Collagen Fiber Realignment Research Articles

Abstract SY21-02: Extracellular matrix density, alignment, and tumor progression

Abstract An increase in the mammographic density of breast tissue is correlated to a four to six-fold increased risk of developing breast carcinoma, making it the single biggest risk factor for breast carcinoma. Increased breast density is associated with a significant increase in the deposition of ECM components, most notably collagen, which leads to an increase in matrix stiffness. In a mouse model of increased collagen deposition, we find that mammary tumor incidence, invasion, and metastasis increased three-fold. Mammary tumors arising in FAK-/- mammary glands were non-invasive and non-metastatic. Gene expression profiling of these tumors identified a metastasis signature regulated by FAK, and further identify novel mechanisms and signaling pathways governing invasion into collagen matrices. Using state-of-the-art non-linear optical imaging techniques (FLIM and SHG), we have imaged collagen and cells at the tumor-stromal boundary in live, unfixed mammary tumors. We find that during tumor progression, the structure and alignment of collagen fibers is altered, such that the perpendicular realignment of collagen fibers is completely coincident with areas of local invasion. A recent analysis of 207 human samples demonstrates that the presence of perpendicular aligned collagen was a novel significant and independent prognostic indicator of poor patient survival, with a risk factor of 4.3. To determine whether fibril alignment facilitates invasion, we engineered aligned matrices, and found that human breast carcinoma cells preferentially invaded along perpendicularly aligned vs. random or parallel collagen fibers. Our results demonstrate collagen fibers serve as a highway to facilitate local invasion, and promote distal metastasis. Citation Format: Patricia J. Keely. Extracellular matrix density, alignment, and tumor progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr SY21-02. doi:10.1158/1538-7445.AM2014-SY21-02

Diabetes alters mechanical properties and collagen fiber re-alignment in multiple mouse tendons.

Tendons function to transfer load from muscle to bone through their complex composition and hierarchical structure, consisting mainly of type I collagen. Recent evidence suggests that type II diabetes may cause alterations in collagen structure, such as irregular fibril morphology and density, which could play a role in the mechanical function of tendons. Using the db/db mouse model of type II diabetes, the diabetic skin was found to have impaired biomechanical properties when compared to the non-diabetic group. The purpose of this study was to assess the effect of diabetes on biomechanics, collagen fiber re-alignment, and biochemistry in three functionally different tendons (Achilles, supraspinatus, patellar) using the db/db mouse model. Results showed that cross-sectional area and stiffness, but not modulus, were significantly reduced in all three tendons. However, the tendon response to load (transition strain, collagen fiber re-alignment) occurred earlier in the mechanical test, contrary to expectations. In addition, the patellar tendon had an altered response to diabetes when compared to the other two tendons, with no changes in fiber re-alignment and decreased collagen content at the midsubstance of the tendon. Overall, type II diabetes alters tendon mechanical properties and the dynamic response to load.

A Method for Predicting Collagen Fiber Realignment in Non-Planar Tissue Surfaces as Applied to Glenohumeral Capsule During Clinically Relevant Deformation

Previously developed experimental methods to characterize micro-structural tissue changes under planar mechanical loading may not be applicable for clinically relevant cases. Such limitation stems from the fact that soft tissues, represented by two-dimensional surfaces, generally do not undergo planar deformations in vivo. To address the problem, a method was developed to directly predict changes in the collagen fiber distribution of nonplanar tissue surfaces following 3D deformation. Assuming that the collagen fiber distribution was known in the un-deformed configuration via experimental methods, changes in the fiber distribution were predicted using 3D deformation. As this method was solely based on kinematics and did not require solving the stress balance equations, the computational efforts were much reduced. In other words, with the assumption of affine deformation, the deformed collagen fiber distribution was calculated using only the deformation gradient tensor (obtained via an in-plane convective curvilinear coordinate system) and the associated un-deformed collagen fiber distribution. The new method was then applied to the glenohumeral capsule during simulated clinical exams. To quantify deformation, positional markers were attached to the capsule and their 3D coordinates were recorded in the reference position and three clinically relevant joint positions. Our results showed that at 60deg of external rotation, the glenoid side of the posterior axillary pouch had significant changes in fiber distribution in comparison to the other sub-regions. The larger degree of collagen fiber alignment on the glenoid side suggests that this region is more prone to injury. It also compares well with previous experimental and clinical studies indicating maximum principle strains to be greater on the glenoid compared to the humeral side. An advantage of the new method is that it can also be easily applied to map experimentally measured collagen fiber distribution (obtained via methods that require flattening of tissue) to their in vivo nonplanar configuration. Thus, the new method could be applied to many other nonplanar fibrous tissues such as the ocular shell, heart valves, and blood vessels.

