Abstract
Background/Aim: Insulin-like growth factor-1 (IGF-1) and macrophage colony-stimulating factor (MCSF) are critical to skeletal homeostasis. We investigated the effects of combined IGF-1 plus MCSF on mice. Materials and Methods: C57BL/6J mice, aged 7 weeks, were assigned to baseline, vehicle, IGF-1, MCSF, or combined IGF-1 plus MCSF (1 mg/kg/day each, n=12-13/group, 28-day duration). Results: IGF-1 or MCSF had no effect on bone formation rate; however, IGF-1 plus MCSF produced a 169% increase in periosteal bone formation rate. Combined therapy increased femoral mechanical properties (+25% elastic force), while IGF-1, and MCSF alone did not. Combined therapy affected trabecular bone volume fraction (+40%), number (+13%), and spacing (−13%). MCSF produced similar trabecular changes, while IGF-1 had no effect. Combined therapy and MCSF alone increased bone mineral content. Conclusion: We have demonstrated the superior effects of combined IGF-1 and MCSF. Together, these agents may promote bone modeling to a greater extent than either therapy alone.
Insulin-like growth factor-1 (IGF) is an abundant intercellular cytokine that is mainly secreted by the liver in response to growth hormone (1), however this factor is also synthesized by osteoblasts, fibroblasts, and other bone cell types (2). IGF-1 is known as a critical factor in the regulation of bone formation, having been shown to stimulate proliferation and survival of osteoblast-like cells (3, 4) and increase collagen formation (5).
Previous studies have documented that IGF-1 generally acts in an anabolic manner on bone. Tobias and colleagues revealed that while rat long bone length and circumference increases with IGF-1 administration, trabecular bone formation may be reduced (6). In a study by another group, ovariectomized rats treated with IGF-1 showed an increase in periosteal bone formation that was greater than the observed increase in endocortical bone formation (7). Previous studies in our laboratory have found that IGF-1 administration to rats yields histomorphometric measurements that are not consistent throughout mid-diaphyseal sections of the femur, tibia, or humerus (8). More recent studies have also highlighted the contribution of IGF-1 to osteoclastogenesis (9), where IGF-1 was shown to promote osteoclast differentiation and play a role in the coupling of osteoclast and osteoblast activity. Further, IGF-1 has been shown to regulate bone shape, size, and composition and is thus implicated in mediating bone's response to mechanical loading (10, 11). Furthermore, IGF-1 has been found to regulate expression of another important mediator of skeletal homeostasis: macrophage-colony stimulating factor (MCSF).
MCSF is a cytokine that was originally defined by its ability to stimulate the differentiation of precursor cells into mature macrophage colonies (12). MCSF is now characterized as a haematopoietic growth factor with an important role in the development of cells from the mononuclear phagocyte lineage (13, 14). MCSF is primarily produced by connective tissue cells, such as stromal cells and osteoblasts (15). This cytokine is a key regulator of osteoclastogenesis both in vitro and in vivo, being necessary for the proliferation and differentiation of osteoclast progenitors (16, 17). MCSF stimulates osteoclastogenesis by promoting entry of progenitor cells into the osteoclast lineage. It has been observed that osteoclasts in culture cannot proliferate in the absence of MCSF producing osteoblasts (18).
Studies subjecting mice to daily injections of MCSF, and investigations of transgenic mice that express MCSF, have revealed increased cortical bone formation and beneficial increases in the material and biomechanical properties of cortical bone (19). Bone-forming osteoblasts play an integral role in the regulation of osteoclast development through both cell–cell interactions and cytokine release (20). MCSF improves the function of both osteoclasts and osteoblasts and is easily detected in models of fracture healing (21-23). Increases in bone formation following MCSF administration are likely induced by the coupling phenomenon that exists between osteoclasts and osteoblasts (24). This coupling mechanism is thought to be mediated either through the release of anabolic factors from the bone matrix (25, 26) or even the osteoclast itself, independently of resorptive activity (27).
