Alexander KA, Chang MK, Maylin ER, Kohler T, Müller R, Wu AC, van Rooijen N, Sweet MJ, Hume DA, Raggatt LJ, Pettit AR.
Osteal macrophages promote in vivo intramembranous bone healing in a mouse tibial injury model.
Journal of Bone and Mineral Research. 2011 Jul;26(7):1517–32.
[toggle_content title=”Abstract”] Bone-lining tissues contain a population of resident macrophages termed osteomacs that interact with osteoblasts in vivo and control mineralization in vitro . The role of osteomacs in bone repair was investigated using a mouse tibial bone injury model that heals primarily through intramembranous ossification and progresses through all major phases of stabilized fracture repair. Immunohistochemical studies revealed that at least two macrophage populations, F4/80 + Mac-2 -/low TRACP – osteomacs and F4/80+Mac-2 hi TRACP – inflammatory macrophages, were present within the bone injury site and persisted throughout the healing time course. In vivo depletion of osteomacs/macrophages (either using the Mafia transgenic mouse model or clodronate liposome delivery) or osteoclasts (recombinant osteoprotegerin treatment) established that osteomacs were required for deposition of collagen type 1 + (CT1 + ) matrix and bone mineralization in the tibial injury model, as assessed by quantitative immunohistology and micro–computed tomography. Conversely, administration of the macrophage growth factor colony-stimulating factor 1 (CSF-1) increased the number of osteomacs/macrophages at the injury site significantly with a concurrent increase in new CT1 + matrix deposition and enhanced mineralization. This study establishes osteomacs as participants in intramembranous bone healing and as targets for primary anabolic bone therapies. [/toggle_content]
[toggle_content title=”Clodronate Liposome Parameters”] [custom_table]
| Clodronate Concentration | Total Lipid Concentration | Lipid Composition | Lipid Mole % | Liposome Type | Control Liposomes |
|---|---|---|---|---|---|
| 7 mg/ml 1 | 23.5 mg/ml | EPC/Chol | 86/14 | MLV | PBS |
1 Based on typical encapsulation results reported by van Rooijen and Sanders (1994).
[/custom_table] [/toggle_content] [toggle_content title=”Animals and Dosing”] [custom_table]| Animal Description | Clodronate Dose | Dosing Method/Site | Target Phagocytes | Systemic Dosing? | Systemic Results |
|---|---|---|---|---|---|
| Mafia 2 mice, 11-12 w | 100 µl | intradefect/bone injury site | F4/80+ osteomacs | yes (i.p. 10 µl/g) | not quantitated |
2 Transgenic mice; C57BL/6 mice were used as controls.
[/custom_table] [/toggle_content] [toggle_content title=”Notes”]- Mice were dosed by injecting 100 µl clodronate liposomes into the tibial bone injury site at the time of surgical injury followed by 10 µl/g clodronate liposomes (200-250 µl) dosed i.p. daily X 9 d.
- FACS analysis of cells isolated from the control bone (contralateral uninjured tibia) showed an average 25.5% depletion in F4/80+ macrophages as a result of the i.p. injections.
- Immunohistochemistry of the injured bone confirmed depletion of F4/80+ macrophages.
- µCT confirmed a statistically significant reduction in bone density in mice treated with clodronate liposomes vs. PBS liposomes.
- Authors did not directly compare osteomac counts in control vs. injured bone, therefore we cannot determine the contribution of the intradefect injection of clodronate liposomes.
- Could pre-treatment of mice 18-24 hours before bone injury have effected the bone healing? Or, could withholding clodronate liposome treatment until 3 days post-surgery after the inflammatory phase have made a difference? Although the study focused on days 4 through 9 after surgery, we wonder how, or if, macrophage depletion during (vs before or after) the inflammatory phase changes the timing or extent of anabolic bone modeling (days 4-7) and catabolic modeling/remodeling (days 8-9).
- Direct delivery of a significant volume (100 µl) of clodronate liposomes to the site of the injury most likely resulted in some free clodronate being released in the region where osteoclasts reside. As free clodronate is known to kill osteoclasts, we believe that a free clodronate control group was critical to this study.