Picrosirius-polarization Method Research Articles

Picrosirius-Polarization Method for Collagen Fiber Detection in Tendons: A Mini-Review.

Although the structure and composition of collagen have been studied by polarized light microscopy since the early 19th century, many studies and reviews have paid little or no attention to the morphological problems of histopathological diagnosis. The morphology of collagen fibers is critical in guiding mechanical and biological properties in both normal and pathological tendons. Highlighting the organization and spatial distribution of tendon‐containing collagen fibers can be very useful for visualizing a tendon's ultrastructure, biochemical and indirect mechanical properties, which benefits other researchers and clinicians. Picrosirius red (PSR) staining, relying on the birefringence of collagen fibers, is one of the best understood histochemical methods that can highly and specifically underline fibers better than other common staining techniques when combined with polarized light microscopy (PLM). Polarized light microscopy provides complementary information about collagen fibers, such as orientation, type and spatial distribution, which is important for a comprehensive assessment of collagen alteration in a tendon. Here, this brief review serves as a simplistic and important primer to research developments in which differential staining of collagen types by the Picrosirius‐polarization method is increasing in diverse studies of the medical field, mainly in the assessment of the morphology, spatial distribution, and content of collagen in tendons.

Determination of age of skin wound by measuring collagen type I and III using picrosirius polarization method

This study was conducted to determine the association between the age of skin wound and percentage of type III and type I collagen using Picrosirius polarization method. Cross-sectional study of 35 samples of skin wound was taken from medicolegal death cases which displayed skin wounds with known time of incident. All the samples were stained with Picrosiriusred solution and type of collagens were assessed at the edge of injury site and percentages were given. Statistical significant differences were found between type III and type I collagen with age of skin wounds. Type I collagen showed good correlations with the age of skin wound.

Histological evidence of reparative activity in chorioamniotic membrane following open fetal surgery for myelomeningocele.

An increased understanding of the reparative process in fetal membrane following surgical techniques may be helpful to decrease the risks to mother and fetus and avoid adverse pregnancy outcomes. The present study discusses histological evaluation of the fetal membrane following open fetal surgery. Chorioamniotic membranes (n=10) were obtained following birth from pregnancies that underwent open fetal surgery for myelomeningocele. The collagen distribution was quantified using picrosirius-polarization method comparing the suture site with non-suture site. The differences between the collagen fiber percentages at the two sites was evaluated by the paired t-test with P<0.05. The mean gestational age of fetal surgery was 26.09±0.3 and 33.81±0.82 weeks at birth. The picrosirius red sign was more intense at the suture site, primarily associated with collagen type 1. Collagen observed in the surgical area was significantly increased (13.22±2.84%) compared with the non-surgical area (6.16±1.09%; P<0.0001). It was observed that the reparative activity at the suture site of the fetal membrane was characterized by a significant increase in collagen fibers. The findings suggest nascent collagen synthesis, tissue remodeling and repair of suture site, a mechanism likely to prevent the amniotic fluid leakage and maintain pregnancy following open fetal surgery.

Qualitative analysis of connective tissue stroma in different grades of oral squamous cell carcinoma: A histochemical study.

Detection of oral cancer at an early stage is of utmost importance to decrease morbidity and mortality. Tumor stroma plays a critical role during carcinogenesis. There is lack of information regarding the characteristics of the stroma in relation to the invading malignant epithelial cells and the interdependence between stroma and tumor cells in different grades of oral squamous cell carcinoma (OSCC). The present study was aimed to analyze and compare the nature of stroma in the vicinity of invading tumor islands in different grades of OSCC, using a histochemical technique picrosirius-polarization method. The present study also evaluated and correlated the possible role of inflammatory response in determining the nature of the stroma. The study included thirty cases of different grades of histologically diagnosed OSCC and ten sections of normal buccal mucosa as a control group. Nature of collagen was analyzed using picrosirius-polarization method, and intensity of inflammatory cell infiltrate was recorded using ImageJ software (1.42q, NIH, USA). The results were tabulated and analyzed statistically. Normal oral mucosa showed predominantly reddish birefringence. All cases of well-differentiated OSCC showed reddish-orange color. Nearly 70% moderately differentiated cases showed yellowish-orange (YO) and 60% of poorly differentiated cases, showed greenish-yellow (GY). The mean inflammatory cell count was highest in well-differentiated group. There was shift to YO and GY collagen when the cell differentiation and inflammatory cell count decreased in moderate and poorly differentiated cases. Both inflammatory cells and tumor cells have a role in determining the nature of the collagen fibers in tumor stroma of OSCC, probably with opposing effects on stromal behavior and hence both are significant in predicting prognosis.