In the present study, we investigated the effects of IGF-1 and MCSF, both alone and in combination, on the microarchitectural, mechanical, and compositional properties of mouse bones. Because these cytokines exert their skeletal effects through differing mechanisms, IGF-1 being more targeted to osteoblast development and MCSF eliciting its effect primarily through coupled activation of osteoclasts and osteoblasts, it is hypothesized that, in tandem, these factors may be be able to increase the efficacy of bone modeling to a greater extent than either could individually.
Materials and Methods
Animals. Sixty-one male C57BL/6J mice (Jackson Labs, Bar Harbor, ME, USA) aged 7 weeks were assigned to one of five groups: baseline (n=12), inert vehicle control (n=12), IGF-1 (1 mg/kg/day; n=12), MCSF (1 mg/kg/day; n=12), or combined IGF-1 and MCSF (1 mg/kg/day each; n=13). Daily injections of vehicle or protein were made into the intraperitoneal cavity of the mice for 28 days. The vehicle was composed of phosphate-buffered saline (PBS; ~0.2 ml/injection). Both IGF-1 and MCSF were dissolved in PBS vehicle. In order to allow for later dynamic histomorphometric analysis, tetracycline bone label (20 mg/kg) was administered to these animals 20 and 2 days prior to sacrifice, while a calcein bone label was administered 14 days prior to sacrifice (28).
Study endpoint. The baseline group of mice was sacrificed on day 0, while the four treatment groups were sacrificed on day 28. In each case, the mice were weighed, anaesthetized with sodium pentobarbital (90 mg/kg, i.p.), and then sacrificed by cervical dislocation. The whole spleen and gastrocnemius muscles were isolated and weighed. Both hindlimbs and forelimbs were removed. The tibiae, femora, and humeri were separated and cleaned of all non-osseous tissue. Right femora and humeri were subjected to mechanical testing and compositional analysis, while the left femora were utilized for quantitative histomorphometry and subsequent microhardness and nanoindentation assays. The right tibiae were subjected to microarchitectural analysis via microcomputed tomography. All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of Colorado (Boulder, CO, USA).
Microcomputed tomography. The right tibiae were fixed in 10% neutral buffered formalin for two days, rinsed with distilled water, and stored in 70% ethanol. Trabecular parameters were obtained from microcomputed tomography (μCT20; Scanco Medical AG, Bassersdorf, Switzerland) scans of 0.9 mm longitudinal trabecular bone sections at the proximal end of the tibia, immediately distal to the epiphyseal plate. A voxel size of 9 μm in all three directions (~15 μm resolution) was utilized. Measured parameters included trabecular bone volume (BV; mm3), total volume (TV; mm2), trabecular number (Tb.N), and trabecular separation (Tb.Sp; mm). BV was normalized to TV in order to obtain the percentage trabecular bone volume fraction (BV/TV×100; %). Tb.N was calculated by taking the inverse of the mean distance between the middle axes of the trabeculae. Tb.Sp was calculated by measuring three-dimensional distances in the trabecular bone network and determining the mean over all voxels.
Mechanical properties. The length of the right femora and humeri were recorded using a Vernier caliper (Fisher Scientific, Hampton, NH, USA) with a 10 μm resolution. The bones were then air dried. Prior to mechanical testing, femora and humeri were rehydrated in PBS for 90 minutes (29). Three-point bending tests were performed using an Instron 1331 and Merlin, Series IX software (Instron Corporation, Norwood, MA, USA). Bones were tested to failure with an 8 mm span length and deflection rate of 5 mm/min. Force (Pe; N) and deflection (δe; mm) were measured at the elastic limit. Maximum and structural failure (fracture) loads were also measured. Stiffness (S; N/mm) was calculated from Pe/δe.
Bone mineral content. Mineral content analysis was performed on femora and humeri fractured during mechanical testing. Prior to analysis, the enlarged ends of the femur were separated where the diaphysis meets the metaphysis. These end portions are hereafter referred to as the epiphyses. Mineral content data were obtained separately from epiphyses and diaphyses. Dry mass (Dry-M; mg) was measured after heating the bones at 105°C for 24 hours to remove water from the bone matrix. Mineral mass (Min-M; mg) was measured after the bones had been heated for an additional 24 hours at 800°C. All masses were obtained using a properly calibrated analytical scale (Denver Instruments, Denver, CO, USA). Percent mineral content (%Min) was calculated as Min-M/Dry-M×100.
