An Improved Methodology to Evaluate Cell and Molecular Signals in the Reparative Callus During Fracture Healing

Abstract

Approximately 5% to 10% of all bone fractures do not heal completely, contributing to significant patient suffering and medical costs. Even in healthy individuals, fracture healing is associated with significant downtime and loss of productivity. However, no pharmacological treatments are currently available to promote efficient bone healing. A better understanding of the underlying molecular mechanisms is crucial for developing novel therapies to hasten healing. The early reparative callus that forms around the site of bone injury is a fragile tissue consisting of shifting cell populations held together by loose connective tissue. The delicate callus is challenging to section and is vulnerable to disintegration during the harsh steps of immunostaining, namely, decalcification, deparaffinization, and antigen retrieval. Here, we describe an improved methodology for processing early-stage fracture calluses and immunofluorescence labeling of the sections to visualize the temporal (timing) and spatial (location) patterns of cellular and molecular events that regulate bone healing. This method has a short turnaround time from sample collection to microscopy as it does not require lengthy decalcification. It preserves the structural integrity of the fragile callus as the method does not entail deparaffinization or harsh methods of antigen retrieval. Our method can be adapted for high-throughput screening of drugs that promote efficacious bone healing.

Keywords: bone matrix; cartilage; chondrocytes; cryosection; fluorescence microscopy; fracture callus; immunofluorescence labeling; osteoblasts; safranin O/fast green.

Figure 1.

Cryosectioning and verifying the structural integrity of fracture callus sections. (A) The OCT block containing the fracture callus is fixed to a circular metal disk attached to the stage of the cryotome. The section is directly collected on an adhesive cryofilm tape, as shown in the bottom of the image. (B) A composite image of a day 7 murine fracture callus (outlined with red dotted lines) from an 8-µm cryosection stained with toluidine blue. Black arrows indicate the site of injury resulting in the Fr. gap. Scale bar shown in (B) is 1 mm. Abbreviations: OCT, optimal cutting temperature; Fr., fracture; BM, bone marrow; Ct.B, old cortical bone.

Figure 2.

Effect of antigen retrieval on IHH immunofluorescence on callus cryosections. Eight-µm cryosections prepared from day 7 fracture calluses were stained directly on the adhesive cryotape with a primary antibody against IHH, followed by a secondary antibody conjugated to Alexa Fluor 488. (A) and (B) IHH immunolabeling in serial sections without or with antigen retrieval, respectively. (C) Secondary antibody–only stained serial section (negative control). Nuclei were stained with DAPI. Representative scale bar is 100 µm in length. Abbreviations: IHH, Indian hedgehog; DAPI, 4′, 6-diamidino-2-phenylindole.

Figure 3.

Spatial and temporal patterns of marker protein expression within the fracture callus. Eight-µm cryosections of fracture calluses were incubated with primary antibodies against EMCN, SOX9, OSX, and COL1A1 and subsequently with Alexa Fluor 594–conjugated (EMCN) or Alexa Fluor 488–conjugated (all others) secondary antibodies. Nuclei were stained with DAPI. Low (100×) and high (400×) magnification images from day 7 and day 14 fracture callus sections are shown to provide an overall view of the pattern of expression of these marker proteins within the callus as healing proceeds. Negative controls are day 7 cryosections stained with only the secondary antibody and DAPI. Scale bars are 100 µm in length. Abbreviations: EMCN, endomucin 1; SOX9, sex-determining region Y box 9; OSX, osterix; COL1A1, type 1 collagen A1; DAPI, 4′, 6-diamidino-2-phenylindole.

Figure 4.

Multiplexing of day 7 postfracture callus sections. Simultaneous staining with primary antibodies against VEGF and OSX (A) or against COL10 and COL1A1 (B) was performed on 8-µm cryosections generated from a day 7 fracture callus. Secondary antibodies conjugated to Alexa Fluor 594 (VEGF and COL10) and Alexa Fluor 488 (OSX and COL1A1) were used. The presence of yellow color in the overlay (far right panel) images indicates colocalization of signals. Negative controls are serial sections stained only with the two secondary antibodies. Images were taken at 200× magnification. Representative scale bar is 100 µm in length. Abbreviations: VEGF, vascular endothelial growth factor; OSX, osterix; COL10, type 10 collagen; COL1A1, type 1 collagen A1.

Figure 5.

Immunofluorescence staining shows patterns of two different MSC populations within the reparative fracture callus. Cryosections (8 µm) from day 7 and day 14 fracture calluses were stained with primary antibodies against SMA or Nestin, followed by a secondary antibody conjugated to Alexa Fluor 488. Nuclei were stained with DAPI. Negative controls are day 7 cryosections (8 µm) stained with only the secondary antibody and DAPI. Images were captured at 100× magnification. Representative scale bar is 100 µm in length. Abbreviations: SMA, smooth muscle actin; MSC, mesenchymal stem cell; DAPI, 4′, 6-diamidino-2-phenylindole.

Figure 6.

Endochondral ossification within a day 7 fracture callus. Representative images of a day 7 fracture callus cryosection of 12 µm thickness stained histologically with safranin O/fast green are shown. Safranin O stains cartilage orange, and fast green stains other tissues including bone green. Images were taken at (A) 40×, (B) 100×, and (C) 200× magnification. The lower magnification images provide an overview of the location and extent of cartilage within the day 7 callus, whereas the higher magnification image provides an appreciation of the morphology of hypertrophic chondrocytes within the central callus. Scale bars are 100 µm in length.