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Published in final edited form as: J Orthop Res. 2015 May 13;33(7):948–956. doi: 10.1002/jor.22816

Fixation stability dictates the differentiation pathway of periosteal progenitor cells in fracture repair

Y Hagiwara *, NA Dyment *, X Jiang *, J Huang *, C Ackert-Bicknell #, DJ Adams ^, DW Rowe *
PMCID: PMC4891973  NIHMSID: NIHMS788637  PMID: 25639792

Abstract

This study compared fracture repair stabilized by intramedullary pin (IMP) or external fixation (EF) in GFP reporter mice. A modified IMP was used as control while EF utilized six needles inserted transversely through the tibia and into a segment of a syringe barrel. X-rays taken at days 0, 14, and 35 showed that IMP resulted in significant three-dimensional deformity with a large callus while EF showed minimal deformity and callus formation. Cryohistological analysis of IMP at day 14 confirmed a large ColX- RFPchry+ callus surrounded by woven bone (Col3.6-GFPcyan) and TRAP+ osteoclasts with mature bone (hOC-GFPtpz) at the base. By day 35, cartilaginous components had been resorbed and an outer cortical shell (OCS) showed evidence of inward modeling. In contrast, the EF at day 14 showed no evidence of cartilage formation. Instead, periosteal-derived osteoblasts (Col3.6-GFPcyan) entered the fracture cleft and formed woven bone that spanned the marrow space. By day 35, mature bone had formed that was contiguous with the opposing cortical bone. Fracture site stability greatly affects the cellular response during repair and must be considered in the preclinical models that test therapies for improving fracture healing.

Keywords: external and internal fracture fixation, closed tibial fracture, GFP reporter mice, cryohistology of non-decalcified bone

INTRODUCTION

Our research group has been examining skeletal repair models that take advantage of GFP reporter mice to enhance the histological interpretation of the cellular repair program. We have previously used the closed long bone fracture model in mice1 because it has been widely used to study the effects of genetic and drug manipulations on fracture healing.2,3 However, in our hands, the tibial fractures treated with IMP often result in a robust, but widely spaced, callus that includes a highly variable large cartilaginous component. The high variability of the callus size is probably due to the inherent rotational instability of the IMP. We considered extending our reporter analysis to fractures stabilized by external fixation (EF) to improve fracture site alignment.4–12 However, there are a number of disadvantages of the EF design including weight, bulk, potential hindrance of limb movement, and radio-opacity, which can interfere with radiographic studies. Internal plate and screw devices have the disadvantages of invasiveness of the surgery, radio-opacity, difficulty of removal, and cost of the fixation devices.

To take advantage of the quantitative potential of GFP reporters as a read out for cellular participation in fracture repair, we wanted to implement a low-cost experimental design that is rapidly performed, reproducible, and amenable to high-quality radiographic and histological assessment. In this study, we utilized a modified IMP method and documented the degree of instability by three radiographic projections. We also describe a new percutaneous EF method that is compact, light-weight, radiolucent, and removable in the living animal. Using these methods, we compared fracture healing of IMP and EF in GFP reporter mice at days 14 and 35 of repair.

MATERIALS AND METHODS

Transgenic Mice

Mice with a combination of three GFP reporters were used for this study (hOC-GFPtpz 1/4 hOC-green,13 Col3.6-GFPcyan 1/4 Col3.6-blue,14 and Col10A1-RFPchry 1/4 ColX-red15). The three-color mice were identified at birth using fluorescent goggles and confirmed by fluorescence imaging of a tail biopsy at weaning. The mice (12 females and 12 males) were evenly distributed between IMP (age: 15.7 ± 3.9 weeks, range: 10–22 weeks) and EF (age: 15.5±4.9, range: 9–22 weeks) groups. The institutional animal care committee approved all aspects of the experimental protocol.

Surgical Model

The details of the method for both IM pinning and EF stabilization that include annotated videos are provided in the supplement material (Figs. S1 and S2 and Videos S1 and S2). The table of materials and costs for the procedures are given in the supplementary Table S1.