Quantitative histomorphometry. Left femora were air dried at 25°C for four days, then embedded in non-infiltrating Epo-Kwick epoxy (Buehler, Lake Bluff, IL, USA). The formed disks were sectioned with a low-speed saw (Buehler; 12.7 cm ×0.5 mm, diamond blade) at the mid-diaphysis of the femur. The sections were wheel-polished to a flat, smooth surface using 350-, 400-, and 600-grit carbide paper followed by polishing with a cloth impregnated with 6 μm diamond paste. Tetracycline and calcein bone labels were visualized, indicating sites of new bone formation between the multiple time points. Digital images of each cross section were captured using an Olympus AHBT-3 microscope (Olympus, Tokyo, Japan) with a CMOS-Pro digital camera (1000×800 pixels) (Sound Vision, Framingham, MA, USA) at ×16.7 initial magnification (violet light at ~400 nm). These digital images were used to quantify femur mid-diaphysis cross-sectional morphology and to perform quantitative histomorphometric analysis (SigmaScan Pro; SPSS Science; Chicago, IL, USA).
Total body, gastrocnemius muscle, and spleen mass of male C57BL/6J mice treated for 28 days with inert vehicle, insulin-like growth factor 1 (IGF-1; 1 mg/kg/day), macrophage colony-stimulating factor (MCSF; 1 mg/kg/day), or combined IGF-1 plus MCSF (1 mg/kg/day each).
Measurements of bone morphology (30) included total bone area enclosed by periosteal perimeter (T.Ar; mm2) and medullary area (Me.Ar; mm2). Cortical area (Ct.Ar; mm2) was calculated as T.Ar – Me.Ar. Cortical thickness (Ct.Th; mm) was also determined. The area between the bone labels was recorded as the bone formation area (BF.Ar; mm2). The length of the labelled perimeter was defined as active mineralizing perimeter (AMPm; mm). Bone formation rate (BFR=BF.Ar/no. days; μm2/day) and mineral apposition rate (MAR=BFR/AMPm; μm/day) were calculated separately for the periosteal (Ps.BFR, Ps.MAR) and endocortical (Ec.BFR, Ec.MAR) surfaces. All bone formation data were collected for the two time-points 20 to 14 days prior to sacrifice and 14 to 2 days prior to sacrifice. Data are presented herein are for the total BFA from days 20 to 2 prior to sacrifice and the mean of the two separate time-points for AMP and MAR.
Two-dimensional, cross-sectional moments of intertia (IX and IY; mm4) were also calculated. These values were calculated using the assumption that the periosteal and endocortical surfaces were in the approximate shape of concentric ellipses (31).
Microhardness and nanoindentation. Femora prepared for histomorphometric analysis were utilized for these assays. Three microhardness indents were placed in extant bone within each sectioned and polished femur cross-section using a pyramid-shaped Vicker's diamond indenter (Fischer Scope-H1100 and WINHCU 1.3 software; Fischer Technology, Windsor, CT, USA) with a 50 g load for 10 s. In order to minimize edge effects, one indent length was maintained between the indent site, sample edges, and visible lacunae (32). Pyramid diagonal lengths were measured (×250), and the Vicker's hardness number (VHN; kgF/mm2) was calculated using the formula: VHN=(2Psin(×/2))/d2, where P=applied load, x=pyramid angle (136°), and d=average measure of the two diagonal lengths.
