Influence of Internal Fixator Stiffness on Murine Fracture Healing: Two Types of Fracture Healing Lead to Two Distinct Cellular Events and FGF-2 Expressions

Abstract

This study aimed to clarify the relationship between the mechanical environment at the fracture site and endogenous fibroblast growth factor-2 (FGF-2). We compared two types of fracture healing with different callus formations and cellular events using MouseFix™ plate fixation systems for murine fracture models. Left femoral fractures were induced in 72 ten-week-old mice and then fixed with a flexible (Group F) or rigid (Group R) Mouse Fix™ plate. Mice were sacrificed on days 3, 5, 7, 10, 14, and 21. The callus volumes were measured by 3D micro-CT and tissues were histologically stained with hematoxylin & eosin or safranin-O. Sections from days 3, 5, and 7 were immunostained for FGF-2 and Proliferating Cell Nuclear Antigen (PCNA). The callus in Group F was significantly larger than that in Group R. The rigid plate allowed bone union without a marked external callus or chondrogenesis. The flexible plate formed a large external callus as a result of endochondral ossification. Fibroblastic cells in the granulation tissue on days 5 and 7 in Group F showed marked FGF-2 expression compared with Group R. Fibroblastic cells showed ongoing proliferation in granulation tissue in group F, as indicated by PCNA expression, which explained the relative granulation tissue increase in group F. There were major differences in early phase endogenous FGF-2 expression between these two fracture healing processes, due to different mechanical environments. Key words: callus formation, fracture, growth factor, internal fixator, mouse

Bone Marrow-Engrafted Cells after Mice Umbilical Cord Blood Transplantation Differentiate into Osteoblastic Cells in Response to Fracture and Placement of Titanium Screws

“…here, GFPpositive cells were clearly observed in the fracture gap and callus, and were positive for aLP and type i collagen staining on day 7. by day 14, GFP-positive cells were observed around the callus and under the periosteum, in concurrence with the aLP and type i collagen staining. Moreover, von Kossa-positive cells were observed on day 14. in our previous studies, similar cellular events were observed within the same time period in wild-type c57bL/6J mice and a GFP bone marrow chimeric mouse [24,25]. taken together, our results suggest that bone marrow-engrafted Ucbcs contribute to fracture healing in a manner similar to endogenous bone marrow-derived cells.…”

“…We used a rigid plate of the MouseFix tM system (ao development institute, davos, switzerland), which is a locking plate system with good angular stability, as an internal fixator of fractured mouse femurs [25]. Prior to the plate implantation and fracture of femurs, a mixture of 1 part midazolam (sandoz, tokyo, Japan), 3 parts domitor tM (nippon Zenyaku Kogyo co., Ltd., Fukushima, Japan), and 1 part Vetorphale tM (Meiji seika Pharma co., Ltd., tokyo, Japan) was intramuscularly injected into the upper limbs of mice at an optimal dose of 0.05 ml/100 g to induce anesthesia.…”

“…here, to clarify the in vivo function of bone marrowengrafted Ucbcs, we investigated whether these cells differentiate into osteoblastic lineages during fracture healing using a mouse Ucb transplantation model, which facilitates studies on the basic biology, engraftment, and therapeutic potential of Ucbs [18,19,23], and mouse fracture models, which are useful for evaluating fracture healing under standardized mechanical and biological conditions [24,25].…”

Distribution of Bone Marrow-Derived Cells in the Fracture Callus during Plate Fixation in a Green Fluorescent Protein-Chimeric Mouse Model

“…1B) configuration. We have already confirmed that we can observe two distinct cellular events at the fracture healing site using two Mouse Fix™ plates with different levels of flexibility [24]. By the same technique, a mixture of 1 part midazolam, 3 parts Domitor™, and 1 part Vetorphale™ was then intramuscularly injected into the upper limbs at an optimal dose of 0.05 ml/100 g. The operation was performed only on the left femur.…”

“…We already reported that the use of different types of plates with the Mouse Fix™ system leads to 2 different types of fracture healing, with differences in the type of callus formation and cellular events [10,24].…”