The concept of distraction histogenesis was introduced by G.A.Ilizarov and classic papers were published in the English literature in 1989[1,2]. Gradual traction on living tissues creates stresses that can stimulate regeneration and maintain active growth of certain tissue structures. Ilizarov designated this principle the Law of Tension-Stress [1]. The clinical applications of this principle in orthopaedics include limb lengthens discrepancy or short stature [3,4], delayed unions and nonunions of fractures [5], limb deformities correction [6], congenital pseudoarthrosis [7], and the treatment of bone defect [8]. The basic research of limb lengthening falls into two aspects. One is the responses of various tissues under tension stress during limb lengthening, including new bone formatted in the distraction gap, muscles, tendons, vessels and nerves [9-11]. The other concerns the underlying molecular mechanism of this principle [12,13]. However, among all the previous investigations, reports related to the deep fascia remain limited.

Congenital and developmental deformities are intriguing and difficult to overcome for orthopedic surgeons. These deformities are closely related to the lesions of soft tissues, mainly dense connective tissue. The deep fascia, belonging to connective tissue in histology, is just one of the most directly related tissues. In the previous study, histological and ultrastructural alterations of deep fascia in response to leg lengthening have been reported [14]. However, the response of extracellular matrix of fascia remains unknown. The aim of this study is to investigate the morphological characteristics of extracellular matrix, including glycosaminoglycans (GAGs) and extracellular matrix proteins, of deep fascia during leg lengthening and to evaluate candidate strategies to improve regeneration of fascia tissues, i.e., distraction fasciogenesis.

Alcian blue stain showed that GAGs of fascia of each group were composed of chondroitin sulphate and heparin sulphate, but not of keratan sulphate.

Figure 1 Fascia distracted at 2 mm/d with 10% increase in tibia length under the polarization microscopy (Original magnification 10 × 10). A high abundance and a wide distribution of collagen type II (yellow) were detected.

Figure 2 Fascia distracted at 1 mm/d with 10% increase in tibia length under the polarization microscopy (Original magnification 10 × 10). The deep fascia mainly consisted of collagen type I (red), and a basal collagen type II (yellow) distribution was (more ...)

Table 2 Relative abundances of collagens in fascia matrix ( ± s)

The distribution and composition of extracellular matrix in tissues play important roles in the etiology, pathology and mechanism of diseases. In particular, the abundance alterations of collagens are closely related to the injury and repairing of tissues, fibrosis pathology and the physiologic process of tissue regeneration. The total amount of collagen type I is approximately equal to that of collagen type III under normal conditions in most organs and tissues. Total collagens increases from 4% in normal liver to 10% in cirrhosis, and the levels of collagen type I reaches 4 times collagen type III as cirrhosis occurs [20]. Similarly, collagen type I increases dramatically while collagen type III decreases in the fibrosis of lung [21]. In contrast, collagen type III but not collagen type I level increases during tissue repair, suggesting a prior role of collagen type III in the regeneration of related tissues.

In this study, a distraction rate of 2 mm/d led to injuries of fascia and increasing amount of collagen type III in the matrix, which represents a repairing process of related tissues. The closest abundance of collagen to that of normal fascia was detected in the matrix of fascia that had been distracted at a rate of 1 mm/d until 20% increase in tibia length was achieved. Combined with our previous findings of increased amount of reticular fibers and ribosome, a nuclear split, the activation of endotheliocyte and newly formed young collagenous fibrils in the same scheme [14], these data indicate that the regeneration of multi-tissues involving deep fascia occurs in animals subjected to distraction-forced leg lengthening. While the distraction rates represent a certain load exerted on legs, the increments in tibia length (10% and 20%) may reflect the duration time of the load. As a result, 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. This may explain why 20% lengthening at a rate of 1 mm per day causes less collagen damage than 10%. Whereas GAGs are essential components of the extracellular matrix in normal, embryonic, or tumor tissues, in our study, a strong staining with Alcian blue in both control and distracted fascias indicated the presence of chondroitin sulphate and heparin sulphate, but not of keratan sulphate. However, the accurate detection of GAGs in biological samples has been precluded by the lack of sensitive methods [22].

Histochemical assays, including Alcian blue stain and picrosinus-polarizition method, are commonly used in the studies of the extracellular matrix, and computer-assisted imagine analysis system is usually imperative for a quantitative analysis of the imaging data. In this study to dissect the matrix alterations following leg lengthening, sections of different fascia regions were prepared, and pixels corresponding to different colors are analyzed to quantify the relative abundances of the major matrix components, collagen types I and III. Available techniques to detect GAGs usually require dissociative extraction of tissues [23]. For histochemistry, Alcian blue staining is generally used, in combination with critical electrolyte conditions at a definite pH [19]. The presence of sulfated GAGs can be demonstrated in the deep fascia using Alcian blue added MgCl₂. Due to currently unavailable quantification methods, the measurement of structural constituents on sections is usually difficult and the results are often doubtful [24]. Further study focusing on more sensitive histochemical methods to quantify GAGs may facilitate the quantitative comparison of extracelular matrix during leg lengthening.

Together, this study investigated the extracellular matrix changes including GAGs and the distribution and abundance of collagens in deep fascia in the context of leg lengthening for the first time according to our knowledge. Although further studies are definitely needed to dissect the synergized cellular and extracellular matrix signaling network responsible for regeneration and repair of the deep fascia, our study provides evidence for a potential clinical application of the Tension-Stress principle to deformity correction and limb lengthening.

The authors declare that they have no competing interests.

ZJL and HQW conceived of the study, participated in the design of the study and performed the statistical analyses. All authors carried out the experiments. HQW drafted the manuscript with the help of ZXW and YYW. All authors have read and approved the final manuscript.

The pre-publication history for this paper can be accessed here:

http://www.biomedcentral.com/1471-2474/9/101/prepub

We would like to thank engineer Jun Wang and Dr. Rong Lv for assistance in imagine analysis.