Gait Analysis

Mobility of the mice was quantified with a Digigait™ Imaging system (Mouse Specifics, Inc., Boston, MA) following the method described by Vincelette et al.16 Tread mill speed was set to 10cm/s (slowly walking speed) for the 2-week analysis and 24cm/s (fast running speed) for the 5-week analysis.

Tissue Analysis

The histological and μCT methods are described in the supplemental materials section. The reader is encouraged to download the higher resolution histological images from the journal website, or the original stacked images from our laboratory webserver (http://ucsci.uchc.edu/yupaperf).

Statistics

All tabulated data were analyzed by Mann–Whitney U test with comparisons made between IMP and EF at given time points or over time (D0 vs. D14 vs. D35) within IMP or EF groups. A bonferroni correction was applied to account for multiple comparisons, with significance level set to p<0.05. Values are expressed as mean ± SD.

RESULTS

Comparison of the Fracture Fixation at Day 0 The location of all fractures was relatively consistent. Measuring proximally from the distal end of the tibia, the fracture was located 45.8 ± 5.5% and 45.1 ± 2.5% of tibial length for IMP and EF fixation, respectively (n1/412 per group). In the IMP group, four legs (33.3%) had a fractured fibula at day 0 just after surgery, while the EF group had one leg with a fibular fracture (8.3%).

Deformity Measurements

The tibial length measurement encompasses the overall deformity and tibial compression at the fracture site (Figs. 1 Fig. 11 and S4). The average value of the IMP group at day 14 was 95.7±3.0% of the original length compared to 100.1 ± 0.5% in the EF group (p < 0.05). These values were consistent at D35 with the IMP group measuring 94.4 ± 2.9% and the EF group measuring 100.1 ± 0.6% (p < 0.05).

Figure 1. Assessing the extent of bone deformity in the IMP and EF models of fracture fixation at day 14 and 35 post fracture.

Figure 1

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Tibial length at day 14 and 35 is expressed as a percentage of bone length immediately after fracture. Posterior tibia slope of the contralateral normal bone (10.9º) is compared to the slope immediately after fracture and at days 14 and 35. Varus deformity is measured just after the fracture and at day 14 and 35. The difference between the day 14 or 35 from the day 0 value is plotted. Rotational deformity is obtained from the axial X-ray and is the angle of external rotation of the foot relative to the femur. Connecting bars indicate significant difference (p<0.05).

The posterior tibial slope reflects the anteriorly directed angular deformity of the tibia fracture site in the sagittal plane. When expressed against the slope of the intact contralateral left legs (10.9° ± 2.8, n 1/4 24), the slope of the IMP group at day 0 was 10.8 ± 2.9° but had increased to 17.9 ± 5.9° at day 14 (p < 0.05) and remained increased at 19.5 ± 6.2° on day 35 (p < 0.05). There was no difference between the days 14 and 35 time points. In comparison, the average value of the EF group at day 0 was 12.5±2.5° (n1/412), 13.2±2.5° at day 14, and 11.3 ± 3.1° at day 35. These differences between the IMF and EF groups relative to the initial angle were significant (p < 0.01) at both time points.

The varus deformity is a measure of lateral displacement of the fracture site. The angle was recorded immediately after fracture and subtracted from the value obtained at days 14 and 35. The average angles of the IMP group at day 14 was 9.2±7.7° and at day 35 was 10.2±7.3°. However, the varus deformity of the EF at day 14 was 2.1±2.2°) and 3.0±2.0° at day 35. The IMP and EF groups had statistically significant (p < 0.05) differences in the varus deformity at both time points.

The axial rotational deformity provided the greatest contrast between the two fixation methods. The average value of the IMP group at day 14 was 28.8±15.5° and it remained the same at day 35 (29.2±12.8°). In contrast, the average value of the EF group was 2.8±3.2° at day 14 and 5.2±1.9° at day 35. The IMP and EF groups had statistically significant (p<0.05) differences in rotational deformity at both time points.