Method for Picrosirius Red-Polarization Detection of Collagen Fibers in Tissue Sections.

The extracellular matrix is critical in guiding cell behavior in normal and pathologic tissues, particularly in cancers and fibrosis. Highlighting the organization (or disorganization) of a collagen-containing matrix can be very useful for understanding or grading pathophysiological conditions. The picrosirius red stain (also called "Sirius red" stain) is one of the best understood histochemical techniques able to selectively highlight collagen networks. Relatively inexpensive, the technique relies on the birefringent properties of collagen molecules. While the picrosirius red stain alone does not selectively bind collagen network, it becomes more specific than the other common collagen stains when combined with polarized light detection. This is why the selective histochemical procedure for collagen detection should be called the picrosirius-polarization method. In this chapter, we will provide essential explanation and detailed protocols and tips to allow collagen researchers not only to better understand how this staining technique works but also to easily apply this technique to their collagen-related research.

Pulsed electromagnetic field (PEMF) promotes collagen fibre deposition associated with increased myofibroblast population in the early healing phase of diabetic wound.

The present study evaluated the effects of PEMF on collagen fibre deposition, collagen fibril alignment and collagen fibre orientation. The potential relationships between collagen fibre deposition and myofibroblast population in diabetic wound healing were also examined. Forty young male streptozotocin-induced diabetic Sprague-Dawley rats were randomly assigned to PEMF group or control group. 2cm×2cm square wounds were made at their back. The PEMF group received daily exposure of PEMF to the wounds, while control group was handled in the same manner except that the PEMF device was not activated. Wound tissues harvested on post-wounding day 7, 10 and 14 were fixed, processed and sectioned. The abundance, fibril alignment and fibre orientation of type I collagen were quantified with picro-sirius polarization method and image analysis software (Nikon NIS Element AR). Myofibroblast population data were adopted from our previous study. Correlation between myofibroblast population and collagen fibre deposition was examined. There was significantly greater abundance of type I collagen fibre in the PEMF group than in the control on day 7 (P=.013), but not on day 10 or 14. No significant between-group differences were found in collagen fibril alignment and collagen fibre orientation at any measured time points. Positive correlation was found between collagen fibre deposition and myofibroblast population only on day 7 (r=.729, P=.007). In conclusion, PEMF can significantly increase collagen fibre in the early phase of diabetic wound healing, which is associated with the enhancement of myofibroblast population.

Development of Collagen Fibres and Lysyl Oxidase Expression in the Presumptive Dermis of Chick Limb Bud

Lysyl oxidase (LOX) plays a critical role in the formation of cross-linkages in extracellular matrix molecules. Thus, it is essential for the biogenesis and homeostasis of the connective tissue matrix. During development, collagen fibres and elastic system fibres emerge and accumulate in a temporospatial manner in the presumptive dermis of chicks. In this study, we investigated LOX mRNA expression by laser capture microdissection and RT-qPCR and LOX protein localization by immunohistochemistry. The picrosirius polarization method was used to investigate a relation between collagen accumulation and LOX expression. PCR analysis showed that the expression of LOX mRNA in the presumptive dermis became apparent at embryonic day 13 and increased considerably by ED17. Immunohistochemical staining for LOX in the dermis was very low at all stages of development. Accumulation of collagen fibres was seen in the dermis on ED10, and higher wavelengths of birefringence became evident by ED13. Our findings suggest that the temporal pattern of LOX mRNA expression correlates with collagen fibre accumulation in the dermis of the developing chick limb bud, whereas LOX expression was relatively constant at the protein level.

