Histochemical and molecular analyses of distraction osteogenesis in a mouse model

Abstract

A tibial lengthening scheme in the mouse was used to study the molecular and cellular events regulating tissue regeneration during distraction osteogenesis. Here, we report on the surgical technique and frame design and describe the histochemical and molecular aspects of distraction during different phases of treatment. A total of 26 mice were used in this study. The treatment protocol was divided into a latency period of 7 days, a phase of active distraction that lasted 10 days with a distraction rate of 0.42 mm/day, and a maturation phase of 9 days. During latency, the distraction site resembled a stabilized fracture callus on both a histochemical and a molecular level. During active distraction, the gap was characterized by a central fibrous interzone bordered by primary matrix fronts, regenerate bone aligned with the distraction force, parallel columns of vascular sinusoids, and a medullary cavity. Alkaline phosphatase activity was detected in the endosteal and periosteal surfaces of the bone ends. Tartrate resistant acid phosphatase staining revealed that osteoclasts remodeled the bone regenerate as it formed. Collagen type I was expressed in the periosteum and the primary matrix front during distraction, whereas collagen type-II transcripts were localized to discrete regions on the periosteal surfaces, immediately adjacent to the osteotomy ends. Collagen type-II transcripts were not detected in the fibrous interzone. During the maturation phase, cells within the fibrous interzone expressed collagen type I and exhibited abundant alkaline phosphatase activity, suggesting that they had begun to terminally differentiate. Collectively, these data demonstrate the utility of a mouse model to study the molecular and cellular bases for the regeneration and remodeling of tissue.

Relationships between tissue dilatation and differentiation in distraction osteogenesis

“…The osteogenic effect of static, equibiaxial tensile stretch applied to osteoblasts in vitro has been shown previously (Fong et al, 2003). These results are consistent with the spatial correspondence observed between the regions experiencing static tensile stretch and those in which membranous bone formation occurs (Choi et al, 2002;Tay et al, 1998;Yasui et al, 1997;Yeung et al, 2002). At present, however, it is not known if it is the static nature of the physical stimulus per se that promotes these osteogenic phenomena.…”

“…As noted in previous studies Loboa et al, in press), these periosteal regions are of interest in that they are the only locations within the callus and gap that experience both positive pressures and tissue compaction (Fig. 3), and they are also the only locations in which cartilage formation predominates (Tay et al, 1998). This cartilage subsequently undergoes endochondral ossification.…”

“…Further, only one set of material properties was used for both the regenerate and medullary tissue. This rendered the analysis most applicable to the early stages of the distraction phase, when only granular and loose connective tissues, rather than mineralized tissues, are present in the regenerate (Rauch et al, 2000;Tay et al, 1998). We also assumed that the tissues did not become damaged during distraction.…”

An externally fixed femoral fracture model for mice

“…Models have also been developed in the murine tibia to examine distraction osteogenesis [5,9]. Both these models appear to permit reasonably satisfactory examination of the biology of osteogenesis, and permit production of a fracture with a closed method.…”

The adaptation of soleus and edl in a rat model of distraction osteogenesis: IGF‐1 and fibrosis

“…One possibility is the use of IGF-1 gene therapy to stimulate muscle growth. Whereas the DO regimen used in small rodents [1,33] is adequate for bone remodeling, it is not known what its effects are in muscle. Our result suggest that in adult rats there is an increase in IGF-1 in lengthened muscle, which seems to be associated with a remodeling of the extracellular matrix and not with a regenerative response in the muscle.…”