The smaller size of a nanoindenter tip permits measurement of bone formed during the treatment period. Nanoindentations (MTS Nanoindenter XP; Oak Ridge, TN, USA) were made using a Berkovich tip to a maximum contact depth of 1500 nm. Elastic modulus values were collected from a fit of the first 80% of the unloading curve using the Oliver and Pharr method with an assumed Poisson's ratio for bone of 0.3 (33). Indents were laid out in rectangular grids (minimum of 5×8 indents; 20 μm spacing in x- and y-directions) to extend across both extant and newly formed bone, as demarcated by fluorescent labels, from the endocortical to the periosteal surface. Indents were classified as falling on extant or newly formed bone. Those indents falling on the labelled region or within two indent widths of cracks, edges, or pores were excluded from this analysis. It should be noted that microhardness indicates resistance to plastic deformation while nanoindentation modulus provides a measurement of elastic properties only.
Statistics. Statistical comparisons were performed between endpoint groups (i.e. vehicle, IGF-1, MCSF, and combined IGF-1 plus MCSF groups) using a one-way analysis of variance (ANOVA) with a Tukey's post-hoc test. A 95% level of significance (type I error) was utilized for all tests. A lettering system was employed to indicate significant differences in all figures and tables. Different letters indicate a significant difference between groups for a given parameter (<0.05); a common letter, or no letter, indicates no significant difference between groups for that parameter (p>0.05). Throughout the text, data are presented as the mean±standard error of the mean (SEM).
Results
Total body, gastrocnemius, and spleen mass. The mass of the whole animal, gastrocnemius muscle, and whole spleen was measured for all treatment groups at the study endpoint (Table I). IGF-1 alone produced no significant change in the final mass of the whole animal, gastrocnemius muscle, or spleen when compared to vehicle control. Mice treated with MCSF alone had a significantly greater final body mass (+9%) and whole spleen mass (+55%) versus control. Gastrocnemius muscle mass was not significantly affected by MCSF alone. Compared to control, combined IGF-1 plus MCSF therapy increased final body mass (+12%) and whole spleen mass (+68%). However, these increases were not different from those obtained using MCSF alone. Combined therapy had no significant effect on the final mass of the gastrocnemius muscle.
Microcomputed tomography. Microcomputed tomography was used to assess the trabecular microarchitectural properties of the right tibiae (Figure 1). IGF-1 alone produced no significant effect on trabecular BV/TV, Tb.N, or Tb.Sp versus vehicle control. Mice treated with MCSF alone had significantly higher BV/TV (+53%), higher Tb.N (+14%), and lowerTb.Sp (-15%) compared to control. There was no difference between the response to IGF-1 alone and MCSF alone for any trabecular parameter. Combined IGF-1 plus MCSF therapy showed a trend of increased trabecular BV/TV (+40%), significantly increased Tb.N (+13%), and significantly decreased Tb.Sp (-13%) versus control. The response to combined therapy was not significantly different from that to IGF-1 or MCSF alone for any of the parameters measured.
Trabecular microarchitecture parameters determined through microcomputed tomography of tibiae collected from male C57BL/6J mice treated for 28 days with inert vehicle, insulin-like growth factor 1 (IGF-1; 1 mg/kg/day), macrophage-colony stimulating factor (MCSF; 1 mg/kg/day), or combined IGF-1 plus MCSF (1 mg/kg/day each). Measured parameters included A: trabecular bone volume fraction (BV/TV), B: trabecular number, and C: trabecular separation. Different letters indicate a significant difference between treatment groups for a given endpoint parameter (<0.05 via one-way ANOVA). A common letter, or no letter, indicates no significant difference. Error bars represent SEM.
Mechanical properties. Three-point bending was used to determine the mechanical properties of isolated femora (Table II) and humeri (Table III) from all groups. Compared to vehicle control, IGF-1 and MCSF, whether alone or in combination, had no effect on the final length of femur or humerus in the population of young, male mice used in this study. IGF-1, and MCSF alone had no significant effect on femoral or humeral stiffness or elastic, maximum, or fracture force versus control. Combined IGF-1 plus MCSF therapy resulted in apparent trends for increased mechanical properties of the femur and humerus, often greater than those found using IGF-1 or MCSF alone. Combined therapy did produce a significant increase in femoral elastic force (+25%).