Construction and Clinical Application of a Human Tissue-Engineered Epidermal Membrane

Prolonged healing times and hypertrophic scarring of the donor site for split-thickness-skin grafts thicker than 0.3 mm are common problems that continue to challenge plastic surgeons in the clinic. As such, a human tissue-engineered epidermal membrane was constructed to promote wound healing and reduce scar hypertrophy. An artificial allogenic epidermis was created in vitro using human keratinocytes and chitosan-gelatin membrane. Split-thickness skin graft donor sites were divided into three treatment groups: those covered with the combined keratinocyte/chitosan-gelatin membrane, those covered with chitosan-gelatin membrane only (control group), and those covered with traditional petroleum jelly gauze (blank group). The degree of wound healing was assessed at various time points after the operation by gross observation, hematoxylin and eosin staining, immunohistochemistry, and an assay of type I collagen using the picrosirius polarization method. Reverse-transcriptase polymerase chain reaction detection of the Y chromosome was also performed to distinguish between sexes. Over a 6-month observation period, treatment with the human tissue-engineered epidermal membrane (keratinocyte/chitosan-gelatin) appeared to decrease donor-site healing time (48 wounds in 24 cases). Average healing time was 8.1 +/- 1.3 days for the keratinocyte/chitosan-gelatin group, 16.4 +/- 1.7 days for the chitosan-gelatin group, and 22.9 +/- 4.2 days for the blank group. The artificial epidermis survived well and maintained a normal structure. Furthermore, hypertrophic scar formation was less severe for these wounds. Keratinocyte/chitosan-gelatin membranes can be constructed in vitro and survive temporarily in vivo. They can be used to promote wound healing and reduce the severity of hypertrophic scarring of skin graft donor sites.

The presence of a vocal ligament in fetuses: a histochemical and ultrastructural study

Although it is currently believed that the vocal ligament of humans undergoes considerable development postnatally, there is no consensus as to the age at which it first emerges. In the newborn infant, the lamina propria has been described as containing a sparse collection of relatively unorganized fibres. In this study we obtained larynges from autopsy of human fetuses aged 7-9 months and used light and electron microscopy to study the collagenous and elastic system fibres in the lamina propria of the vocal fold. Collagen fibres were viewed using the Picrosirius polarization method and elastic system fibres were stained using Weigert's resorcin-fuchsin after oxidation with oxone. The histochemical and electron microscopic observations were consistent, showing collagen populations with an asymmetric distribution across different compartments of the lamina propria. In the central region, the collagen appeared as thin, weakly birefringent, greenish fibres when viewed using the Picrosirius polarization method, whereas the superficial and deep regions contained thick collagen fibres that displayed a strong red or yellow birefringence. These findings suggest that the thin fibres in the central region consist mainly of type III collagen, whereas type I collagen predominates in the superficial and deep regions, as has been reported in studies of adult vocal folds. Similarly, elastic system fibres showed a differential distribution throughout the lamina propria. Their distribution pattern was complementary to that of collagen fibres, with a much greater density of elastic fibres apparent in the central region than in the superficial and deep regions. This distribution of collagen and elastic fibres in the fetal vocal fold mirrors that classically described for the adult vocal ligament, suggesting that a vocal ligament has already begun to develop by the time of birth. The apparently high level of organization of connective tissue components in the newborn is in contrast to current hypotheses that argue that the mechanical stimuli of phonation are essential to the determination of the layered structure of the lamina propria and suggests that genetic factors may play a more significant role in the development of the vocal ligament than previously believed.

Lamina propria of the human vocal fold: histomorphometric study of collagen fibers

Since the seminal work of M. Hirano, which defined the three-layered lamina propria of the human vocal fold, there has been confusion in the labeling of each layer. Recent studies described the composition of fibers and interstitial molecules within the lamina propria leading to various biomechanical properties. However, collagen fibers appear as the most important structure component. We used an optical analysis and the picrosirius-polarization method to describe collagen fibers from six adult and two fetal human larynges fixed in formalin and frontally sectioned in the middle part of the vocal fold. The deep layer of the lamina propria is the most densely organized band of collagen fibers penetrating the superficial muscle bundles of the vocal muscle. The mean thickness of this layer is about 36% of the lamina propria and shows a network of strongly birefringent fibers (collagen type I and III). The superficial layer of the lamina propria is a narrow band of collagen fibers immediately below the basement membrane of the epithelium. The mean thickness of this layer is about 13% of the lamina propria and shows strong birefringent fibers. The intermediate layer is the less densely organized band between the deep and superficial layers. The mean thickness of this layer is about 51% of the lamina propria and shows clear, green weakly birefringent fibers characterized as collagen type III. The fetal lamina propria contains only a monolayer distribution of loose collagen fibers between the epithelium and the vocal muscle. These results help describe the distribution of collagen fibers within the lamina propria of the human vocal fold and have implications to understand the cover-body theory of voice production both in the adult and newborn.

