Rejuvenation of the inflammatory system stimulates fracture repair in aged mice
Introduction
Bone has the ability to regenerate after fracture1. However, the capacity for repair declines with age. In humans, and in experimental models, bone repair has been shown to be delayed and protracted in middle-aged animals compared to juveniles and healing continues to decline as animals age26. The mechanisms responsible for this decline in healing potential remain unknown. A number of studies have examined potential mechanisms that contribute to delayed healing in aged animals. We have shown that angiogenesis is decreased during fracture healing in elderly animals7. Other investigators have examined elderly mice for changes in expression patterns of genes that are required for bone and cartilage formation during fracture healing. These studies indicate that age-dependent changes in expression of genes required for bone and cartilage formation do not seem to underlie delayed fracture repair, and these authors concluded that deficits may lie outside of the skeletal tissues 8.
Recent work has suggested that components of the inflammatory response may be altered in aged animals during fracture repair9. This is an interesting observation, because inflammation is an important part of bone repair10, and chronic inflammation is thought to underlie problems associated with aging11. For example, expression of pro-inflammatory cytokines are increased as a result of aging12, and increased inflammation as a result of age has been linked to changes in the skeleton1315. However, the effect that age-related inflammation has on fracture healing is not known.
Accumulating evidence suggests that aging is associated with sustained inflammation after injury. While inflammation is an essential component of the healing process, the alterations that occur within the inflammatory response in aged animals are known to hinder tissue repair. IL-6 levels are higher in older patients after acute myocardial infarction, and these elevations are associated with exaggerated ventricular remodeling16. Further, studies have shown that plasma levels of IL-6 are significantly increased in older trauma patients compared to juveniles, and this increase correlates with higher morbidity1718.
We hypothesized that age-related changes in the inflammatory system were associated with protracted bone repair in middle-aged animals compared to juveniles. To test this, we used a mouse model of bone marrow transplantation after lethal irradiation19. In chimeric mice, the fracture callus is comprised of bone and cartilage that are formed by host cells, while inflammatory cells are derived from the donor. To examine the effect of aging inflammatory cells on fracture repair we created heterochronic chimeras comprised of either juvenile or middle-aged that were reconstituted with juvenile or middle aged bone marrow-derived cells or cells, and then we compared fracture healing in the heterochronic chimeras.
Materials and Methods
Design and creation of chimeric mice
All procedures were approved by the Institutional Animal Care and Use Committee at the University of California at San Francisco. To create chimeric mice19, bone marrow was collected from Juvenile (4-week-old) or middle-aged (12-month-old) male Rosa26 mice (Jackson Laboratory, Bar Harbor, Maine, USA). Rosa26 mice were chosen because they have ubiquitous expression of the LacZ reporter gene that can be detected with X-gal staining. Red blood cells were lysed (Sigma-Aldrich, St. Louis, Missouri, USA), remaining cells were washed with RPMI-1640, and suspended in PBS. Cells were transplanted within 4 hours of collection. Congenic, male C57BL/6J mice, juvenile (4-week-old) or middle-aged (12-month-old), were irradiated with 6 Gray using Cs. Then donor cells (10) were injected via the tail vein. Thus, 4 groups of mice were created: middle-aged donor into middle-aged host (MA:MA), juvenile donor into middle-aged host (Ju:MA), juvenile donor into juvenile host (Ju:Ju), and middle-aged donor into juvenile host (MA:Ju).
Neomycin (2mg/ml) was provided beginning 2 days before irradiation and continuing for 2 weeks after irradiation. Body weight and activity of chimeras were monitored. Blood was collected from the retro-orbital plexus 2 weeks before irradiation, and at 1 and 4 weeks after irradiation. Blood cells were counted using a hematological cell counter.
Creation of non-stabilized fractures
Four weeks after bone marrow transplantation, fractures were created in the diaphysis of the right tibia in anesthetized mice (2% avertin) by three-point-bending20. Fractures were not stabilized. Analgesic (1% buprenex) was injected twice a day for 2 days after fracture. Mice were sacrificed at 7, 14, 21 and 28 days after fracture.
Tissue processing
After sacrifice, fractured legs were collected, fixed in 0.4% paraformaldehyde, and decalcified in 19% EDTA at 4°C. The decalcified tissues were infused with 30% sucrose, embedded in Tissue-Tek OCT, and sectioned (10μm) through the whole callus.
Histology and histomorphometry
To visualize cartilage in fracture callus, immunostaining using an anti-type II collagen antibody (Chondrex Inc., Redmond, Washington, USA) was performed on serial sections (300μm apart) through the whole callus. After being treated with 0.2% Hyaluronidase for 30 minutes for antigen retrieval, sections were incubated with the primary antibody (1:50, 1 hour), washed 3 times, and then incubated with an HRP-conjugated second antibody at room temperature (1 hour). The immune complex was visualized with DAB. Newly formed bone was detected with Modified Milligan's Trichrome. Slides were examined with a Leica DMRB microscope. Images were captured using Adobe Photoshop. The volume of callus, cartilage, and bone was estimated using Cavalieri's principle621.
X-gal staining
To detect donor cells or donor cell-derived cells in the host, X-gal staining was performed on tissue sections22. This staining detects β-galactosidase activity. Cells derived from donor cells will be stained blue. Sections were counterstained with hematoxylin without treatment with tap water which stains tissues pink. To visualize donor-derived inflammatory cells, sections that were stained with X-gal were then reacted with macrophage and neutrophil-specific antibodies as described below.