Erratum: “Effect of Age and Proteoglycan Deficiency on Collagen Fiber Re-Alignment and Mechanical Properties in Mouse Supraspinatus Tendon” [Journal of Biomechanical Engineering, 135(2), p. 021019

Erratum: “Effect of Age and Proteoglycan Deficiency on Collagen Fiber Re-Alignment and Mechanical Properties in Mouse Supraspinatus Tendon” [Journal of Biomechanical Engineering, 135(2), p. 021019

Effect of Age and Proteoglycan Deficiency on Collagen Fiber Re-Alignment and Mechanical Properties in Mouse Supraspinatus Tendon

Collagen fiber realignment is one mechanism by which tendon responds to load. Re-alignment is altered when the structure of tendon is altered, such as in the natural process of aging or with alterations of matrix proteins, such as proteoglycan expression. While changes in re-alignment and mechanical properties have been investigated recently during development, they have not been studied in (1) aged tendons, or (2) in the absence of key proteoglycans. Collagen fiber re-alignment and the corresponding mechanical properties are quantified throughout tensile mechanical testing in both the insertion site and the midsubstance of mouse supraspinatus tendons in wild type (WT), decorin-null (Dcn(-/-)), and biglycan-null (Bgn(-/-)) mice at three different ages (90 days, 300 days, and 570 days). Percent relaxation was significantly decreased with age in the WT and Dcn(-/-) tendons, but not in the Bgn(-/-) tendons. Changes with age were found in the linear modulus at the insertion site where the 300 day group was greater than the 90 day and 570 day group in the Bgn(-/-) tendons and the 90 day group was smaller than the 300 day and 570 day groups in the Dcn(-/-) tendons. However, no changes in modulus were found across age in WT tendons were found. The midsubstance fibers of the WT and Bgn(-/-) tendons were initially less aligned with increasing age. The re-alignment was significantly altered with age in the WT tendons, with older groups responding to load later in the mechanical test. This was also seen in the Dcn(-/-) midsubstance and the Bgn(-/-) insertion, but not in the other locations. Although some studies have found changes in the WT mechanical properties with age, this study did not support those findings. However, it did show fiber re-alignment changes at both locations with age, suggesting a breakdown of tendon's ability to respond to load in later ages. In the proteoglycan-null tendons however, there were changes in the mechanical properties, accompanied only by location-dependent re-alignment changes, suggesting a site-specific role for these molecules in loading. Finally, changes in the mechanical properties did not occur in concert with changes in re-alignment, suggesting that typical mechanical property measurements alone are insufficient to describe how structural alterations affect tendon's response to load.

The failure response of the human cervical facet capsular ligament during facet joint retraction

Studies implicate the cervical facet joint and its capsule as a primary anatomical site of injury during whiplash exposures to the neck. Although the facet joint is known to undergo stretch as the superior vertebra is retracted relative to the inferior vertebra during the whiplash kinematic, the response of the facet capsular ligament and its microstructure during failure in joint retraction is unknown. Polarized light imaging and vector correlation analysis were used to measure the collagen fiber alignment in the human capsular ligament, together with traditional mechanical metrics, during joint retraction sufficient to induce ligament failure. Anomalous fiber realignment occurs at 2.95±1.66mm of displacement, which is not different from the displacement when the ligament first yields (2.77±1.55mm), but is significantly lower (p=0.016) than the displacement at tissue failure (5.40±1.65mm). The maximum principal strain at the first detection of anomalous fiber realignment (0.66±0.39) also is significantly lower (p=0.046) than the strain at failure (1.39±0.64), but is not different from the strains at yield or partial failure. The onset of collagen fiber realignment determined in this study corresponds to the ligament's yielding and supports assertions that the facet capsule can undergo tissue injury during joint retraction. Further, such microstructural responses may indicate tissue damage in the absence of rupture.