Effect of reconstruction on medial collateral ligament healing of knee in rabbits

Objective To provide histological evidence for clinic tendon reconstruction on severe medial collateral ligament injury in rabbits. Methods Medial collateral ligaments of two knee joints were completely incised to create own control model of MCL rupture. The operation group intimated the procedure of human being MCL reconstruction with semitendinous tendon. In the 4th,12th,16th,24th weeks, healing of MCL was observed by anatomy and the picrosirius-polarization method. Results In the 4th week, the MCL healing tissue of ex-periment group is more than control group. The average ratios of type Ⅲ collagen to Ⅰ, Ⅲ collagen in the experiment group [(0.263±0.075), (0.235±0.041), (0.210±0.045), (0.197±0.029)] were significantly lower than those in control group [(0.310±0.072), (0.286±0.045), (0.261±0.046), (0.236±0.043) ] in the 4th, 12th, 16th, 24th weeks, respectively (P < 0.05). Conclusions The semitendinons tendon reconstruction can improve MCL healing in early and middle period after injury. Key words: Medial collateral ligament; knee/SU; Tendons/SU

Disarrangement of Collagen Fibers in Reinke's Edema

To describe the arrangement of collagen fibers in the superficial layer of the lamina propria of the vocal folds with Reinke' edema. Cross sectional analysis of the lamina propria of the vocal folds with Reinke's edema (RE). The picrosirius polarization method was used to study the arrangement of collagen fiber. Findings of collagen disarrangement were categorized semiquantitatively and correlated with RE severity, age, cigarette smoking and duration of dysphonia. Analysis of 20 specimens of vocal folds with RE showed that the intertwined network of collagen fibers resembling a wicker-basket normally observed in vocal folds was disarranged in RE. The collagen fibers were loosely arranged, fragmented and intermixed with varying amounts of myxoid stroma. Moderate and large areas of disarrangement (90% of cases) predominated. Collagen fiber arrangement in the region underneath the epithelium was better preserved when compared with fibers in the deeper region of the superficial layer of the lamina propria. There was a statistical difference in collagen disarrangement between grade II and grade III severity (P = .007) that appeared to be due to the large areas of disarrangement observed in 73% of patients with grade III severity and in 44% of grade II severity. Age was the only variable correlated with collagen fiber disarrangement (r = 0.47, P = .037). Our findings suggest that the flexible framework which maintains the uniformity of the lamina propria was lost in RE caused by the disarrangement of the collagen fibers.

The effect on the extracellular matrix of the deep fascia in response to leg lengthening.

BackgroundWhereas the alterations of diverse tissues in cellular and molecular levels have been investigated during leg lengthening via microscopy and biochemical studies, little is known about the response of deep fascia. This study aims to investigate the changes of the extracellular matrix in deep fascia in response to leg lengthening.MethodsAnimal model of leg lengthening was established in New Zealand white rabbits. Distraction was initiated at a rate of 1 mm/day and 2 mm/day in two steps, and preceded until increases of 10% and 20% in the initial length of tibia had been achieved. Alcian blue stain and picrosirius-polarization method were used for the study of the extracellular matrix of deep fascia samples. Leica DM LA image analysis system was used to investigate the quantitative changes of collagen type I and III.ResultsAlcian blue stain showed that glycosaminoglycans of fascia of each group were composed of chondroitin sulphate and heparin sulphate, but not of keratan sulphate. Under the polarization microscopy, the fascia consisted mainly of collagen type I. After leg lengthening, the percentage of collagen type III increased. The most similar collagen composition of the fascia to that of the normal fascia was detected at a 20% increase in tibia length achieved via a distraction rate of 1 mm/d.ConclusionThe changes in collagen distribution and composition occur in deep fascia during leg lengthening. Although different lengthening schemes resulted in varied matrix changes, the most comparable collagen composition to be demonstrated under the scheme of a distraction rate of 1 mm/day and 20% increase in tibia length. Efficient fascia regeneration is initiated only in certain combinations of the leg load parameters including appropriate intensity and duration time, e.g., either low density distraction that persist a relatively short time or high distraction rates.