Detection of macrophages and neutrophils at the fracture site
Immunostaining was performed to detect macrophages (F4/80, eBioscience, San Diego, California, USA) and neutrophils (MCA771G, Serotec, Raleigh, North Carolina, USA) on tissue sections. Briefly, 3 to 5 sections were systematically and randomly selected from each sample of day 7 fractures for immunostaining. Sections were incubated with primary antibodies (1:100 dilution for F4/80 and MCA771G) at 4°C overnight or at room temperature for 1 hour. Sections were then incubated with a second antibody. The immune complex was visualized with DAB and sections were counterstained with hematoxylin. No antigen retrieval was required. The cellular density of macrophages and neutrophils in the fracture callus were estimated using an Olympus CAST system (Olympus, Center Valley, PA) and Visiopharm software (Visiopharm, Hørsholm, Denmark). On each section, the fracture callus was outlined and fields covering 20% of the outlined callus were randomly selected. A counting frame probe was applied to quantify the number of macrophages and neutrophils within these fields and the area of each field. Values were expressed as number of macrophages or neutrophils per mm callus tissue.
Detection of Osteoclasts in fracture callus
TRAP staining (leukocyte Acid Phosphatase kit, Sigma-Aldrich, St. Louis, Missouri, USA) was performed on frozen sections at day 28 after fracture to visualize osteoclasts. The cell density of osteoclast in fracture callus was determined using stereology as described above.
Assessing the systemic inflammatory response
At day 7 after fracture, plasma was collected from the chimeric mice via heart puncture immediately after euthanasia (8-week and 12-month-old). The plasma levels of IL-6 and TNF-alpha were determined using ELISA kits following the company's instruction (eBioscience, San Diego, California, USA).
Statistical analysis
Data are expressed as mean +/- SD. Two-way ANOVA was performed to test the main effects of age of bone marrow, healing time, and the interaction between age of bone marrow and healing time on formation of callus, new bone and new cartilage. Student's t test was used to compare the IL-6 and TNF-α levels. P<0.05 is considered significant.
Design and creation of chimeric mice
All procedures were approved by the Institutional Animal Care and Use Committee at the University of California at San Francisco. To create chimeric mice19, bone marrow was collected from Juvenile (4-week-old) or middle-aged (12-month-old) male Rosa26 mice (Jackson Laboratory, Bar Harbor, Maine, USA). Rosa26 mice were chosen because they have ubiquitous expression of the LacZ reporter gene that can be detected with X-gal staining. Red blood cells were lysed (Sigma-Aldrich, St. Louis, Missouri, USA), remaining cells were washed with RPMI-1640, and suspended in PBS. Cells were transplanted within 4 hours of collection. Congenic, male C57BL/6J mice, juvenile (4-week-old) or middle-aged (12-month-old), were irradiated with 6 Gray using Cs. Then donor cells (10) were injected via the tail vein. Thus, 4 groups of mice were created: middle-aged donor into middle-aged host (MA:MA), juvenile donor into middle-aged host (Ju:MA), juvenile donor into juvenile host (Ju:Ju), and middle-aged donor into juvenile host (MA:Ju).
Neomycin (2mg/ml) was provided beginning 2 days before irradiation and continuing for 2 weeks after irradiation. Body weight and activity of chimeras were monitored. Blood was collected from the retro-orbital plexus 2 weeks before irradiation, and at 1 and 4 weeks after irradiation. Blood cells were counted using a hematological cell counter.
Creation of non-stabilized fractures
Four weeks after bone marrow transplantation, fractures were created in the diaphysis of the right tibia in anesthetized mice (2% avertin) by three-point-bending20. Fractures were not stabilized. Analgesic (1% buprenex) was injected twice a day for 2 days after fracture. Mice were sacrificed at 7, 14, 21 and 28 days after fracture.
Tissue processing
After sacrifice, fractured legs were collected, fixed in 0.4% paraformaldehyde, and decalcified in 19% EDTA at 4°C. The decalcified tissues were infused with 30% sucrose, embedded in Tissue-Tek OCT, and sectioned (10μm) through the whole callus.
Histology and histomorphometry
To visualize cartilage in fracture callus, immunostaining using an anti-type II collagen antibody (Chondrex Inc., Redmond, Washington, USA) was performed on serial sections (300μm apart) through the whole callus. After being treated with 0.2% Hyaluronidase for 30 minutes for antigen retrieval, sections were incubated with the primary antibody (1:50, 1 hour), washed 3 times, and then incubated with an HRP-conjugated second antibody at room temperature (1 hour). The immune complex was visualized with DAB. Newly formed bone was detected with Modified Milligan's Trichrome. Slides were examined with a Leica DMRB microscope. Images were captured using Adobe Photoshop. The volume of callus, cartilage, and bone was estimated using Cavalieri's principle621.
X-gal staining
To detect donor cells or donor cell-derived cells in the host, X-gal staining was performed on tissue sections22. This staining detects β-galactosidase activity. Cells derived from donor cells will be stained blue. Sections were counterstained with hematoxylin without treatment with tap water which stains tissues pink. To visualize donor-derived inflammatory cells, sections that were stained with X-gal were then reacted with macrophage and neutrophil-specific antibodies as described below.
Detection of macrophages and neutrophils at the fracture site
Immunostaining was performed to detect macrophages (F4/80, eBioscience, San Diego, California, USA) and neutrophils (MCA771G, Serotec, Raleigh, North Carolina, USA) on tissue sections. Briefly, 3 to 5 sections were systematically and randomly selected from each sample of day 7 fractures for immunostaining. Sections were incubated with primary antibodies (1:100 dilution for F4/80 and MCA771G) at 4°C overnight or at room temperature for 1 hour. Sections were then incubated with a second antibody. The immune complex was visualized with DAB and sections were counterstained with hematoxylin. No antigen retrieval was required. The cellular density of macrophages and neutrophils in the fracture callus were estimated using an Olympus CAST system (Olympus, Center Valley, PA) and Visiopharm software (Visiopharm, Hørsholm, Denmark). On each section, the fracture callus was outlined and fields covering 20% of the outlined callus were randomly selected. A counting frame probe was applied to quantify the number of macrophages and neutrophils within these fields and the area of each field. Values were expressed as number of macrophages or neutrophils per mm callus tissue.
