Creating Rigidly Stabilized Fractures for Assessing Intramembranous Ossification, Distraction Osteogenesis, or Healing of Critical Sized Defects

Summary

This article describes a method for stabilizing long bone fractures that is based on the application of modified Ilizarov external fixators ^1-3. After application of the fixators and creation of the bone injury, healing can be assessed, distraction osteogenesis can be performed, or non-union or critical sized defect can be created and used to study therapeutic interventions.

Abstract

Assessing modes of skeletal repair is essential for developing therapies to be used clinically to treat fractures. Mechanical stability plays a large role in healing of bone injuries. In the worst-case scenario mechanical instability can lead to delayed or non-union in humans. However, motion can also stimulate the healing process. In fractures that have motion cartilage forms to stabilize the fracture bone ends, and this cartilage is gradually replaced by bone through recapitulation of the developmental process of endochondral ossification. In contrast, if a bone fracture is rigidly stabilized bone forms directly via intramembranous ossification. Clinically, both endochondral and intramembranous ossification occur simultaneously. To effectively replicate this process investigators insert a pin into the medullary canal of the fractured bone as described by Bonnarens⁴. This experimental method provides excellent lateral stability while allowing rotational instability to persist. However, our understanding of the mechanisms that regulate these two distinct processes can also be enhanced by experimentally isolating each of these processes. We have developed a stabilization protocol that provides rotational and lateral stabilization. In this model, intramembranous ossification is the only mode of healing that is observed, and healing parameters can be compared among different strains of genetically modified mice ^5-7, after application of bioactive molecules ^8,9, after altering physiological parameters of healing ¹⁰, after modifying the amount or time of stabilization ¹¹, after distraction osteogenesis ¹², after creation of a non-union ¹³, or after creation of a critical sized defect. Here, we illustrate how to apply the modified Ilizarov fixators for studying tibial fracture healing and distraction osteogenesis in mice.

Protocol

All Procedures were approved by the UCSF Institutional Animal Care and Use Committee and conform to national guidelines.

1. Preparation of Fixators Prior to Surgery

1. Before creating stabilized fracture, one needs to assemble the custom-design external fixation device. The custom-designed external fixation frames consisted of two aluminum rings stabilized by three stainless-steel #0/56 threaded rods, 8 #2/56 hexagonal nuts, and 17 matching bolts (Small Part Inc., Miami Lakes, FL³).
2. Two aluminum rings are needed per fracture. To one ring assemble 4 #2/56 hexagonal nuts (small Part Inc., Miami Lakes, FL) and matchi....

Discussion

Bones heal by two different modalities depending on mechanical stability (reviewed in: ¹⁴). When left unstable a large cartilage template forms in the fracture gap that is replaced by bone to bridge the two ends of broken bone. Proximally and distally to the break, bone forms directly by intramembranous ossification within the periosteum and endosteum. In contrast, in stable fractures healing occurs exclusively via intramembranous ossification ³. However, the specific mechanisms that regulate the sw.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.25mm insect pin	Fine Science Tools	26000-25	Blacked Anodized Steel, 0.25mm rod diameter, 4cm length
Stainless Steel Hex Nut	Small Parts, Inc.	#2-56	1/8" length, 56 threads per inch
Stainless Steel Hex Nut	Small Parts, Inc.	#0-80	1/8" length, 80 threads per inch
Stainless Steel Machine Screw	Small Parts, Inc.	#0-80	1/8" length, 80 threads per inch
Stainless Steel Machine Cut Threaded Rod	Small Parts, Inc.	#0-80	6" length, 80 threads per inch
18-8 Stainless Steel Head Machine Screw	McMaster-Carr		2-56 Threads, 3/6" length
External Fixation Device	Machine shop	Custom-designed