Mineral content analysis. Compositional analysis was carried out in order to assess the effects of protein treatment on the percent mineral content of the femur (Table II) and humerus (Table III). IGF-1 alone elicited no significant change in the mineral content of any femur component (i.e. epiphysis, diaphysis, or whole femur) versus vehicle control. The percentage mineral content of the whole humerus was significantly increased with IGF-1 alone (+2%). Mice treated with MCSF alone had significantly greater mineral content in the whole femur (+2%), as well as of the femoral epiphysis (+4%) and diaphysis (+2%), versus control. MCSF alone increased total humeral mineral content (+1%) versus control. Combined IGF-1 plus MCSF therapy significantly increased the percentage mineralization of the whole femur (+2%), as well as of the femoral epiphysis (+4%) and diaphysis (+2%), versus vehicle control. The response to the combined therapy in the femur was significantly greater than the response to IGF-1 alone for the whole femur and epiphysis. In the humerus, the total mineral content increased (+5%) versus control.
Quantitative histomorphometry. IGF-1, and MCSF administered alone resulted in no significant changes in morphology of the femur mid-diaphysis (Table IV). However, there were trends indicating that mice treated with combined IGF-1 plus MCSF therapy had increased cortical area, cortical thickness, and calculated cross-sectional moments of inertia when compared to vehicle control.
Length, composition, and mechanical properties of femora collected from male C57BL/6J mice treated for 28 days with inert vehicle, insulin-like growth factor 1 (IGF-1; 1 mg/kg/day), macrophage colony-stimulating factor (MCSF; 1 mg/kg/day), or combined IGF-1 plus MCSF (1 mg/kg/day each).
Length, composition, and mechanical properties of humeri collected from male C57BL/6J mice treated for 28 days with inert vehicle, insulin-like growth factor 1 (IGF-1; 1 mg/kg/day), macrophage colony-stimulating factor (MCSF; 1 mg/kg/day), or combined IGF-1 plus MCSF (1 mg/kg/day each).
Dynamic quantitative histomorphometry parameters of the femur were obtained following administration of bone labels 20, 14, and 2 days prior to animal sacrifice (Figure 2). Significant effects of IGF-1, and MCSF, alone and in combination, were limited to the periosteal bone surface (i.e. BFR). No significant differences were seen at the endocortical surface for any parameter. At the periosteal surface, IGF-1 alone tended to have a greater effect than MCSF alone. Compared to vehicle control, IGF-1 significantly increased Ps.MAR (+24%). However, combined IGF-1 plus MCSF was able to produce significant changes at periosteal bone sites, even in cases where neither of the agents acting alone did. Combined IGF-1 plus MCSF significantly increased both Ps.AMPm (+77%) and Ps.MAR (+32%) versus vehicle control.
The effects of combined IGF-1 plus MCSF therapy were best illustrated by the observed changes in BFR at the periosteal surface (Figure 2A). Compared to the vehicle control, IGF-1, and MCSF alone did not significantly increase BFR at this site; however, combination therapy produced a highly significant 169% increase in BFR (<0.001).
Microhardness and nanoindentation. The effects of the individual and combined IGF-1 plus MCSF did not change the material behavior of extant or newly formed femoral cortical bone as measured via microhardness and nanoindentation assays (Table V). Measurements of Vicker's hardness for all groups treated with individual or combined therapies were maintained at values similar to bone in the vehicle control group (1.04±0.02 kgF/mm2). Similarly, the elastic modulus, as determined by nanoindentation in newly-formed femoral bone, did not vary significantly from values measured in extant bone (23.4±0.7 GPa) from the vehicle control group. Newly-formed bone also showed no significant differences in the measured indentation modulus from values obtained from femurs in the vehicle control group (23.7±0.4 GPa). Elastic modulus was not altered between measurements of extant and newly-formed bone.