Detection of Osteoclasts in fracture callus
TRAP staining (leukocyte Acid Phosphatase kit, Sigma-Aldrich, St. Louis, Missouri, USA) was performed on frozen sections at day 28 after fracture to visualize osteoclasts. The cell density of osteoclast in fracture callus was determined using stereology as described above.
Assessing the systemic inflammatory response
At day 7 after fracture, plasma was collected from the chimeric mice via heart puncture immediately after euthanasia (8-week and 12-month-old). The plasma levels of IL-6 and TNF-alpha were determined using ELISA kits following the company's instruction (eBioscience, San Diego, California, USA).
Statistical analysis
Data are expressed as mean +/- SD. Two-way ANOVA was performed to test the main effects of age of bone marrow, healing time, and the interaction between age of bone marrow and healing time on formation of callus, new bone and new cartilage. Student's t test was used to compare the IL-6 and TNF-α levels. P<0.05 is considered significant.
Results
Donor marrow cells re-established the hematopoietic system in chimeras by 4 weeks
To ensure that juvenile and middle-aged bone marrow cells reconstituted the hematopoietic system equally well, we assessed recovery of mice from the radiation and bone marrow transplantation by examining various physiological parameters. By four weeks all mice exhibited similar recovery from the irradiation injury. There was no decrease in body weight in either group (Fig. 1A). White blood cells in the peripheral circulatory system had recovered significantly by this time in all groups (Fig. 1B). Red blood cells decreased slightly after irradiation, and there was no significant change in platelet number after bone marrow transplantation (Fig. 1C,1D). By 4 weeks there was no significant difference in the total number of each blood cell type (Fig. 1), or in differential counts among white blood cells (Fig. 1E) between middle-aged hosts receiving bone marrow from middle-aged donors (MA:MA) or receiving bone marrow from juvenile donors (Ju:MA). Similar findings were observed between juvenile hosts receiving bone marrow from middle-aged donors (MA:Ju) or receiving bone marrow from juvenile donors (Ju:Ju). Thus, we created fractures at this time.
(A) 4 weeks after BMT, there was no difference in weight, (B) white blood cell (WBC), (C) red blood cell (RBC), (D) platelet (PLT), and (E) differential counts of white blood cells among the chimeric and control groups (MA:MA, n = 22 at each time point; Ju:MA mice, n = 21 before BMT, n = 19 at 1wk or 4wk; Ju:Ju and MA:Ju, n=8/time point).
Donor-derived cells were restricted to hematopoietic derivatives
We examined the distribution of donor derived cells in the fracture site using X-gal staining. We first confirmed that in donor Rosa26 mice, cells in bone marrow, bone, and periosteum (Fig. 2A), growth plate (Fig. 2B), muscle (Fig. 2C), and the fracture callus (Fig. 2D) all stained positively for β-galactosidase activity. At 4 weeks after bone marrow transplantation, bone marrow of the chimeric mice was comprised of donor-derived cells, but no donor-derived cells were detected in the growth plate, bone, periosteum, or muscle fibers (Fig. 2E, 2F). At 7 days after fracture, none of the new chondrocytes and osteoblasts in fracture callus was derived from the donor bone marrow (Fig. 2G, 2H). However, a large number of donor-derived cells were detected in the granulation tissue at this time (Fig. 2I, 2J). Double staining using immunohistochemistry of macrophage and neutrophils markers and X-gal staining revealed that these cells were macrophages and neutrophils (Fig. 2K, 2L).
(A) Cells in bone marrow, (B) periosteum, growth plate, (C) muscle fibers, and (D) chondrocytes in the fracture callus were stained blue by X-gal staining in ROSA26 mice. (E) In chimeric mice, donor cells were not observed in growth plate, (F) cortical bone, periosteum, muscle, and (G) cartilage and (H) bone in the callus. (I) 7 days after fracture donor cells (blue) were present at the fracture site and (J) in granulation tissue. (K) Donor cells were stained with F4/80 or (L) MCA771G. (Scale bar = 50μm)
Recruitment of inflammatory cells to the fracture site was not affected by the age of the donor cells
We assessed whether inflammatory cells derived from juvenile or middle-aged donor animals exhibited fundamental differences in response to the fracture. We quantified neutrophils and macrophages in fracture calluses in the chimeric mice at 7 days after injury. There were more macrophages and neutrophils at the fracture site in juvenile hosts compared to middle-aged hosts, regardless of the age of the donors (p<0.001 macrophages and neutrophils). However, there was no difference in the numbers of inflammatory cells in middle-aged animals that received juvenile or middle-aged marrow, and cell numbers were not different in juvenile hosts that received different aged marrow (Fig. 3).
7 days after fracture, there was no difference in the densities of macrophages and neutrophils in fracture callus between Ju:MA and MA:MA as well as between MA:Ju and Ju:Ju. However, the cell densities were significantly higher in juvenile hosts than middle-aged hosts regardless of the ages of donors (p<0.001 for both cell types; n = 6 each chimeric group)
Rejuvenation of inflammatory cells alters the inflammatory response
We characterized the inflammatory response by comparing plasma levels of IL-6 and TNF-alpha in juvenile mice, middle-aged mice, and the chimeric mice after fracture. At 7 days after fracture, plasma levels of IL-6 were significantly higher in 12-month-old mice than juveniles (Fig. 4, p<0.01). We next determined that the levels of IL-6 in Ju:Ju was not different than in the Ju animals (p=0.985). While the MA:MA mice had higher levels of IL-6 than the MA mice, this only approached statistical significance (p=0.089). However, when we replaced the hematopoietic system of middle-aged mice with juvenile bone marrow (Ju:MA), we observed a significant decrease in the plasma level of IL-6 after fracture compared to controls (MA:MA) (Fig. 4, p=0.014). Plasma levels of TNF-alpha were lower in juvenile mice compared to middle-aged mice and slightly decreased in middle-aged mice with juvenile marrow, but again the differences only approached significance (Fig. 4, p=0.099, p=0.0503, respectively).