Characterizing local collagen fiber re-alignment and crimp behavior throughout mechanical testing in a mature mouse supraspinatus tendon model

BackgroundCollagen fiber re-alignment and uncrimping are two postulated mechanisms of tendon structural response to load. Recent studies have examined structural changes in response to mechanical testing in a postnatal development mouse supraspinatus tendon model (SST), however, those changes in the mature mouse have not been characterized. The objective of this study was to characterize collagen fiber re-alignment and crimp behavior throughout mechanical testing in a mature mouse SST. Method of approachA tensile mechanical testing set-up integrated with a polarized light system was utilized for alignment and mechanical analysis. Local collagen fiber crimp frequency was quantified immediately following the designated loading protocol using a traditional tensile set up and a flash-freezing method. The effect of number of preconditioning cycles on collagen fiber re-alignment, crimp frequency and mechanical properties in midsubstance and insertion site locations were examined. ResultsDecreases in collagen fiber crimp frequency were identified at the toe-region of the mechanical test at both locations. The insertion site re-aligned throughout the entire test, while the midsubstance re-aligned during preconditioning and the test's linear-region. The insertion site demonstrated a more disorganized collagen fiber distribution, lower mechanical properties and a higher cross-sectional area compared to the midsubstance location. ConclusionsLocal collagen fiber re-alignment, crimp behavior and mechanical properties were characterized in a mature mouse SST model. The insertion site and midsubstance respond differently to mechanical load and have different mechanisms of structural response. Additionally, results support that collagen fiber crimp is a physiologic phenomenon that may explain the mechanical test toe-region.

Effect of Preconditioning and Stress Relaxation on Local Collagen Fiber Re-Alignment: Inhomogeneous Properties of Rat Supraspinatus Tendon

Repeatedly and consistently measuring the mechanical properties of tendon is important but presents a challenge. Preconditioning can provide tendons with a consistent loading history to make comparisons between groups from mechanical testing experiments. However, the specific mechanisms occurring during preconditioning are unknown. Previous studies have suggested that microstructural changes, such as collagen fiber re-alignment, may be a result of preconditioning. Local collagen fiber re-alignment is quantified throughout tensile mechanical testing using a testing system integrated with a polarized light setup, consisting of a backlight, 90 deg-offset rotating polarizer sheets on each side of the test sample, and a digital camera, in a rat supraspinatus tendon model, and corresponding mechanical properties are measured. Local circular variance values are compared throughout the mechanical test to determine if and where collagen fiber re-alignment occurred. The inhomogeneity of the tendon is examined by comparing local circular variance values, optical moduli and optical transition strain values. Although the largest amount of collagen fiber re-alignment was found during preconditioning, significant re-alignment was also demonstrated in the toe and linear regions of the mechanical test. No significant changes in re-alignment were seen during stress relaxation. The insertion site of the supraspinatus tendon demonstrated a lower linear modulus and a more disorganized collagen fiber distribution throughout all mechanical testing points compared to the tendon midsubstance. This study identified a correlation between collagen fiber re-alignment and preconditioning and suggests that collagen fiber re-alignment may be a potential mechanism of preconditioning and merits further investigation. In particular, the conditions necessary for collagen fibers to re-orient away from the direction of loading and the dependency of collagen reorganization on its initial distribution must be examined.

The Application of Cell Material in Tendon Injuries for Exercise Training with Biological Materials

Traditional tendon repair methods mainly used autogenous tendon transplantation and autogenous tendon transfer, but the development of two repair methods may be greatly limited due to the limited donor site and the added trauma. At the present time, the research on tissue-engineered tendon has made significant progress. The sources of seed cells for tendon repair cannot be limited to the autogenous tendon cells; we should make gene transformation to mechanocyte through gene chips method in order to resolve the problems related with sources of seed cells of tissue engineered tendon. The well-balanced mechanical stimulation is very important in tendon regeneration process. This stimulation on realignment of neonatal tendon collagen fibers and improve mechanical strength is necessary, which can guarantee implementation of early functional exercise, reducing tendon adhesion, and make tendon repair process into a virtuous circle.