Dynamic histomorphometry parameters obtained from femur mid-diaphysis cross-sections collected from male C57BL/6J mice treated for 28 days with inert vehicle, insulin-like growth factor 1 (IGF-1; 1 mg/kg/day), macrophage colony-stimulating factor (MCSF; 1 mg/kg/day), or combined IGF-1 plus MCSF (1 mg/kg/day each). Bone labels were administered 20, 14, and 2 days prior to sacrifice to label areas of active bone formation. Measured parameters included A: total bone formation rate (BFR; μm2/day), B: mean active mineralizing perimeter (AMPm; mm), and C: mean mineral apposition rate (MAR; μm/day) at both the periosteal and endocortical surfaces. Different letters indicate a significant difference between treatment groups for a given endpoint parameter (<0.05 via one-way ANOVA). A common letter, or no letter, indicates no significant difference. Error bars represent SEM.
Discussion
Although IGF-1 and MCSF administered individually have anabolic-like effects on the skeletal system of mice, these agents were shown here to have greater effects on bone when co-administered. Combined IGF-1 plus MCSF therapy was found to increase periosteal bone formation, leading to an increase in mechanical strength parameters. Similar effects on trabecular microarchitecture were also observed with combined IGF-1 plus MCSF. Indeed, increases in trabecular bone volume fraction and trabecular number were much greater than for IGF-1 alone. While this study cannot confirm the exact mechanisms by which this new bone was formed, it does highlight the interacting effects of combined IGF-1 plus MCSF on murine skeletal physiology.
A combination of IGF-1 and MCSF, two endogenous cytokines required for the maintenance of skeletal homeostasis, was investigated because of the differences in the mechanisms by which they exert their pharmacological effects. IGF-1 was chosen to more directly target bone-building osteoblasts (i.e. IGF-1 actively promotes osteoblast activation and differentiation) (3, 4). Conversely, MCSF produces its anabolic effects indirectly through coupled activation of osteoblasts via osteoclasts (19, 34). This type of mechanism is consistent with our previous studies of high-dose MCSF in mice (35). In tandem, these two factors were hypothesized to increase the efficacy of bone modeling, and possibly increase both the amount and quality of newly formed bone – the present study indicates this may be the case in young mice. It is likely, however, that IGF-1 and MCSF direct, rather than induce, bone formation through promotion of the existing modeling processes.
We have demonstrated the ability of MCSF to modulate bone modeling in a potentially cooperative manner with IGF-1. While IGF-1 alone tended to have a greater effect than MCSF alone on measured dynamic histomorphometric parameters at the periosteal surface (i.e. Ps.BFR, Ps.AMPm, and Ps.MAR), combined therapy had an even greater impact, and significantly increased all measured parameters at this site. The percentage increase in periosteal active mineralizing perimeter and bone formation rate with combined IGF-1 plus MCSF was approximately two times greater than that produced by individual therapies, highlighting the combined effects of these agents. The change in mineral apposition rate from vehicle control was also greater in the combined therapy group. Because the effects of combined IGF-1 plus MCSF are significant for many parameters, the mechanisms causing the increased modeling from each cytokine are likely different. This is consistent with the aforementioned in vitro and in vivo studies that have investigated their mechanisms of action.
Bone morphological parameters obtained from femur mid-diaphysis cross-sections collected from male C57BL/6J mice treated for 28 days with inert vehicle, insulin-like growth factor 1 (IGF-1; 1 mg/kg/day), macrophage colony-stimulating factor (MCSF; 1 mg/kg/day), or combined IGF-1 plus MCSF (1 mg/kg/day each).
The quality of the newly formed bone was consistent with extant bone despite the increased rate of formation. Microhardness measurements, extending up to approximately 80 μm in diameter, likely sampled regions of both extant and newly formed bone. Nanoindentation measurements sampled effective contact areas of only several micrometers in diameter and were able to distinguish between extant and newly formed bone. The lack of change from the vehicle group as well as comparisons between extant and newly formed bone indicates that bone quality was maintained at control levels in all groups. Furthermore, the significantly increased rate of bone formation in treatment groups, and especially with the combined IGF-1 plus MCSF therapy, did not ostensibly affect bone mineralization and local measurements of bone quality.