At day 7 after fracture, middle-aged mice (MA, n=6) had significantly higher plasma IL-6 than juvenile mice (Ju, n=6, p<0.01). After rejuvenation of bone marrow, plasma IL-6 in middle-aged mice (Ju:MA, n=6) was significantly lower than in controls (MA:MA, n=7, p=0.014). Plasma IL-6 in juvenile mice with aged marrow (MA:Ju, n=4) was higher than controls (Ju:Ju, n=5), but not significant. TNF-alpha exhibited the same trend (* p>0.05)
Rejuvenation of inflammatory system accelerated bone repair in middle-aged mice
Next, we determined that the process of fracture healing was significantly accelerated in Ju:MA mice compared to MA:MA mice (Fig. 5A-C). One week after injury, Ju:MA mice formed a larger callus (p=0.014) that was comprised of more bone (p=0.005) than the MA:MA mice. Between day 14 and 28 the callus size declined indicating that remodeling had occurred during this time. At day 14 time no differences in bone and cartilage were detected between these groups. Remodeling was protracted in the MA:MA mice; bone volume continued to increase through 28 days after injury in these mice. By day 28, the callus in Ju:MA group had been significantly reduced compared to MA:MA group (p=0.001). At this time, endochondral ossification was complete in Ju:MA chimeras; cartilage had been removed in all of these mice (n=6/6). However the majority (n=5/7) of MA:MA mice still had cartilage in the fracture callus. We did not detect differences in the density of osteoclasts in the callus at day 28 in MA:MA and Ju:MA chimeras (38±11 vs. 43±8/mm, p=0.384).
(A) Callus size and (B) volume of new bone were significantly larger in Ju:MA chimeras at day 7, and were significantly decreased at day 28 compared to MA:MA mice. (C) There was no difference in the volume of cartilage between these groups at day 7 and day14. At day 28, 5/7 MA:MA mice had cartilage in the callus. (* p<0.05). (D) There was no difference in Callus size, (E) volume of new bone, and (F) new cartilage between Ju:Ju and MA:Ju mice at day 7 (Ju:Ju, n=8, MA:Ju,n=8), day14 (Ju:Ju, n=7, MA:Ju, n=6), and day21 (Ju:Ju, n=7, MA:MA, n=7) after fracture.
Aging of the inflammatory system did not alter bone repair in juvenile mice
Finally, we replaced the bone marrow of juvenile mice (4 week old) with that derived from 12-month-old donors (MA:Ju) and we compared healing in these animals to juvenile mice that received bone marrow from Juvenile donors (Ju:Ju). While the plasma levels of IL-6 and TNF-alpha were increased at day 7 in the MA:Ju animals compared to controls (Ju:Ju) these changes were not statistically significant (Fig. 4, p=0.459, p=0.185, respectively). Histomorphometry revealed that there was no significant difference in the total callus size and amounts of bone or cartilage between MA:Ju mice and Ju:Ju mice (Fig. 5D-F), indicating that callus formation and remodeling was not affected in juvenile mice by the presence of inflammatory cells from middle-aged animals.
Donor marrow cells re-established the hematopoietic system in chimeras by 4 weeks
To ensure that juvenile and middle-aged bone marrow cells reconstituted the hematopoietic system equally well, we assessed recovery of mice from the radiation and bone marrow transplantation by examining various physiological parameters. By four weeks all mice exhibited similar recovery from the irradiation injury. There was no decrease in body weight in either group (Fig. 1A). White blood cells in the peripheral circulatory system had recovered significantly by this time in all groups (Fig. 1B). Red blood cells decreased slightly after irradiation, and there was no significant change in platelet number after bone marrow transplantation (Fig. 1C,1D). By 4 weeks there was no significant difference in the total number of each blood cell type (Fig. 1), or in differential counts among white blood cells (Fig. 1E) between middle-aged hosts receiving bone marrow from middle-aged donors (MA:MA) or receiving bone marrow from juvenile donors (Ju:MA). Similar findings were observed between juvenile hosts receiving bone marrow from middle-aged donors (MA:Ju) or receiving bone marrow from juvenile donors (Ju:Ju). Thus, we created fractures at this time.
(A) 4 weeks after BMT, there was no difference in weight, (B) white blood cell (WBC), (C) red blood cell (RBC), (D) platelet (PLT), and (E) differential counts of white blood cells among the chimeric and control groups (MA:MA, n = 22 at each time point; Ju:MA mice, n = 21 before BMT, n = 19 at 1wk or 4wk; Ju:Ju and MA:Ju, n=8/time point).
Donor-derived cells were restricted to hematopoietic derivatives
We examined the distribution of donor derived cells in the fracture site using X-gal staining. We first confirmed that in donor Rosa26 mice, cells in bone marrow, bone, and periosteum (Fig. 2A), growth plate (Fig. 2B), muscle (Fig. 2C), and the fracture callus (Fig. 2D) all stained positively for β-galactosidase activity. At 4 weeks after bone marrow transplantation, bone marrow of the chimeric mice was comprised of donor-derived cells, but no donor-derived cells were detected in the growth plate, bone, periosteum, or muscle fibers (Fig. 2E, 2F). At 7 days after fracture, none of the new chondrocytes and osteoblasts in fracture callus was derived from the donor bone marrow (Fig. 2G, 2H). However, a large number of donor-derived cells were detected in the granulation tissue at this time (Fig. 2I, 2J). Double staining using immunohistochemistry of macrophage and neutrophils markers and X-gal staining revealed that these cells were macrophages and neutrophils (Fig. 2K, 2L).
