprecht et al18 in their research in cartilage of funnel chest, also remained the same as in the control group. Unlike the report in a previous study,18 stating that the number of the chondrocytes strongly increased within the single chondrons with rising age, we found that the number of the cartilage cells remains constant. This difference might find its explanation in the different mean age of the patients in our research and the previous study. The collagen in DZ areas, however, has shown a significant difference between control and experimental groups by transmission electron microscopy and immunohistochemistry. The disturbance of the distribution and the arrangement of the collagen can be seen by transmis-
sion electron microscopy. Moreover, this abnormality mainly results from the disturbance of the distribution and the arrangement of the type II collagen shown by immunohistochemistry. The different properties of collagen meshwork, found by other investigators, may well affect the results of the tensile or compressive test.19 For example, the size and number of the individual fibrils might be related to the strength of the cartilage. Meanwhile, tensile stiffness might reflect interactions between collagen and the noncollageous matrix. The degree of binding between macromolecules can be expected to have a bearing on how fast the network can rearrange itself when subjected to stress. Kempson et al20 regarded that the tensile stiffness of the cartilage at low values of applied stress was largely dependent on 2 factors. First, it depended on the extent to which the collagen fibers were aligned initially in the direction of the applied stress and second, it depended on the resistance that fibers experience when they aligned in the direction of the stress. The properties of the collagen also influences the biomechanical property in compression.19-23 If the collagen meshwork has been dysarranged, the collagen fibers would fail to restrain the imbibition of the water by the hydrophilic proteoglycans, and the total water content of the cartilage could be expected to increase, whereas the compressive modulus might decrease. The disturbance of the distribution and arrangement of the collagen in cartilage therefore is responsible for the damage of the
Table 6. The Intensity and Distribution of the Proteoglycan of the Sixth Costal Cartilages Between Control and Experimental Group (Safranin-O Staining) Group
ARLS
ARDS
GRLS
GRDS
MGR
Experimental Control
12.520 ⫾ 2.005 13.617 ⫾ 2.640
20.647 ⫾ 7.145 22.203 ⫾ 5.015
169.729 ⫾ 30.070 165.942 ⫾ 20.086
160.474 ⫾ 33.521 154.997 ⫾ 29.568
164.137 ⫾ 32.134 158.807 ⫾ 30.247
NOTE. P ⬎ .05. Abbreviations: RALS, area ratio of light staining; ARDS, area ratio of deep staining; GRLS, gray scale of light staining; GRDS, gray scale of deep staining; MGR, mean gray scale.
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Table 7. The Intensity and Distribution of the Proteoglycan of the Sixth Costal Cartilages Between Control and Experimental Group (PAS Staining) Group
ARLS
ARDS
GRLS
GRDS
MGR
Experimental Control
18.805 ⫾ 3.451 17.765 ⫾ 4.394
13.305 ⫾ 2.947 11.205 ⫾ 3.175
142.246 ⫾ 28.360 151.675 ⫾ 19.528
128.347 ⫾ 23.980 130.714 ⫾ 30.505
137.388 ⫾ 40.136 142.474 ⫾ 35.919
NOTE. P ⬎ .05. Abbreviations: RALS, area ratio of light staining; ARDS, area ratio of deep staining; GRLS, gray scale of light staining; GRDS, gray scale of deep staining; MGR, mean gray scale.
biomechanical properties of the cartilage from the children with PE. Proteoglycan, the other important element of the matrix of the cartilage, also contributes to the cartilage biomechanics. When a large proportion of the total proteoglycan content of the articular cartilage is degraded and released from the matrix by the action of either cathepsin D or cathpsin B1, the compressive stiffness of the tissue is reduced significantly.20 Similar reduction in the tensile stiffness of cartilage at low stresses can be found using the same way to deal with the cartilage.20 But the distribution and the content of the proteoglycans of the cartilage from the experimental group, shown by Safranin-O and PAS staining, are the same as in the control group. These results may indicate that the biomechanical properties of the costal
cartilage in PE have been damaged by disturbance of the distribution and arrangement of the collagen rather than disturbance of the proteoglycan. Many factors might influence the distribution and arrangement of the collagen in cartilage. Further investigations will determine in which way a particular factor may contribute to the disturbance of the collagen in PE in the future research. ACKNOWLEDGMENTS The authors wish thank Professor Song ZM for his help in the biomechanical tests, Professor GUO X and Zhang ZT for their assistance with Safranin-O staining, Professor Yang XY for her help in immunohistochemistry and PAS staining, Dr Zhang J for his help in transmission electron microscopy, and Dr Neubauer H for his help in the revised form of the manuscript.
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