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Knockdown of toll-like receptor 4 signaling pathways ameliorate bone graft rejection in a mouse model of allograft transplantation

View Article: PubMed Central - PubMed

ABSTRACT

Non-union occurring in structural bone grafting is a major problem in allograft transplantation because of impaired interaction between the host and graft tissue. Activated toll-like receptor (TLR) induces inflammatory cytokines and chemokines and triggers cell-mediated immune responses. The TLR-mediated signal pathway is important for mediating allograft rejection. We evaluated the effects of local knockdown of the TLR4 signaling pathway in a mouse segmental femoral graft model. Allografts were coated with freeze-dried lentiviral vectors that encoded TLR4 and myeloid differentiation primary response gene 88 (MyD88) short-hairpin RNA (shRNA), which were individually transplanted into the mice. They were assessed morphologically, radiographically, and histologically for tissue remodeling. Union occurred in autografted but not in allografted mice at the graft and host junctions after 4 weeks. TLR4 and MyD88 expression was up-regulated in allografted mice. TLR4 and MyD88 shRNAs inhibited TLR4 and MyD88 expression, which led to better union in the grafted sites. More regulatory T-cells in the draining lymph nodes suggested inflammation suppression. Local inhibition of TLR4 and MyD88 might reduce immune responses and ameliorate allograft rejection.

No MeSH data available.


Related in: MedlinePlus

Establishment of a murine femoral allograft model.Mice with femoral bone autografts and allografts were euthanized at 2 (WK 2) and 4 weeks (WK 4) after surgery. (a) A schematic figure of the mouse femoral bone transplantation. (b) Representative radiographs of autografted, frozen allografted, allografted, fractured, and normal mice at WK 2 and at WK 4 after surgery. White arrows indicate callus formation on the cortical bone surface of the autograft or the frozen allograft at WK 2 or WK 4. (c) Representative H&E and alcian blue-stained sections of autografted, frozen allografted, and allografted mice at WK 2 and at WK 4 after surgery. The black arrow indicates periosteal intramembranous bone formation, and the blue arrow indicates new bone-marrow cavity formation. Alcian blue-positive areas can be identified in the frozen allografted mice at WK 4 and autografted mice at WK 2. Scale bars represent 200 μM at 100× magnification.
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f1: Establishment of a murine femoral allograft model.Mice with femoral bone autografts and allografts were euthanized at 2 (WK 2) and 4 weeks (WK 4) after surgery. (a) A schematic figure of the mouse femoral bone transplantation. (b) Representative radiographs of autografted, frozen allografted, allografted, fractured, and normal mice at WK 2 and at WK 4 after surgery. White arrows indicate callus formation on the cortical bone surface of the autograft or the frozen allograft at WK 2 or WK 4. (c) Representative H&E and alcian blue-stained sections of autografted, frozen allografted, and allografted mice at WK 2 and at WK 4 after surgery. The black arrow indicates periosteal intramembranous bone formation, and the blue arrow indicates new bone-marrow cavity formation. Alcian blue-positive areas can be identified in the frozen allografted mice at WK 4 and autografted mice at WK 2. Scale bars represent 200 μM at 100× magnification.

Mentions: To determine and clarify the role of TLR4 signaling in bone-graft healing, we set up an in vivo murine femoral transplantation model17. We first removed a 4-mm section from the middle of the femoral diaphysis of C57BL/6 mice and then placed them back in the original C57BL/6 mice as an autograft or in the allogenic BALB/C mice as an allograft (Fig. 1a). Callus formation started at 2 weeks, and a new bone collar encircled the graft by 4 weeks during autograft healing. In contrast, this did not occur at 2 or 4 weeks during allograft healing, except slightly in the frozen allograft healing at 4 weeks (Fig. 1b). Accordingly, the autograft healing through endochondral ossification at the junctions with intramembranous ossification along the periosteum of the cortex of the graft can be observed after 2 or 4 weeks. Healing was completed at the graft and host junctions, and a new bone marrow cavity had formed after 4 weeks. Notably, neither endochondral nor intramembranous ossification can be identified in the allograft healing (Fig. 1c).