(A) Cells in bone marrow, (B) periosteum, growth plate, (C) muscle fibers, and (D) chondrocytes in the fracture callus were stained blue by X-gal staining in ROSA26 mice. (E) In chimeric mice, donor cells were not observed in growth plate, (F) cortical bone, periosteum, muscle, and (G) cartilage and (H) bone in the callus. (I) 7 days after fracture donor cells (blue) were present at the fracture site and (J) in granulation tissue. (K) Donor cells were stained with F4/80 or (L) MCA771G. (Scale bar = 50μm)
Recruitment of inflammatory cells to the fracture site was not affected by the age of the donor cells
We assessed whether inflammatory cells derived from juvenile or middle-aged donor animals exhibited fundamental differences in response to the fracture. We quantified neutrophils and macrophages in fracture calluses in the chimeric mice at 7 days after injury. There were more macrophages and neutrophils at the fracture site in juvenile hosts compared to middle-aged hosts, regardless of the age of the donors (p<0.001 macrophages and neutrophils). However, there was no difference in the numbers of inflammatory cells in middle-aged animals that received juvenile or middle-aged marrow, and cell numbers were not different in juvenile hosts that received different aged marrow (Fig. 3).
7 days after fracture, there was no difference in the densities of macrophages and neutrophils in fracture callus between Ju:MA and MA:MA as well as between MA:Ju and Ju:Ju. However, the cell densities were significantly higher in juvenile hosts than middle-aged hosts regardless of the ages of donors (p<0.001 for both cell types; n = 6 each chimeric group)
Rejuvenation of inflammatory cells alters the inflammatory response
We characterized the inflammatory response by comparing plasma levels of IL-6 and TNF-alpha in juvenile mice, middle-aged mice, and the chimeric mice after fracture. At 7 days after fracture, plasma levels of IL-6 were significantly higher in 12-month-old mice than juveniles (Fig. 4, p<0.01). We next determined that the levels of IL-6 in Ju:Ju was not different than in the Ju animals (p=0.985). While the MA:MA mice had higher levels of IL-6 than the MA mice, this only approached statistical significance (p=0.089). However, when we replaced the hematopoietic system of middle-aged mice with juvenile bone marrow (Ju:MA), we observed a significant decrease in the plasma level of IL-6 after fracture compared to controls (MA:MA) (Fig. 4, p=0.014). Plasma levels of TNF-alpha were lower in juvenile mice compared to middle-aged mice and slightly decreased in middle-aged mice with juvenile marrow, but again the differences only approached significance (Fig. 4, p=0.099, p=0.0503, respectively).
At day 7 after fracture, middle-aged mice (MA, n=6) had significantly higher plasma IL-6 than juvenile mice (Ju, n=6, p<0.01). After rejuvenation of bone marrow, plasma IL-6 in middle-aged mice (Ju:MA, n=6) was significantly lower than in controls (MA:MA, n=7, p=0.014). Plasma IL-6 in juvenile mice with aged marrow (MA:Ju, n=4) was higher than controls (Ju:Ju, n=5), but not significant. TNF-alpha exhibited the same trend (* p>0.05)
Rejuvenation of inflammatory system accelerated bone repair in middle-aged mice
Next, we determined that the process of fracture healing was significantly accelerated in Ju:MA mice compared to MA:MA mice (Fig. 5A-C). One week after injury, Ju:MA mice formed a larger callus (p=0.014) that was comprised of more bone (p=0.005) than the MA:MA mice. Between day 14 and 28 the callus size declined indicating that remodeling had occurred during this time. At day 14 time no differences in bone and cartilage were detected between these groups. Remodeling was protracted in the MA:MA mice; bone volume continued to increase through 28 days after injury in these mice. By day 28, the callus in Ju:MA group had been significantly reduced compared to MA:MA group (p=0.001). At this time, endochondral ossification was complete in Ju:MA chimeras; cartilage had been removed in all of these mice (n=6/6). However the majority (n=5/7) of MA:MA mice still had cartilage in the fracture callus. We did not detect differences in the density of osteoclasts in the callus at day 28 in MA:MA and Ju:MA chimeras (38±11 vs. 43±8/mm, p=0.384).
(A) Callus size and (B) volume of new bone were significantly larger in Ju:MA chimeras at day 7, and were significantly decreased at day 28 compared to MA:MA mice. (C) There was no difference in the volume of cartilage between these groups at day 7 and day14. At day 28, 5/7 MA:MA mice had cartilage in the callus. (* p<0.05). (D) There was no difference in Callus size, (E) volume of new bone, and (F) new cartilage between Ju:Ju and MA:Ju mice at day 7 (Ju:Ju, n=8, MA:Ju,n=8), day14 (Ju:Ju, n=7, MA:Ju, n=6), and day21 (Ju:Ju, n=7, MA:MA, n=7) after fracture.
Aging of the inflammatory system did not alter bone repair in juvenile mice
Finally, we replaced the bone marrow of juvenile mice (4 week old) with that derived from 12-month-old donors (MA:Ju) and we compared healing in these animals to juvenile mice that received bone marrow from Juvenile donors (Ju:Ju). While the plasma levels of IL-6 and TNF-alpha were increased at day 7 in the MA:Ju animals compared to controls (Ju:Ju) these changes were not statistically significant (Fig. 4, p=0.459, p=0.185, respectively). Histomorphometry revealed that there was no significant difference in the total callus size and amounts of bone or cartilage between MA:Ju mice and Ju:Ju mice (Fig. 5D-F), indicating that callus formation and remodeling was not affected in juvenile mice by the presence of inflammatory cells from middle-aged animals.
Discussion
Bone marrow harbors both hematopoietic stem cells and mesenchymal stem cells. We did not observe a contribution of bone marrow derived cells to formation of new bone or cartilage during fracture healing, which agrees with a previous report from our colleagues 19. By changing the age of the host and donor mice used for marrow transplantation, we created an in vivo environment in which the age of skeletogenic cells and inflammatory cells were manipulated separately. In this work, we determined that age-related changes in the inflammatory system were associated with protracted bone repair in middle-aged animals compared to juveniles. Reconstitution of the aged hematopoietic stem cell compartment with juvenile bone marrow significantly reduced systemic inflammation in response to injury and accelerated bone repair. This work agrees with previous research that suggests age-related changes in inflammatory cells underlie deficiencies in healing of cutaneous wounds23, and the observation that juvenile macrophages can accelerate healing cutaneous wounds in aged mice24.
