Mice with a targeted null mutation in the periostin gene (Postn ^{tm1.1(KOMP)Vlcg}) were purchased from Jackson Laboratory (Stock #024186, Bar Harbor, ME). Homozygous Postn ^{tm1.1(KOMP)Vlcg} null mice (PostnKO) are viable and fertile but with reduced mechanical integrity in connective tissues [35, 36]. The Postn ^{tm1.1(KOMP)Vlcg} mice were bred with C57BL/6 mice to produce normal (WT), Postn ^{tm1.1(KOMP)Vlcg} heterozygous (PostnHET), and PostnKO mice.

Mice with a targeted null mutation in the osteopontin gene (Spp1 ^tm1Blh) were purchased from Jackson Laboratory (Stock #004936, Bar Harbor, ME) [22]. The Spp1 ^tm1Blh null mice (Spp1KO) have a C57BL/6 genetic background and were maintained by homozygous null breeding to produce Spp1KO mice. Mice were genotyped via allele-specific PCR amplification of DNA extracted from tail clip biopsies (Table 1). Immunohistochemistry was conducted to confirm loss of Postn and Spp1 protein expression in the fracture calluses of homozygous null mice. Six to fourteen mice were used for each genotype at each time point of which at least 3 were males. All animal procedures were approved by the Rutgers-New Jersey Medical School Institutional Animal Care and Use Committee (IACUC; protocol #201800006).

Male and female mice aged 15 weeks were anesthetized by intraperitoneal injection of ketamine and xylazine (0.1 and 0.01 mg/g body weight, respectively) prior to retrograde insertion of 0.01-inch diameter stainless steel pin in the right femoral canal to stabilize the impending fracture. At 16 weeks of age, the mice were anesthetized again and underwent a closed, diaphyseal fracture of the right femur using a custom-made, three-point controlled impact device (BBC Specialty Automotive Center, Linden, NJ) as described previously [37]. For all genotypes, male mice weighed significantly more than the females.

C57BL/6 and Spp1KO mice had significantly higher body weights than PostnKO mice. C57BL/6 mice weighed 23.17 ± 3.70 g (mean ± SD), Spp1KO 24.42 ± 3.23 g (mean ± SD), PostnKO 21.28 ± 3.19 g (mean ± SD) and PostnHET 22.47 ± 3.89 g (mean ± SD). Ventral-dorsal radiographs of the mice were made using an XPERT80 digital radiography cabinet (KUBTEC, Stratford, CT) with exposure settings of 65 kV, 90 µA, and 8 s immediately after fracture and then at 7, 10, 14, 21, and 28 days after fracture (dpf) or until the endpoint (see Supplementary Figure S1). Fixed femurs (see below) were stored in 70% ethanol prior to high-resolution computerized tomography (µCT) scanning. Specimens were scanned using a Bruker Skyscan 1275 system (Micro Photonics Inc., Allentown, PA) at 73 kVp, with an intensity of 133 µA, a voxel size of 12 μm isotropic, and with a 0.5 mm aluminum filter. Scanner images were reconstructed (NRecon), analyzed (CTan), and viewed (CTvol) using software from the manufacturer (Bruker, Kontich, Belgium). A detailed description of the method used to measure fracture callus volume and fracture callus bone (calcified tissue) volume can be found in the Supplementary Material (see Supplementary Figure S2).

After euthanasia, femurs were resected and then fixed in an aqueous solution of bronopol (3% w/v), diazolidinyl urea (3% w/v), zinc sulfate hepta-hydrate (1.2% w/v), sodium citrate (0.29% w/v), ascorbic acid (0.025% w/v), and 20% (v/v) ethanol for 2 days at room temperature. The specimens were then either immediately decalcified in 0.5 M disodium EDTA for 2 weeks at 4°C or analyzed by µCT before decalcification. The decalcified specimens were embedded in paraffin, cut into 5 µm thick longitudinal sections parallel to the intramedullary canal in the dorsal-ventral plane, and mounted onto TruBond 380 glass slides (Newcomer Supply, Middleton, WI). Mounted sections were deparaffinized using three changes of xylene and rehydrated in a graded ethanol series. Osteoclasts were detected by tartrate-resistant acid phosphatase (TRAP) staining with a hematoxylin counterstain. Cartilage was visualized by safranin-O staining with fast green and hematoxylin counterstaining. Bone was visualized using aniline blue staining with Biebrich scarlet-acid fuchsin and hematoxylin counterstaining (Masson’s trichrome staining). Sections were cover-slipped with Cytoseal mounting medium (Fisher Scientific, Waltham, MA). Unless otherwise indicated, stains and reagents were from Sigma-Aldrich (St. Louis, MO).