Knockdown of toll-like receptor 4 signaling pathways ameliorate bone graft rejection in a mouse model of allograft transplantation
Establishment of a murine femoral allograft model.Mice with femoral bone autografts and allografts were euthanized at 2 (WK 2) and 4 weeks (WK 4) after surgery. (a) A schematic figure of the mouse femoral bone transplantation. (b) Representative radiographs of autografted, frozen allografted, allografted, fractured, and normal mice at WK 2 and at WK 4 after surgery. White arrows indicate callus formation on the cortical bone surface of the autograft or the frozen allograft at WK 2 or WK 4. (c) Representative H&E and alcian blue-stained sections of autografted, frozen allografted, and allografted mice at WK 2 and at WK 4 after surgery. The black arrow indicates periosteal intramembranous bone formation, and the blue arrow indicates new bone-marrow cavity formation. Alcian blue-positive areas can be identified in the frozen allografted mice at WK 4 and autografted mice at WK 2. Scale bars represent 200 μM at 100× magnification.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5385519&req=5

f1: Establishment of a murine femoral allograft model.Mice with femoral bone autografts and allografts were euthanized at 2 (WK 2) and 4 weeks (WK 4) after surgery. (a) A schematic figure of the mouse femoral bone transplantation. (b) Representative radiographs of autografted, frozen allografted, allografted, fractured, and normal mice at WK 2 and at WK 4 after surgery. White arrows indicate callus formation on the cortical bone surface of the autograft or the frozen allograft at WK 2 or WK 4. (c) Representative H&E and alcian blue-stained sections of autografted, frozen allografted, and allografted mice at WK 2 and at WK 4 after surgery. The black arrow indicates periosteal intramembranous bone formation, and the blue arrow indicates new bone-marrow cavity formation. Alcian blue-positive areas can be identified in the frozen allografted mice at WK 4 and autografted mice at WK 2. Scale bars represent 200 μM at 100× magnification.
Mentions: To determine and clarify the role of TLR4 signaling in bone-graft healing, we set up an in vivo murine femoral transplantation model17. We first removed a 4-mm section from the middle of the femoral diaphysis of C57BL/6 mice and then placed them back in the original C57BL/6 mice as an autograft or in the allogenic BALB/C mice as an allograft (Fig. 1a). Callus formation started at 2 weeks, and a new bone collar encircled the graft by 4 weeks during autograft healing. In contrast, this did not occur at 2 or 4 weeks during allograft healing, except slightly in the frozen allograft healing at 4 weeks (Fig. 1b). Accordingly, the autograft healing through endochondral ossification at the junctions with intramembranous ossification along the periosteum of the cortex of the graft can be observed after 2 or 4 weeks. Healing was completed at the graft and host junctions, and a new bone marrow cavity had formed after 4 weeks. Notably, neither endochondral nor intramembranous ossification can be identified in the allograft healing (Fig. 1c).

View Article: PubMed Central - PubMed

ABSTRACT

Non-union occurring in structural bone grafting is a major problem in allograft transplantation because of impaired interaction between the host and graft tissue. Activated toll-like receptor (TLR) induces inflammatory cytokines and chemokines and triggers cell-mediated immune responses. The TLR-mediated signal pathway is important for mediating allograft rejection. We evaluated the effects of local knockdown of the TLR4 signaling pathway in a mouse segmental femoral graft model. Allografts were coated with freeze-dried lentiviral vectors that encoded TLR4 and myeloid differentiation primary response gene 88 (MyD88) short-hairpin RNA (shRNA), which were individually transplanted into the mice. They were assessed morphologically, radiographically, and histologically for tissue remodeling. Union occurred in autografted but not in allografted mice at the graft and host junctions after 4 weeks. TLR4 and MyD88 expression was up-regulated in allografted mice. TLR4 and MyD88 shRNAs inhibited TLR4 and MyD88 expression, which led to better union in the grafted sites. More regulatory T-cells in the draining lymph nodes suggested inflammation suppression. Local inhibition of TLR4 and MyD88 might reduce immune responses and ameliorate allograft rejection.

No MeSH data available.


Related in: MedlinePlus