Collagen Fiber Re-Alignment in a Neonatal Developmental Mouse Supraspinatus Tendon Model

Collagen fiber re-alignment is one postulated mechanism of tendon structural response to load. While collagen fiber distribution has been shown to vary by tendon location in the supraspinatus tendon (SST), changes in local re-alignment behavior have not been examined throughout postnatal development. Postnatal tendons, with immature collagen fibrils, may respond to load in a much different manner than collagen fibers with mature fiber-fiber and fiber-matrix connections. Local collagen fiber re-alignment is quantified throughout tensile mechanical testing in a developmental mouse SST model and corresponding mechanical properties measured. Collagen fiber re-alignment occurred during preconditioning for 28 day old tendons, at the toe-region for 10 day tendons and at the linear-region for 4 day tendon midsubstance. Mechanical properties increased with developmental age. Linear modulus was lower at the insertion site compared to the midsubstance location at all time points. Local differences in collagen fiber distributions were found at 10 and 28 days for all mechanical testing points (except the 10 day transition point). This study found that collagen fiber re-alignment depends on developmental age and suggests that collagen fibrillogenesis may influence the tendon's ability to structurally respond to load. Additionally, results indicate that the insertion site and tendon midsubstance locations develop differently.

Different Power Settings of LLLT on the Repair of the Calcaneal Tendon

The purpose of this study was to evaluate the effect of an 830-nm GaAlAs diode laser operating at output powers of 40, 60, 80, and 100 mW and energy density of 30 J/cm(2) on the repair of partial calcaneal tendon ruptures in rats. A partial tendon rupture was induced in all animals, which were treated with laser irradiation for 5 consecutive days. Six days after injury, the injured tendons were removed and examined by polarized light microscopy. Collagen fiber organization was evaluated by birefringence measurements, and collagen content was determined by Picrosirius Red staining. It was observed that the higher the output power (60-100 mW) the greater the amount of type III collagen (p<0.01). The amount of type I collagen was significantly greater (p=0.05) in the 80 mW group than in the control group (sham stimulation). A non-statistically significant improvement in the realignment of collagen fibers was observed in the irradiated groups. Low-level laser therapy resulted in significantly greater amounts of type III collagen (output powers of 60 mW or more) and type I collagen (output power of 80 mW), however, no significant differences between groups were found in the realignment of collagen fibers.

Realignment of Collagen Fibers in Mouse Skin under Biaxial Loads

Realignment of Collagen Fibers in Mouse Skin under Biaxial Loads

Vector correlation technique for pixel-wise detection of collagen fiber realignment during injurious tensile loading

Excessive soft tissue loading can produce adverse structural and physiological changes in the absence of any visible tissue rupture. However, image-based analysis techniques to assess microstructural changes during loading without any visible rupture remain undeveloped. Quantitative polarized light imaging (QPLI) can generate spatial maps of collagen fiber alignment during loading with high temporal resolution and can provide a useful technique to measure microstructural responses. While collagen fibers normally realign in the direction that tissue is loaded, rapid, atypical fiber realignment during loading may be associated with the response of a local collagenous network to fiber failure. A vector correlation technique was developed to detect this atypical fiber realignment using QPLI and mechanical data collected from human facet capsular ligaments (n=16) loaded until visible rupture. Initial detection of anomalous realignment coincided with a measurable decrease in the tissue stiffness in every specimen and occurred at significantly lower strains than those at visible rupture (p<0.004), suggesting this technique may be sensitive to a loss of microstructural integrity. The spatial location of anomalous realignment was significantly associated with regions where visible rupture developed (p<0.001). This analysis technique provides a foundation to identify regional differences in soft tissue injury tolerances and relevant mechanical thresholds.