Collagen Fiber and Versican Distribution Within the Lamina Propria of Fetal Vocal Folds

To analyze the presence and distribution of collagen fibers and versican in human vocal fold lamina propria of fetal larynges. Cross sectional analysis of cadaveric vocal folds of human fetuses. Seven fetal larynges obtained from 28- to 36-week-old fetuses were analyzed with the Picrosirius-polarization method, immunohistochemistry, and image analysis. Collagen fibers within the lamina propria exhibited a monolaminar distribution pattern and spatial arrangement in "wicker basket." Versican distribution was larger in the superficial and intermediate layers when compared to the deep layer. Our findings suggest that collagen and versican distribution and arrangement within the lamina propria in the developing fetus are important for vocalization at birth.

Polarization-sensitive optical coherence tomography for imaging human atherosclerosis

Polarization-sensitive optical coherence tomography (PS-OCT) combines the advantages of OCT with image contrast enhancement, which is based on its ability to detect phase retardation and the fast-axis angle. Both PS-OCT images and histopathology have demonstrated similar features that allowed differentiation of atherosclerotic structures (i.e., plaques) from normal tissue. Moreover, the picrosirius polarization method was used to confirm PS-OCT assessment of collagen in the fibrous cap of atherosclerotic plaques, and high-frequency (40 MHz) ultrasound images were used to identify calcium in the vessel wall. Our preliminary ex vivo investigation of human aortic specimens indicated that PS-OCT might help to identify atherosclerotic lesions.

Idiopathic and Collagen Vascular Disease Nonspecific Interstitial Pneumonia: Clinical Significance of Remodeling Process

Recently, active remodeling may indicate a good prognosis in idiopathic interstitial pneumonias. In this study we sought to validate the importance of the collagen/elastic system in the extracellular matrix remodeling and to study the relationships between the collagen/elastic system in nonspecific interstitial idiopathic pneumonia (NSIP) and collagen vascular disease associated with nonspecific interstitial idiopathic pneumonia (CVD-NSIP). We examined collagen/elastic system fibers in open lung biopsies of 20 idiopathic NSIP and 21 CVD-NSIP patients. The clinical features were analyzed with respect to age, gender, pulmonary functional tests, chest X-ray and computed tomography, treatment, and survival. We used the picrosirius polarization method and Weigert's resorcin-fuchsin histochemistry and morphometric analysis to evaluate the amount of collagen/elastic system fibers and their association with the NSIP histologic pattern. No differences in clinical features and pulmonary function tests were observed between idiopathic NSIP and CVD-NSIP, but a significantly higher collagen and elastic fiber proliferation was detected in CVD-NSIP lungs and fibrosing NSIP histologic pattern. Multivariate Cox model analysis demonstrated that sex and quantitative elastic fiber staining added important prognostic information (p=0.01) and was indicative of a worse prognosis than collagen staining. A cut point at the mean staining of 1.5% for elastic fibers divided the patients into two groups with distinctive survival times. Those with elastic fibers greater than 1.5% had a median survival time of just 52 months. We concluded that idiopathic NSIP and CVD-NSIP were clinically similar but pathologically different, suggesting that different remodeling profiles in NSIP may represent evolutionary adapted responses to injury grade, which depend, at least in part, on the extent of elastic extracellular matrix deposition. Patients with greater than 1.5% of elastic fibers comprise a subset with a high risk for dying due to NSIP and may be an appropriate target for prospective studies.

Picrosirius-polarization staining method as an efficient histopathological tool for collagenolysis detection in vesical prolapse lesions

The Picrosirius-polarization method has been indicated as a selective histochemical stain for collagen detection in tissue sections. This method can also be of value for studying collagen degradation given that, under polarized light, collagen displays birefringence due to its molecular order. The aim of this study is to highlight this staining method as an additional instrument for a rapid and excellent confirmatory diagnosis of the presence of collagenolysis in connective tissue in the vaginal wall with vesical prolapse lesion, in tissue sections. Dramatic changes in collagen morphology were found in vaginal mucosa in vesical prolapse disorder: they were weakly stained by Sirius red and under polarized light appeared as thin, pale (weakly birefringent), greenish, and with fibers more scattered, while the histoarchitecture of the organ showed a disrupted appearance. Thus, in the present study, we showed in vaginal mucosa in the vesicle prolapse that corroded collagenous framework appears as fragmentary and irregularly separated collagenous structures, that are weakly birefringent, corresponding to a molecular disorganization of these fibers caused by collagenolysis.