Another complication of the IM fixation procedure is an increased incidence of delayed fibula fracture (Table S2). Of 12 fibulae in the IMP group, eight did not have a fracture at day 0. However, by day 14, seven of these remaining eight (87.5%) had newly developed fractures. In comparison, 11 fibulae were intact at day 0 in the EF group, and 2 out of the 11 (18.2%) developed a new fibula fracture by day 14. There were no new fractures between days 14 and 35 in either group. In addition, comminution of bony fragments within the fracture field appeared more frequently in the less stable IMP fixation procedure. In the IMP group, the 12 tibiae did not have bony fragments at day 0 but they developed in nine tibiae (75%) by day 14. In contrast, of the 11 tibiae of EF group without bony fragment at day 0, no (0%) newly separated fragments were apparent at day 14. There were no newly developed bony fragments at day 35 in either group.

CT Images at Day 35

The μCT scan at day 35 showed no visible fracture line in either group. In the IMP group, a mineralized callus was seen around the fracture site (Fig. S5). However, in the EF group, there was no visible callus. In all specimens in the EF group, six needle holes were seen, confirming proper placement of EF.

Animal Health

Both the IMP and EF group mice started to use their fractured right legs immediately after surgery. The average body weight of the IMP group at day 14 had decreased from day 0 by _4.5 ± 5.3% versus EF group of _4.9 ± 6.9%, while at 5 weeks, it was _1.3 ± 6.1% and _0.45 ± 4.4%, respectively. These differences were not statistically significant. Their gait was analyzed using the Digigait walking analyzer. At both day 14 and day 35, there was no significant differences in swing, brake and propel phases and total stride time between the IMP and EF groups or between left limb and right limb of both groups (Fig. S6).

Histological Evaluation

We have previously described the use of our GFP reporters and fluorescent stains in the IMP model at days 7, 14, 21, and 35 of repair.1 For this study, we selected day 14 to represent the stage of maximal hypertrophic callus formation and early development of the outer cortical shell (OCS), while day 35 reflects the inward remodeling phase of fracture callus resolution.

IMP, Day 14

The fracture site of the IMP group is compressed with a bony fragment and displaced cortical bone in the marrow space and a healing fracture of the fibula (Fig. 2). Despite these features of instability, the fracture site is bridged by a large cartilage callus. hOC-green cells (mature osteoblasts) are observed at the base of the callus while Col3.6-blue cells (early osteoblasts) are extending into the cartilage core and partially over the surface of the cartilage core, which is the beginnings of the OCS. The Col3.6-blue cells are AP-red positive and the later signal is particularly useful for following the formation of the OCS as a strong red line delineating the entire callus (Fig 2D, arrow). The hypertrophic chondrocytes are ColX-red and also AP-red but with a different morphology than osteoblast AP. However the central region of the cartilage core which is not yet hypertrophic is sapharin-O positive but does not express either the ColX-red or AP red signal. It is at the interface between Col3.6-blue and ColX-red cells that lines of TRAP+ osteoclasts initially develop. The TRAP+ osteoclasts persist in the Col3.6blue-generated woven bone that will eventually be replace by bone marrow. The progressive invasion of woven bone and osteoclasts into the hypertrophic cartilage moved in a centripedal direction while at the same time the OCS is forming over the residual cartilage.

Figure 2. IMP fixation on Day 14.

Figure 2

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A. Low magnification of the fractured tibia and fibula. The location of the region shown in panels B–E is boxed. B. Darkfield showing the mineralized regions (grey color *), ColX-red in hypertrophic chondrocytes (#), Col3.6-blue of the woven bone invading the hypertrophic zone (Δ) and hOC-green in the more mature bone at the base of the callus (x). C. Same as B with darkfield removed and TRAP stain (yellow) added. TRAP is distributed as a line of osteoclasts (arrows) between the advancing bone and hypertrophic cells and within the remodeling woven bone (Δ). D. AP and DAPI. AP stain showing activity in the woven bone (red,Δ), hypertrophic region (#) and bone beginning to extend over the cartilage core (arrows). E. Safranin-O stain identifying the cartilage core. The red color of the surrounding tissue is derived from the AP stain that was performed in the previous imaging step (panel D). Scale bar=1mm.

