OTA 1997 Posters - Scientific Basis for Fracture Care

Mouse Distraction Osteogenesis: A Novel Model to Study the Molecular Mechanisms of Bone Healing and Regeneration

Bobby K-B Tay, MD, Anh S. Le, MD, Stephen E. Gould, PhD, Jill A. Helms, DDS, PhD

San Francisco, California, USA

Background and Hypothesis: The method by which bone regenerates under gradual distraction was first described by Alessandro Codvilla in 1905. It was not until the mid 1940's that basic cellular processes which occurred during distraction osteogenesis were systematically studied by Dr. Ilizarov in a canine model. Although there is a plethora of information available to describe the process at a gross, cellular, and biochemical level, there is no data that depicts the molecular mechanisms which regulate distraction osteogenesis in its four distinct phases: latency, early distraction, mid-distraction, and maturation. This paucity of molecular information comes from the inavailability of suitable molecular probes for the animal models which are currently in use. We describe a simple and effective model to study distraction osteogenesis using a mouse tibial lengthening scheme. Based upon previously reported histologic observations, we hypothesize that genes involved in embryonic bone formation and osteoblast differentiation are re-induced at the distraction gap.

Methods: Fifteen Swiss Webster mice were used in our initial study. A custom-made ring fixator was applied to the tibia of each animal after which an osteotomy was created at the metaphyseal/diaphyseal junction in the proximal tibia via a small anterior incision. Following a latency period of 7 days, the mice were distracted twice daily at a total rate of 0.42 mm/day. Mice were distracted for a maximum of 10 days. At specific timepoints during the latency, distraction, and maturation phases the distraction gap was characterized radiographically, histologically and biochemically to detect alkaline phosphatase and tartrate resistant acid phosphatase activity (a marker for osteoclasts) and on a molecular level by in-situ hybridization to antisense riboprobes corresponding to collagens I and II, indian hedgehog, bmp-6, and pth-receptor.

Results: By day 7 of the latency period, immature cartilage was seen in the subperiosteal areas at the site of the osteotomy. Membranous bone was observed filling the medullary cavity in the same area. During active distraction, the regenerate was arranged in a linear fashion parallel to the axis of distraction and formed towards a central fibrous interzone area. The bone that formed appeared to do so via an intramembranous route with minimal endochondral ossification limited to the periphery of the osteotomy ends. Histologic analyses showed no evidence of cartilage in the interzone area. This data was confirmed by the absence of col II expression in the interzone. Col I expression was predominately localized to the distraction gap. Col I expression was detected in the periosteal sleeve around the distraction gap and at the interface between the fibrous interzone and the front of regenerate bone. During active distraction, ihh expression was not detected in the distraction gap nor in the cartilaginous collections at the osteotomy ends. Pth-r transcripts were observed in osteoblasts at the primary matrix front. Alkaline phosphatase activity was also detected in the same group of cells which expressed pth-r. During maturation, the regenerate bone reformed a medullary cavity and bone islands began to appear in the interzone area. Alkaline phosphatase activity, which was limited to the regenerate bone during active distraction, was now detected within the interzone. Col I expression, which was limited to the primary matrix front during active distraction, expands into the cells of the fibrous interzone. During both distraction and maturation the activity of osteoclasts within the gap was localized to the regenerate bone adjacent to the primary matrix front.

Discussion and Conclusions: Our molecular analysis showed an induction of genes in the distraction gap which are normally observed during embryonic bone formation. Bmp-6 was expressed in the cartilagenous collections at the periphery of the distraction gap. In addition, the temporal and spatial expression of col I changes as we go from the distraction phase into the maturation phase providing the earliest marker of the initiation of cellular differentiation in the distraction gap. Ihh, a gene which plays a pivotal role in the maturation of chondrocytes in the embryonic limb, was not detected in the cartilage around the distraction gap during active distraction. Despite this, these chondrocytes continued to mature and hypertrophy. This observation suggests that another pathway may be involved in chondrocyte maturation in adult tissues. The localization of osteoclasts to the regenerate bone adjacent to the primary matrix front suggests that regenerate bone is immediately remodeled once it is formed. Thus the creation of regenerate bone at the distraction site over the entire process of distraction osteogenesis is a balance between bone formation stimulated by distraction and immediate and continuous bone remodeling by osteoclasts.

In conclusion, the distraction gap is an excellent model to study the interplay between endochondral and intramembranous bone formation. When compared with the mouse fracture model, the increased stability of the external fixator frame favors osteogenesis via an intramembranous route over an endochondral bone pathway. Molecular evidence using col I and col II gene expression indicates that the regenerate bone is formed directly over a collagen type I scaffold. Finally, the ability to detect changes in the temporal and spatial patterns of gene expression provides a mechanism to detect the earliest events in cellular differentiation occurring during distraction osteogenesis.