Date Published: March 28, 2017
Publisher: Public Library of Science
Author(s): Jose Luis Ramirez-GarciaLuna, Daniel Chan, Robert Samberg, Mira Abou-Rjeili, Timothy H. Wong, Ailian Li, Thorsten B. Feyerabend, Hans-Reimer Rodewald, Janet E. Henderson, Paul A. Martineau, Valérie Geoffroy.
In the adult skeleton, cells of the immune system interact with those of the skeleton during all phases of bone repair to influence the outcome. Mast cells are immune cells best known for their pathologic role in allergy, and may be involved in chronic inflammatory and fibrotic disorders. Potential roles for mast cells in tissue homeostasis, vascularization and repair remain enigmatic. Previous studies in combined mast cell- and Kit-deficient KitW-sh/W-sh mice (KitW-sh) implicated mast cells in bone repair but KitW-sh mice suffer from additional Kit-dependent hematopoietic and non- hematopoietic deficiencies that could have confounded the outcome. The goal of the current study was to compare bone repair in normal wild type (WT) and Cpa3Cre/+ mice, which lack mast cells in the absence of any other hematopoietic or non- hematopoietic deficiencies. Repair of a femoral window defect was characterized using micro CT imaging and histological analyses from the early inflammatory phase, through soft and hard callus formation, and finally the remodeling phase. The data indicate 1) mast cells appear in healing bone of WT mice but not Cpa3Cre/+ mice, beginning 14 days after surgery; 2) re-vascularization of repair tissue and deposition of mineralized bone was delayed and dis-organised in Cpa3Cre/+ mice compared with WT mice; 3) the defects in Cpa3Cre/+ mice were associated with little change in anabolic activity and biphasic alterations in osteoclast and macrophage activity. The outcome at 56 days postoperative was complete bridging of the defect in most WT mice and fibrous mal-union in most Cpa3Cre/+ mice. The results indicate that mast cells promote bone healing, possibly by recruiting vascular endothelial cells during the inflammatory phase and coordinating anabolic and catabolic activity during tissue remodeling. Taken together the data indicate that mast cells have a positive impact on bone repair.
It has been proposed that the discreet phases of bone repair in response to injury recapitulate those during development that give rise to the adult skeleton . It was recognized decades ago that cells of the immune system interact with those of the skeletal system during development and in the adult bone healing micro-environment. The term “osteoimmunology” was coined to define these complex interactions between lymphocytes, macrophages, mast cells, osteoclasts, osteoblasts and others . In the long bones of the adult skeleton the bone healing cascade is initiated with a blood clot and an inflammatory response during which cells migrate to the site of injury . The hematoma is replaced by granulation tissue to form a soft callus, metalloproteases cleave collagen and stored growth factors and cytokines are released. Angiogenic factors attract vascular endothelial cells, which form vessels throughout the repair tissue. Bone anabolic agents such as Wnt ligands, parathyroid hormone (PTH) and related protein (PTHrP) and bone morphogenetic proteins (BMPs) induce differentiation of mesenchymal stromal cells (MSC) into osteoblasts. Woven bone is deposited by these cells in and around the soft callus to form a hard callus, which is then remodeled by osteoclasts delivered through the new vessels.
The goal of the current study was to characterize the impact of mast cell deficiency on the repair of cortical bone defects using adult mast cell-deficient Cpa3Cre/+ mice. The Cpa3Cre/+ strain is constitutively devoid of mast cells in connective and mucosal tissues and it has no known alterations in other cell lineages involved in bone repair. In WT but not in Cpa3Cre/+ mice mast cells appeared in the repair tissue from 14d to 56d PO. Interestingly, bridging of the bone defect was complete in all (6/6) WT mice at 56d PO but only 3/8 Cpa3Cre/+ mice. This incomplete bridging was associated with disruption of re-vascularization and impaired bone mineralization. Osteoclast activity was reduced in Cpa3Cre/+ mice in the early phase of repair but increased at later stages, with no clear differences in macrophage activity. Taken together, the results indicate that mast cells have a positive impact on bone repair that is mediated in part by recruitment of vascular endothelial cells, as also suggested by the previous work of Boesiger et al (1998) , and in part by altered metabolism of newly formed bone. It was proposed more than two decades ago that mast cells are involved in tissue digestion and re-vascularization, which are early and essential steps in the bone healing cascade . Mast cells reside over the long term in connective tissues where they are available locally and for potential trafficking via the vascular and lymphatic systems to sites of tissue injury and repair . The surgical intervention used in this study would have temporarily disrupted existing hind limb vessels and caused local ischemia and hypoxia, which are the major stimuli for re-vascularization . Mast cells, identified by the use of metachromatic staining with aTB, were first seen in WT bone at 5d PO, which was the earliest time at which the soft callus could be preserved intact for histological analyses. At this time they were localized adjacent to vessels in muscle, in bone marrow of the femur proximal to the site of injury, and at the periosteal junction between soft tissue and bone. The appearance of aTB positive cells at the proximal, rather than distal, end of the femur suggests they migrated to the wound from soft tissue stores via the arterial or lymphatic vessels that supply the hind limb. This conjecture was supported by micro CT analyses of vessels in regenerating bone showing initiation of re-vascularization at 5d PO at the proximal end of the defect in WT bones. Quantitative analyses revealed peak numbers of aTB positive mast cells in the defect/medulla of WT bones at 14d PO, and in the cortex at 28d PO. This timeframe was supported by qualitative data showing MC tryptase positive cells in bone marrow starting at 14d in WT but not Cpa3Cre/+ mice. Peak numbers of mast cells in regenerating bone coincided with peak numbers of vessels visualized by micro CT and with CD34 immunostaining of vascular endothelial cells.