Effects of Progesterone on iNOS, COX-2, and Collagen Expression in the Cervix

This study examines the relationship between inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in the control of cervical ripening and parturition under normal (normal term pregnancy) and abnormal (preterm labor and prolongation of pregnancy) conditions by (a) measuring changes in the collagen both visually and quantitatively, (b) localizing and characterizing iNOS and COX-2 under normal conditions, and (c) characterizing the changes in iNOS and COX-2 under abnormal conditions. Cervices are obtained from estrus and timed pregnant Sprague-Dawley rats (n=4-10 per group). Preterm labor is induced with Onapristone (3 mg/rat; progesterone antagonist) and the prolongation of pregnancy with progesterone (2.5 mg, twice daily). Collagen changes are measured and visualized with the picrosirius polarization method. RT-PCR is used to characterize the mRNA expression (p<0.05), and immunohistochemistry is used to localize the protein expression for iNOS and COX-2. The organization and birefringence of the collagen during pregnancy decreased and is supported by changes in the luminosity (p<0.001). The iNOS and COX-2 enzymes were localized in cervical smooth muscle, vascular smooth muscle, and epithelium. Under normal conditions, iNOS mRNA levels decreased as COX-2 mRNA levels increased demonstrating an inverse correlation (Spearman r = -0.497; p=0.00295). Onapristone stimulated preterm labor, increasing the iNOS and COX-2 mRNA (p<0.05). The increase demonstrated a positive correlation (Spearman r = 0.456; p=0.03). Progesterone prolonged pregnancy, decreasing the iNOS and COX-2 mRNA (p=0.036). In conclusion, there may be an interaction between the nitric oxide and prostaglandin pathways in cervical ripening and parturition.

Collagen and elastic system in the remodelling process of major types of idiopathic interstitial pneumonias (IIP)

Structural remodelling in acute and chronic idiopathic interstitial pneumonia (IIP) has been extensively investigated, but little attention has been directed to the elastic tissue in these situations. The aim of this study was to determine whether elastic deposition accompanies collagen deposition in the four major histological patterns of IIP: diffuse alveolar damage (DAD), organizing pneumonia (OP), non-specific interstitial pneumonia (NSIP) and usual interstitial pneumonia (UIP). We measured, by image analysis, the content of fibres of the collagenous and elastic systems of the alveolar septum in histological slides of open lung biopsies, using the picrosirius-polarization method and Weigert's resorcin-fuchsin stain, respectively. Five groups were studied: 10 cases of DAD; nine cases of OP; nine cases of NSIP; and 10 cases of UIP. Four normal lungs were used for comparison. The content of collagen fibres was significantly higher in UIP when compared to DAD, NSIP, OP and normal lung. The content of elastic fibres was increased in comparison with normal lungs but this was not significantly different among the histological patterns. Acute and chronic IIP cause a similar increase in the collagen and elastic contents of the lungs, representing a process of 'fibroelastosis' rather than an exclusive process of fibrosis. A profibrogenic mechanism is responsible for the unparallelled collagen augmentation observed in UIP subjects, the nature of which is yet to be determined.

Histochemical and ultrastructural study of collagen fibers in mouse pubic symphysis during late pregnancy

Reference is usually made to the parallel orientation towards the main line of exerted tension at the pubic joint in mice, for supporting forces applied to the joint. Despite the wealth of morphological information about the extracellular matrix in this joint, little is known regarding the involvement of the crimp of collagen fibers in the dramatic transformations occurring in this region during the last 3 days of pregnancy. Examination of the collagenous architecture suggests that the biomechanical properties are directly related to fibril diameters, composition of ground substance and changes in the bundle morphology, particularly in the crimp structure. The purpose of this study was to further describe the transformation of the collagen fibers of the pubic symphysis during late mouse pregnancy. We examined the architecture of collagen fibers in the symphysis and pubic ligament through the Picrosirius-polarization method and also through scanning electron microscopy to directly visualize and measure the crimping from pregnant and virgin mice. The crimp angle and the length of five consecutive crimps were measured according to Patterson-Kane et al. [Connect. Tissue Res. 36 (1997) 253]. It could be demonstrated that the angles progressively decreased and the crimp length increased, denoting that the fibers have untwisted during the relaxation process. Our findings suggest that a disruption of the helical arrangement of the collagen containing fibers may contribute to explaining the rapid remodeling that occurs at the end of pregnancy and that is responsible for an increase in pliancy and length of the pubic ligament in mice.