Juvenile versus aged inflammatory response
Many studies have illustrated that the inflammatory response in juvenile animals is restrained compared to older animals25. Here we showed that middle-aged mice had a pro-inflammatory systemic environment in response to fracture compared to juvenile mice. We also showed that chimeras made with juvenile inflammatory cells had a reduced systemic inflammatory response. However, the reason for reduced inflammation is not known. One possibility is that the young inflammatory cells simply produced a more controlled inflammatory response. Reducing chronic inflammation prior to injury has been shown to enhance healing in other models26. In our work, the young donor cells may have reduced the chronic inflammation that is present in aging animals27.
When we created chimeras with middle-aged bone marrow in juvenile hosts, we did not observe delays in fracture healing. This could have resulted from not achieving a complete replacement of the young hematopoietic stem cells with the aged donor cells. Thus, the healing outcome may simply reflect the presence of juvenile inflammatory cells. However, we observed a similar nadir and rebound of circulating blood cells independent of host age, indicating that the host cells were similarly ablated by irradiation.
Juvenile mice responded differently to injury. After transplantation of middle-aged inflammatory cells into juvenile hosts we observed low levels of IL-6 and TNF-α compared to middle aged hosts. A recent report suggests that the major source of IL-6 in aged animals may be adipose tissue 28. In this study, the authors found that fat from aged animals produced higher levels of IL-6 than young animals after stimulation with inflammatory agents. Thus, juvenile mice may not be prone to producing sustained systemic levels of IL-6 in response to inflammation, and the presence of the middle-aged inflammatory cells in our experiments may not have been able to evoke the same response from juvenile hosts as from middle-aged hosts.
We also observed a better recruitment of inflammatory cells to the fracture site in young hosts irrespective of the age of the donor cells. These observations could have resulted from differential engraftment of the middle-aged cells into the juveniles, but other possibilities also exist. The young host may have stimulated a stronger chemotactic response after injury, or, due to increased vascularization in the young animals 7, more inflammatory cells may have been recruited to the fracture site. Distinguishing among these possibilities will allow us to better define the interactions between the age of the host and donor tissues in the future.
Studies have shown that inflammatory factors including IL-6 and TNF-alpha can be utilized to improve bone fracture healing 2931. However, the effects of these molecules are dose and time-dependent and inflammatory molecules may produce anabolic or catabolic effects on the skeleton. For instance, IL-6 may stimulate or inhibit proliferation of osteoblasts 32 and may produce catabolic or anabolic effects on bone33 depending on concentration. Studies illustrate that excessive inflammation hinders repair. Recent work indicates that over-expression of IL-5 significantly delays healing of cutaneous wounds34, while suppression of inflammation by estrogens in cutaneous wounds enhances healing35. The higher levels of IL-6 in older trauma patients correlates with higher morbidity1718. Our results suggest that rejuvenating the inflammatory system in aged animals may optimize the inflammatory response to fracture, and thereby accelerate healing. However, this must be viewed with some caution, because we did not measure IL-6 at the fracture site directly. Rather our measurements are of circulating IL-6 levels.
Callus remodelling was accelerated in Ju:MA chimeras
In addition to the formation of a larger callus during early stages of healing, more rapid remodeling of the callus was observed in middle-aged animals with juvenile bone marrow. The number of osteoclasts in the fracture callus was not significantly different, suggesting that the accelerated remodeling was due to improved functional status of the osteoclasts derived from juvenile bone marrow. Alternatively, earlier stabilization of the fracture that was promoted by rapid formation of callus could have indirectly accelerated the remodeling phase.
In summary, our results indicate that inflammatory cells play important roles in regulating differentiation and/or proliferation of mesenchymal stem cells during fracture healing. However, this interaction is dependent on the age of mesenchymal stem cells and hematopoietic stem cells. Autonomous changes in mesenchymal stem cells coupled with age-related changes in the environment lead to problematic bone repair in middle-aged animals. While studies on aging of mesenchymal stem cells may reveal important mechanisms underlying the delayed fracture healing in aged animals, we conclude that fracture healing in aged animals can be enhanced by altering the environment including the inflammatory system in which mesenchymal stem cells function. Identifying the molecular differences among inflammatory cells derived from different ages of hematopoietic stem cells will help us understand the mechanisms of bone repair and may provide new ways to promote fracture healing.
Juvenile versus aged inflammatory response
Many studies have illustrated that the inflammatory response in juvenile animals is restrained compared to older animals25. Here we showed that middle-aged mice had a pro-inflammatory systemic environment in response to fracture compared to juvenile mice. We also showed that chimeras made with juvenile inflammatory cells had a reduced systemic inflammatory response. However, the reason for reduced inflammation is not known. One possibility is that the young inflammatory cells simply produced a more controlled inflammatory response. Reducing chronic inflammation prior to injury has been shown to enhance healing in other models26. In our work, the young donor cells may have reduced the chronic inflammation that is present in aging animals27.
When we created chimeras with middle-aged bone marrow in juvenile hosts, we did not observe delays in fracture healing. This could have resulted from not achieving a complete replacement of the young hematopoietic stem cells with the aged donor cells. Thus, the healing outcome may simply reflect the presence of juvenile inflammatory cells. However, we observed a similar nadir and rebound of circulating blood cells independent of host age, indicating that the host cells were similarly ablated by irradiation.
Juvenile mice responded differently to injury. After transplantation of middle-aged inflammatory cells into juvenile hosts we observed low levels of IL-6 and TNF-α compared to middle aged hosts. A recent report suggests that the major source of IL-6 in aged animals may be adipose tissue 28. In this study, the authors found that fat from aged animals produced higher levels of IL-6 than young animals after stimulation with inflammatory agents. Thus, juvenile mice may not be prone to producing sustained systemic levels of IL-6 in response to inflammation, and the presence of the middle-aged inflammatory cells in our experiments may not have been able to evoke the same response from juvenile hosts as from middle-aged hosts.