Increases in bone were not limited to cortical bone. Increases in trabecular bone volume fraction, trabecular number, and separation, significant for the latter two, were also observed. Observed gains in these parameters were dependent on the presence of MCSF, suggesting that trabecular bone may be less sensitive to the anabolic effects of IGF-1. Although mechanical strength in three-point bending is primarily dependent on cortical bone growth, trabecular bone also makes a meaningful contribution. The highly significant 40% gains in BV/TV likely contributed to the increase in bone mechanical properties that were observed.
Increased bone formation occurred primarily at the periosteal surface of the femur. For IGF-1, this is consistent with previous findings in rats (8). Adding material onto the periosteal rather than the endocortical surface is the most effective means of increasing the cross-sectional moment of inertia and mechanical strength of a cylindrical-like shape (32). Increases in bone formation and cortical area with combined therapy translated into significant gains in the mechanical properties of the femur and humerus, particularly elastic force parameters. These increases occurred even in cases where individual cytokine treatment was not able to produce a significant effect.
Microhardness (Vicker's hardness number; VHN) and elastic modulus from nanoindentation testing obtained from femur mid-diaphysis cross-sections collected from male C57BL/6J mice treated for 28 days with inert vehicle, insulin-like growth factor 1 (IGF-1; 1 mg/kg/day), macrophage colony-stimulating factor (MCSF; 1 mg/kg/day), or combined IGF-1 plus MCSF (1 mg/kg/day each).
Bone mass and percentage mineralization were also significantly increased by treatment with MCSF (individually, and combined with IGF-1). The increase in percentage mineralization of the primarily trabecular bone of the femoral epiphysis may be indicative of improved mineralization of newly formed bone, or of an antiresorptive effect, as noted in by Fuller and colleagues (17); however, as noted, there were no observed differences between the extant and newly formed bone for the microhardness or nanoindentation assays.
The contribution of animal mass is an important consideration in the present study. Although the IGF-1 and MCSF-treated mice did experience greater weight gain over the course of the study, their final body mass was only 7-12% greater than that of vehicle controls, with little difference between protein treatment groups. Although we certainly cannot discount the contribution of this increased mass to the enhanced trabecular bone fraction (+53%) and bone formation (+169%), it is unlikely that such large skeletal effects can be attributed to such modest changes in body mass.
Similarly, the relatively young animals employed in the present study (7-week-old males) do present a limitation to the interpretation and application of our results. Growing animals of this age would be undergoing a significant amount of de novo bone formation, rather than simple remodeling of pre-existing bone. It is possible that the increase in trabecular bone fraction observed herein was due to an inhibition of osteoclast-mediated bone resorption, as observed in previous in vitro, high-dose MCSF studies (36). A similar effect is also observed following administration of bisphosphonate antiresorptive drugs (37). The lack of broad changes in bone mineral content and the large increases in femoral BFR would seem to contradict this conclusion, however, and suggest a primarily anabolic effect. Although it was not possible in the present study due to inadequate preservation of bone samples, a full histological analysis would reveal changes in bone cell number and distribution that could provide insight into these effects.
In summary, we have demonstrated the skeletal effects of IGF-1 and MCSF. Co-administration of IGF-1 and MCSF increased the rate of new bone formation to a greater extent than either agent alone, highlighting their differing mechanisms of action. Similar advantages of co-administration were seen through gains in percentage mineral content, mechanical strength, and bone morphological properties. It is possible that simultaneous stimulation of bone formation, without undue inhibition of normal bone resorption, may prove to be an advantageous pharmacological objective of future research into treatments for pathological bone loss.
Acknowledgements
The Authors would like to acknowledge and thank Ting Wang, Erin Smith, and Dr Pamela Diggle. This work was supported by Novartis, Inc. (formerly Chiron Corporation) and a Co-operative Agreement NCC8-131 from the National Aeronautics and Space Administration (NASA). Additional support was provided by the National Space Biomedical Research Institute (NSBRI) through NASA NCC 9-58.
- Received August 19, 2010.
- Revision received March 2, 2011.
- Accepted March 3, 2011.
- Copyright © 2011 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
