2010-winkler-4815

On Thu, 12-Jul-2012   /   Intravenous Injection, Retro-orbital  
Winkler IG, Sims NA, Pettit AR, Barbier V, Nowlan B, Helwani F, Poulton IJ, van Rooijen N, Alexander KA, Raggatt LJ, Levesque, JP. Bone marrow macrophages maintain hematopoietic stem cell (HSC) niches and their depletion mobilizes HSCs. Blood. 2010 Aug 16;116(23):4815–28.

Abstract

In the bone marrow (BM), hematopoietic stem cells (HSC) reside in specific niches near osteoblast-lineage cells at the endosteum. To investigate regulation of these endosteal niches, we studied mobilization of HSC into the bloodstream in response to granulocyte colonystimulating factor (G-CSF). We report that G-CSF mobilization rapidly depletes endosteal osteoblasts leading to suppressed endosteal bone formation and decreased expression of factors required for HSC retention and self-renewal. Importantly, G-CSF administration also depleted a population of trophic endosteal macrophages (osteomacs) that support osteoblast function. Osteomac loss, osteoblast suppression and HSC mobilization occurred concomitantly, suggesting that osteomac loss could disrupt endosteal niches. Indeed in vivo depletion of macrophages, in either macrophage Fas-induced apoptosis (Mafia) transgenic mice or by administration of clodronate-loaded liposomes to wild-type mice, recapitulated the i) loss of endosteal osteoblasts, ii) marked reduction of HSC-trophic cytokines at the endosteum, with iii) HSC mobilization into the blood as observed during G-CSF administration. Together these results establish that BM macrophages are pivotal to maintain the endosteal HSC niche and that the loss of such macrophages leads to the egress of HSC into the blood.[/toggle_content] [toggle_content title=”Clodronate Liposome Parameters”] [custom_table]

Clodronate Concentration Total Lipid Concentration Lipid Composition Lipid Mole % Liposome Type Control Liposomes
5 mg/ml 23.4 mg/ml EPC/Chol 84/16 MLV PBS
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Animal Description Clodronate Dose Dosing Method/Site Target Phagocytes Local Dosing? Local Results
C57BL/6, female mice, 8-12 w 250 µl/25 g retro-orbital F4/80+Ly-6G+CD11b+ osteomacs no N/A
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Notes

  1. Liposome prep method cited — van Rooijen N, Sanders A. Liposome mediated depletion of macrophages: mechanism of action, preparation of liposomes and applications. Journal of Immunological Methods. 1994 Sep 14;174(1–2):83–93.
  2. Animals were dosed with clodronate (or control) liposomes on days 2 and 4.

Results

  1. >95% of the F4/80+Ly-6G+CD11b+ osteomacs and other bone marrow macrophages were depleted by day 2; depletion continued throughout experiment (6 d).
  2. Effects on endosteal niches began within 24 h of clodronate liposome dosing.
  3. “Substantial reduction” in osteocalcin+ osteoblasts by 48 h presumably due to osteomac depletion rather than direct effect of clodronate liposomes on osteoblasts.
  4. We are curious about the mechansim of osteomac depletion by liposomal clodronate since the osteomacs are shown to exclusively associate with osteoblasts which are remotely located from the sinusoid in the bone marrow. How does the liposome enter the marrow from sinusoid and migrate through the central bone marrow to the endosteum where the osteomacs are located? Since liposomes have been shown to predominantly remain near the capillaries rather than migrating into tissue (other than those carried by macrophages), we wonder if depletion of other macrophages located perisinally elicit migration of the osteomacs toward the sinusoid.

 

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2011-alexander-1511

On Mon, 02-Jul-2012   /   Intradefect (Bone)  
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).

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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.

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  1. 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.
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  1. 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.
  2. Immunohistochemistry of the injured bone confirmed depletion of F4/80+ macrophages.
  3. µCT confirmed a statistically significant reduction in bone density in mice treated with clodronate liposomes vs. PBS liposomes.
  4. 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.
  5. 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).
  6. 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.
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