We also observed a better recruitment of inflammatory cells to the fracture site in young hosts irrespective of the age of the donor cells. These observations could have resulted from differential engraftment of the middle-aged cells into the juveniles, but other possibilities also exist. The young host may have stimulated a stronger chemotactic response after injury, or, due to increased vascularization in the young animals 7, more inflammatory cells may have been recruited to the fracture site. Distinguishing among these possibilities will allow us to better define the interactions between the age of the host and donor tissues in the future.
Studies have shown that inflammatory factors including IL-6 and TNF-alpha can be utilized to improve bone fracture healing 2931. However, the effects of these molecules are dose and time-dependent and inflammatory molecules may produce anabolic or catabolic effects on the skeleton. For instance, IL-6 may stimulate or inhibit proliferation of osteoblasts 32 and may produce catabolic or anabolic effects on bone33 depending on concentration. Studies illustrate that excessive inflammation hinders repair. Recent work indicates that over-expression of IL-5 significantly delays healing of cutaneous wounds34, while suppression of inflammation by estrogens in cutaneous wounds enhances healing35. The higher levels of IL-6 in older trauma patients correlates with higher morbidity1718. Our results suggest that rejuvenating the inflammatory system in aged animals may optimize the inflammatory response to fracture, and thereby accelerate healing. However, this must be viewed with some caution, because we did not measure IL-6 at the fracture site directly. Rather our measurements are of circulating IL-6 levels.
Callus remodelling was accelerated in Ju:MA chimeras
In addition to the formation of a larger callus during early stages of healing, more rapid remodeling of the callus was observed in middle-aged animals with juvenile bone marrow. The number of osteoclasts in the fracture callus was not significantly different, suggesting that the accelerated remodeling was due to improved functional status of the osteoclasts derived from juvenile bone marrow. Alternatively, earlier stabilization of the fracture that was promoted by rapid formation of callus could have indirectly accelerated the remodeling phase.
In summary, our results indicate that inflammatory cells play important roles in regulating differentiation and/or proliferation of mesenchymal stem cells during fracture healing. However, this interaction is dependent on the age of mesenchymal stem cells and hematopoietic stem cells. Autonomous changes in mesenchymal stem cells coupled with age-related changes in the environment lead to problematic bone repair in middle-aged animals. While studies on aging of mesenchymal stem cells may reveal important mechanisms underlying the delayed fracture healing in aged animals, we conclude that fracture healing in aged animals can be enhanced by altering the environment including the inflammatory system in which mesenchymal stem cells function. Identifying the molecular differences among inflammatory cells derived from different ages of hematopoietic stem cells will help us understand the mechanisms of bone repair and may provide new ways to promote fracture healing.
Acknowledgments
We are grateful for the support and contribution of the Orthopaedic Trauma Institute at UCSF. This work was funded by the Hellman Family Foundation to R.M., the NIH (R01-AR053645-01) to T.M., and a Trauma Fellowship from Synthes to Z.X.
Abstract
Age significantly reduces the regenerative capacity of the skeleton, but the underlying causes are unknown. Here, we tested whether the functional status of inflammatory cells contributes to delayed healing in aged animals. We created chimeric mice by bone marrow transplantation after lethal irradiation. In this model chondrocytes and osteoblasts in the regenerate are derived exclusively from host cells while inflammatory cells are derived from the donor. Using this model, the inflammatory system of middle-aged mice (12-month-old) was replaced by transplanted bone marrow from juvenile mice (4-week-old), or age-matched controls. We found that the middle-aged mice receiving juvenile bone marrow had larger calluses and more bone formation during early stages and faster callus remodeling at late stages of fracture healing, indicating that inflammatory cells derived from the juvenile bone marrow accelerated bone repair in the middle-aged animals. In contrast, transplanting bone marrow from middle-age mice to juvenile mice did not alter the process of fracture healing in juvenile mice. Thus, the roles of inflammatory cells in fracture healing may be age-related, suggesting the possibility of enhancing fracture healing in aged animals by manipulating the inflammatory system.