IMP, Day 35

At this stage, all of the woven bone and hypertrophic chondrocytes that were present at day 15 have been replaced by bone marrow and a completely mineralized OCS has been formed (Fig. 3). hOC-green mature osteoblasts are embedded in the solid mineral at the sides of the callus while active osteoblasts (Col3.6blue/ hOCgreen) are forming an appositional matrix line on the bone facing the marrow spaces. These Col3.6blue/ hOCgreen cells are most prominent on the inner surface of the OCS. They are strongly AP positive and overlie a bone surface that stained with the mineralization dye, alizarin complexone (AC), indicating that bone matrix is being made on the endocortical side of the cortical shell. Osteoclasts are also prominent in three distinct areas: (i) the remnants of the original cortical bone that has to be resorbed for complete healing; (ii) trabecular-like bone within the callus that also will be removed; and (iii) on the periosteal side of the OCS. It is the coordinated activity of the osteoclasts on the outer surface and osteoblasts on the inner surface that results in the inward migration of the OCS to regenerate an intact cortical bone.

Figure 3. IMP fixation on Day 35.

Figure 3

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A. Low power view of the fractured tibia from which the higher power views were derived (box). ColX-red activity is present in the growth plate (arrows). B. Darkfield showing the mineralized regions (grey color *) with expression of hOC-green mature osteoblasts at the base of the callus. A band of mineralized bone (OCS, arrow head) unites the two halves of the fracture and is characterized by a strong line of Col3.6-blue osteoblasts (arrows) overlying a red AC mineralization line. C. AP and DAPI stain shows the strongest staining to be associated with the Col3.6-blue osteoblasts on the endocortical surface of the OCS and facing the newly developed bone marrow (blue, μ). D, E have TRAP activity superimposed on B, C and show the osteoclasts that line the periosteal side of the OCS (yellow arrows). Osteoclasts are also active on the segments of trabecular bone beneath the OCS (Δ) as well as the original cortical bone (#), which will eventually be resorbed to accommodate the inward migrating OCS. F. Safranin-O stain is negative for remaining chondrocytic tissue, which was also evident by the lack of ColX-red activity in the callus area (panel B) despite strong activity in the growth plate chondrocytes (panel A, arrows). G. Composite aligning all the signals of bone formation and resorption. Scale bar=1mm.

EF, Day 14

Although the fracture site is minimally displaced, it is not compressed and both fractured edges kept their original shape (Fig. 4). A small bone fragment, which might have been made by the percutaneous osteotomy step of the fracture protocol, is observed inside the bone marrow. Much of this fragment is undergoing osteoclastic resorption. Five of the six needle holes are seen in the section and they have elicited an osteogenic response at sites where the cortical bone was interrupted. A new layer of periosteal bone has formed in the region flanking the fracture site. On the fibular side, the new bone emits the hOC-green/Col3.6-blue signal of mature osteoblasts and has sparse osteoclastic activity indicating a rather mature bone matrix. However, on the opposite side, a thickened layer of periosteal cells appears to be streaming between the ends of the fracture bone, and across the marrow space to the opposite cortical bone. Over the proximal cortical surface, these cells strongly express the Col3.6blue reporter indicative of active new bone formation. These cells are strongly AP+ and have intense TRAP+ activity. The periosteal cells that extend into the marrow space show weak Col3.6-blue expression and are not yet strongly AP+ indicative of a preosteoblastic cell. No detectable chondrocytes (ColX-red or Safranin-O) is present within the fracture repair field. Thus the progenitors that arose from the periosteum and migrated across the fracture site do not go through a cartilage intermediate stage prior to bone formation as occurs in IMF callus.

Figure 4. EF on Day 14.

Figure 4

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A. Low magnification showing the three needle holes in the metaphyseal region and 2 holes in the distal tibial region. The fractured region is the large boxed area and it contains a bone fragment (Δ). The insert (#) is derived from the small boxed region to demonstrate that ColX-red is expressed in this animal. B. Darkfield of the boxed region showing strong Col3.6-blue activity extending around the ends of the upper cortical bone while low intensity Col3.6-blue cells traverse the bone marrow space to the opposite cortical bone. The bone fragment is identified with Δ. No ColX-red activity is seen in the repair field. C. TRAP stain and hOC-green expression. Intense TRAP activity is seen within the newly formed bone on the cortical surface and on the bone fragment within the marrow space (Δ). Note the hOC-green mature osteoblast activity within the newly formed periosteal bone on the lower cortical bone (arrows). D. Fusion of B and C with the mineralized bone removed showing the relationship between Col3.6blue, hOC-green and TRAP+ cells. E. AP and DAPI. AP is strongest over the areas of intense Col3.6-blue cell activity but is absent where the Col3.6blue activity is weak. In contrast with the periosteal new bone that is both strongly Col3.6-blue and AP positive, there is dissociation between the AP and TRAP activity surrounding the bone fragment indicating that the fragment is being resorbed rather than being remodeled. F. Safranin-O stain does not detect chrondrocytic tissue. The red color is derived from the AP stain. G. Merging of images from B–F. Scale bar=1mm.

EF, Day 35

Bony union was achieved without obvious deformity (Fig. 5). The cortical bone is regenerated and populated mostly with hOC-green mature osteoblasts and minimal osteoclastic activity. However, the bone crossing the marrow space and joining the opposing cortical bone shows islands of marrow with active endocortical bone remodeling. The bone surfaces show Col3.6/hOC osteoblastic cells with underlying AC mineralization lines and strong AP-red activity, all indicative of active bone formation. These surfaces are also lined with osteoclasts, suggesting that eventually this inter-cortical bone will be resorbed. An unanticipated finding was increased osteoclastic activity on the surface of endocortical bone distal to the fracture site as well as punctate TRAP activity within the endocortical osteocytes (Fig. 4C, arrow and arrow head). This osteoclastic activity is not associated with intense osteoblastic activity, suggesting it may represent an osteolytic process potentially associated with the stress shielding effect of the EF protocol. Again, no evidence of residual chondrogenic cells is found in the defect space.

Figure 5. EF on Day 35.

Figure 5

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A. Low magnification of the bone showing the 3 needles holes in the proximal bone while the distal holes are less well defined. The healing fracture zone is boxed. Note the ColX-red activity in the growth plate region (arrows). B. Darkfield view of fracture region showing Col3.6blue overlying a red AC mineralization line on the bone surface of the bone that extends between the two cortical surfaces. hOC-green osteoblasts predominate on the cortical bone that spans the fracture site. No ColX-red cells are found in the fracture site. C. Same as B with darkfield removed and TRAP added. TRAP activity is found in the endocortical region away from the fracture site (Δ) as well as in the osteocytes in the subcortical bone (arrows). Less intense TRAP activity is associated with the endocortical bone surfaces of the bone that extends across the fracture zone (*). D. AP/DAPI stain shows the most intense activity on the endocortical bone surfaces. E. Safranin-O stain that does not detect evidence of residual chondrocytic tissue. Scale bar= 1 mm

DISCUSSION

The small size of the mouse should not preclude it as a highly informative, low cost, and clinically relevant model of skeletal repair and regeneration.17 This study was initiated to model and contrast two fundamentally different methods of fracture stabilization. In both models it was important to document the degree of deformity that developed, which indirectly reflects the mechanical instability of the repair site. We were surprised that the lateral X-ray images did not adequately report the extent of deformity in the IMP group that was gained with the PA and Ax images. Similarly, the temporal X-rays showed continuing mechanical stress to the fibula and cortical bone over time that resulted in fibula fractures and cortical bone fragments. None of these features should be acceptable in a clinical model of fracture repair even though the gait analysis did not show a difference in the animal’s adaption to these deformities. This variability may make it difficult to compare studies performed by different research groups particularly if the degree of deformity is not adequately documented. Furthermore, it confounded our development of an image analysis method for quantitative assessment18 of fracture repair as the size and shape of the callus varied tremendously.

In contrast to the IMP method of fixation, the EF method did maintain alignment and stability of the fracture site that persisted for the 5 weeks of the study. Not surprisingly, a fracture callus was not evident by X-ray or by μCT, which corresponds to the majority of clinical fracture repair outcomes, and has been achieved in rodent models as well.5 This outcome provided the opportunity to contrast the cellular repair program between a callus-forming and callus-independent repair using our GFP reporter mice and fluorescent staining methods.