References
- 1. Einhorn TAEnhancement of fracture-healing. J Bone Joint Surg Am. 1995;77:940–56.[PubMed][Google Scholar]
- 2. Meyer RA, Meyer MH, Phieffer LS, Banks DMDelayed union of femoral fractures in older rats:decreased gene expression. BMC Musculoskelet Disord. 2001;2:2.[Google Scholar]
- 3. Meyer J, Ralph A, Tsahakis PJ, Martin DF, et al Age and ovariectomy impair both the normalization of mechanical properties and the accretion of mineral by the fracture callus in rats. Journal of Orthopaedic Research. 2001;19:428–435.[PubMed][Google Scholar]
- 4. Meyer RA, Jr, Meyer MH, Tenholder M, et al Gene expression in older rats with delayed union of femoral fractures. J Bone Joint Surg Am. 2003;85-A:1243–54.[PubMed][Google Scholar]
- 5. Desai BJ, Meyer MH, Porter S, et al The effect of age on gene expression in adult and juvenile rats following femoral fracture. J Orthop Trauma. 2003;17:689–98.[PubMed][Google Scholar]
- 6. Lu C, Miclau T, Hu D, et al Cellular basis for age-related changes in fracture repair. J Orthop Res. 2005;23:1300–7.[Google Scholar]
- 7. Lu C, Hansen E, Sapozhnikova A, et al Effect of age on vascularization during fracture repair. J Orthop Res 2008[Google Scholar]
- 8. Meyer RA, Jr, Desai BR, Heiner DE, et al Young, adult, and old rats have similar changes in mRNA expression of many skeletal genes after fracture despite delayed healing with age. J Orthop Res. 2006;24:1933–44.[PubMed][Google Scholar]
- 9. Naik AA, Xie C, Zuscik MJ, et al Reduced COX-2 expression in aged mice is associated with impaired fracture healing. J Bone Miner Res. 2009;24:251–64.[Google Scholar]
- 10. Mountziaris PM, Mikos AGModulation of the Inflammatory Response for Enhanced Bone Tissue Regeneration. Tissue Eng Part B Rev 2008[Google Scholar]
- 11. Chung HY, Cesari M, Anton S, et al Molecular inflammation: underpinnings of aging and age-related diseases. Ageing Res Rev. 2009;8:18–30.[Google Scholar]
- 12. Sarkar D, Fisher PBMolecular mechanisms of aging-associated inflammation. Cancer Lett. 2006;236:13–23.[PubMed][Google Scholar]
- 13. Podichetty VKThe aging spine: the role of inflammatory mediators in intervertebral disc degeneration. Cell Mol Biol (Noisy-le-grand) 2007;53:4–18.[PubMed][Google Scholar]
- 14. Lim WH, Toothman J, Miller JH, et al IL-1beta inhibits TGFbeta in the temporomandibular joint. J Dent Res. 2009;88:557–62.[Google Scholar]
- 15. Benatti BB, Silverio KG, Casati MZ, et al Inflammatory and bone-related genes are modulated by aging in human periodontal ligament cells. Cytokine. 2009;46:176–81.[PubMed][Google Scholar]
- 16. Mahara K, Anzai T, Yoshikawa T, et al Aging adversely affects postinfarction inflammatory response and early left ventricular remodeling after reperfused acute anterior myocardial infarction. Cardiology. 2006;105:67–74.[PubMed][Google Scholar]
- 17. Miller RR, Cappola AR, Shardell MD, et al Persistent changes in interleukin-6 and lower extremity function following hip fracture. J Gerontol A Biol Sci Med Sci. 2006;61:1053–8.[PubMed][Google Scholar]
- 18. Schneider CP, Schwacha MG, Chaudry IHImpact of sex and age on bone marrow immune responses in a murine model of trauma-hemorrhage. J Appl Physiol. 2007;102:113–21.[PubMed][Google Scholar]
- 19. Colnot C, Huang S, Helms JAnalyzing the cellular contribution of bone marrow to fracture healing using bone marrow transplantation in mice. Biochem Biophys Res Commun. 2006;350:557–61.[PubMed][Google Scholar]
- 20. Thompson Z, Miclau T, Hu D, Helms JAA model for intramembranous ossification during fracture healing. J Orthop Res. 2002;20:1091–8.[PubMed][Google Scholar]
- 21. Gundersen HJ, Jensen EBThe efficiency of systematic sampling in stereology and its prediction. J Microsc. 1987;147:229–63.[PubMed][Google Scholar]
- 22. Colnot C, Lu C, Hu D, Helms JADistinguishing the contributions of the perichondrium, cartilage, and vascular endothelium to skeletal development. Dev Biol. 2004;269:55–69.[PubMed][Google Scholar]
- 23. Lloberas J, Celada AEffect of aging on macrophage function. Exp Gerontol. 2002;37:1325–31.[PubMed][Google Scholar]
- 24. Danon D, Kowatch MA, Roth GSPromotion of wound repair in old mice by local injection of macrophages. Proc Natl Acad Sci U S A. 1989;86:2018–20.[Google Scholar]
- 25. Kovacs EJAging, traumatic injury, and estrogen treatment. Exp Gerontol. 2005;40:549–55.[PubMed][Google Scholar]
- 26. Gomez CR, Plackett TP, Kovacs EJAging and estrogen: modulation of inflammatory responses after injury. Exp Gerontol. 2007;42:451–6.[Google Scholar]
- 27. Khansari N, Shakiba Y, Mahmoudi MChronic inflammation and oxidative stress as a major cause of age-related diseases and cancer. Recent Pat Inflamm Allergy Drug Discov. 2009;3:73–80.[PubMed][Google Scholar]
- 28. Starr ME, Evers BM, Saito HAge-associated increase in cytokine production during systemic inflammation: adipose tissue as a major source of IL-6. J Gerontol A Biol Sci Med Sci. 2009;64:723–30.[Google Scholar]
- 29. Rozen N, Lewinson D, Bick T, et al Fracture repair: modulation of fracture-callus and mechanical properties by sequential application of IL-6 following PTH 1-34 or PTH 28-48. Bone. 2007;41:437–45.[PubMed][Google Scholar]
- 30. Gerstenfeld LC, Cho TJ, Kon T, et al Impaired fracture healing in the absence of TNF-alpha signaling: the role of TNF-alpha in endochondral cartilage resorption. J Bone Miner Res. 2003;18:1584–92.[PubMed][Google Scholar]
- 31. Xie C, Ming X, Wang Q, et al COX-2 from the injury milieu is critical for the initiation of periosteal progenitor cell mediated bone healing. Bone. In Press, Corrected Proof.
- 32. Frost A, Jonsson KB, Nilsson O, Ljunggren OInflammatory cytokines regulate proliferation of cultured human osteoblasts. Acta Orthop Scand. 1997;68:91–6.[PubMed][Google Scholar]
- 33. Blanchard F, Duplomb L, Baud'huin M, Brounais BThe dual role of IL-6-type cytokines on bone remodeling and bone tumors. Cytokine Growth Factor Rev. 2009;20:19–28.[PubMed][Google Scholar]
- 34. Leitch VD, Strudwick XL, Matthaei KI, et al IL-5-overexpressing mice exhibit eosinophilia and altered wound healing through mechanisms involving prolonged inflammation. Immunol Cell Biol. 2009;87:131–40.[PubMed][Google Scholar]
- 35. Ashcroft GS, Mills SJ, Lei K, et al Estrogen modulates cutaneous wound healing by downregulating macrophage migration inhibitory factor. J Clin Invest. 2003;111:1309–18.[Google Scholar]