The IMP-treated group generated the widely studied fracture callus that is initially bridged by the hypertrophic chondrocytes, over which a cortical shell develops that is buttressed to the cortical bone. Once stability is obtained with this structure, the hypertrophic chondrocytes are replaced by bone marrow and the OCS begins its inward remodeling process. These cellular features are reflected in the GFP reporters for the hypertrophic chondrocytes (ColX-red), the mature osteoblasts (hOC) in the cortical buttress area and the matrix-forming osteoblasts (Col3.6/hOC double positive) on the endocortical surfaces that face bone marrow. Osteoclasts play an essential role in the remodeling process, first by removing the cartilage matrix produced by the hypertrophic chondrocytes and the woven bone made by immature osteoblasts (Col3.6-blue) and subsequently by removing bone from the outer surface of the inward migrating OCS.

The EF elicits a completely different response. The periosteal bone near the fracture expands and osteogenic (Col3.6/AP+) cells originating from the periosteum enter the fracture gap space and begin to form bone in the marrow space. By 5 weeks, a unified segment of new bone unites the opposing periosteal surfaces through the new bone that formed across the marrow space. The chondrocytic lineage does not appear to contribute to this process and the new bone formed by bone marrow-derived bone progenitor cells appears to be very limited. Osteoclasts do participate in the endochondral remodeling process, but to a far lesser extent than is seen in the remodeling fracture callus. However, one of the limitations of the EF model is stress shielding that can lead to bone resorption, which may explain the more intense concentration of osteoclasts in the endocortical bone surface adjacent to the fracture site as well as the appearance of TRAP positive regions at the periphery of the subendosteal osteocytes. A similar distribution of osteoclasts and TRAP positive osteocytes develops an EF non-fractured bone (Fig. S7). These are features that have been previously seen in rodent unloading studies19,20 and in lactating females.21 In future studies, the percutaneous pins of the EF can be sequentially removed or the rigidity of the fixation device can be modulated to overcome this unwanted aspect of fracture repair. While none of the histological findings of the EF model are unexpected, the model does provide a platform to study the cellular events that mediate this type of repair response.

In either method of fixation, the periosteum provides the majority of the progenitor cells that will ultimately heal the fracture. Little visual evidence of muscle or bone marrow contribution was observed, although genetic tools for identifying a minor contribution were not employed. In the case of an unstable and loaded fracture, the progenitors differentiate into a fibrocartilage lineage, which serves to provide the initial stabilization necessary for the OCS to form. However, when the fracture is adequately stabilized by the EF procedure, the progenitors differentiate directly to osteoblasts without utilizing a chondrocyte intermediate.5,22 Since these common progenitors can be isolated from the periosteal surface at these early time points,23 there is an opportunity to explore how the mechanical forces promote one lineage option over the other.

Because IMF utilizes bone remodeling for fracture repair, while EF relies primarily on bone modeling, it might be anticipated that drugs which affect these osteogenic mechanisms would have a different impact on fracture healing. Thus in the IMF model, bisphosphonates have been shown to increase fracture strength by inhibiting resorption of the mineralized callus and by retarding the inward remodeling of the OCS.24,25 This increase in experimental strength at the expense of delay in fracture resolution would not be clinically desirable. In contrast, antiresorptives would probably have little impact in the EF fracture healing and might be protective against the cortical bone loss due to stress shielding. In the case of anabolic agents that promote bone modeling, an incremental positive effect of fracture healing in the EF relative to IMP might be expected. These theoretical outcomes need to be tested26 in reproducible and quantitative murine models to identify the optimal agents for influencing the genetic repair program of the periosteal-derived progenitor cells.

Supplementary Material

Supplemental Information

Acknowledgments

This work was supported by Department of Defense W81XWH-11-1-0262 and USPHS R01-AR052374. Training Grant T90-DE021989 (support for NAD).

Footnotes

SUPPORTING INFORMATION

Additional supporting information may be found in the online version of this article at the publisher’s website.

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