Antibodies used are listed in Table 2. Tissue sections were deparaffinized and rehydrated as described above before antigen retrieval in 10 mM sodium citrate pH 6.0 buffer (70–80°C, 1 h) for COX-2, CD31, POSTN, SPP1, F4/80 and Cathepsin K (CTSK) detection, or in phosphate-buffered saline (PBS) with 25 mg/mL testicular hyaluronidase (37°C for 1 h; Worthington Biochemical Corporation, Lakewood, NJ) for MMP-13 and Collagen X detection. Endogenous peroxidases were quenched with 3% H₂O₂ in PBS (30 min at room temperature) followed by non-specific epitope blocking with SuperBlock (room temperature for 1 h; ThermoFisher, Waltham, MA). After washing with PBS, 150 µL of primary antibody diluted in Antibody Diluent pH7.4 (IHC World, Woodstock, MD) was applied to each histological section and incubated overnight in a humidified chamber at 4°C. See Table 2 for antibody dilutions. Following several washes in PBS, sections were incubated in POLINK-2 Plus Rabbit polymeric HRP secondary antibody per the manufacturer’s instructions (IHC World) and then washed with PBS before colorimetric detection using diaminobenzidine (GBI Labs, Bothell, WA). Sections were counterstained with methyl green to detect cell nuclei.

Digital images of callus fracture sections were captured using an Olympus BX53 microscope and DP73 camera (Olympus Corporation of America, Center Valley, PA). Cartilage and bone areas were measured for each specimen using OsteoMeasure Software (OsteoMetrics Inc., Decatur, GA) from safranin-O and trichrome stained sections, respectively, and normalized as a percentage of total callus area. TRAP positive cells were manually counted using OsteoMeasure and divided by callus area to generate the density of TRAP positive cells per mm². Similar procedures for image collection and analysis were conducted with the IHC samples to determine COX-2, Cathepsin K (CTSK), MMP-13, CD31, Collagen X (COL10A1), and F4/80 positive areas or cell numbers in the callus. Examples showing how MMP-13 expressing chondrocytes and vascular lumens surrounded by CD31 expressing cells were counted are shown in Supplementary Figure S3. A comparison between using TRAP staining and IHC detection of CTSK to count callus osteoclasts showed a high degree of correlation (R² = 0.91, see Supplementary Figure S4).

Fracture calluses were resected at 14 days after fracture (dpf) from at least 3 male and 3 female mice of each genotype. Calluses were flash-frozen in liquid nitrogen and stored at −80°C until RNA extraction [38]. Briefly, each callus was homogenized in Trizol Reagent (1 mL per 50 to 100 mg callus weight) using a Precellys 24 Omni Bead Homogenizer (Bertin Technologies SAS; Montigny-le-Bretonneux, France). After Trizol extraction, the RNA was further purified using a Qiagen RNeasy mini kit as per the manufacturer’s instructions (Qiagen, Hilden, Germany). RNA concentration and integrity were determined by absorbance and agarose gel electrophoresis, respectively.

Target mRNA levels were measured using RT-qPCR as follows. cDNA was prepared from each RNA preparation using an Applied Biosystems High-Capacity cDNA kit (Waltham, MA). The cDNA from 0.1 µg of total RNA along with 1 µM of each primer were used in each 25 µL qPCR reaction (SYBR Green PCR Master Mix, Applied Biosystems 4309155). Amplifications were performed using a Bio-Rad Laboratories CFX96 Real-Time System (Hercules, CA). At least 3 qPCR reactions were performed with each cDNA preparation for every target mRNA and the mean threshold cycle (Ct) value was used for further comparisons. At least 6 callus RNA preparations (3 male and 3 female) were measured for each genotype and for each target mRNA. Target mRNA levels were normalized to corresponding β-actin mRNA Ct values. Relative gene expression was determined using the 2^−ΔΔCT method [39].

Statistical analyses were performed using OriginPro 2022b (OriginLab Corp., Northhampton, MA). Callus histomorphometry data, including the number of TRAP positive+ cells, cartilage area, bone area, and all immunohistochemistry gene quantifications were analyzed by 3-way ANOVA using genotype, days post-fracture (dpf) and gender as independent variables. Post-hoc tests utilized Tukey or Holm-Sidak corrections to identify significant differences between groups. Detailed information regarding the statistical analyses is shown in the Supplementary Material.

µCT was performed on femurs resected at 14, 21, and 28 days post-fracture (dpf) to visualize and quantify morphological differences in the facture calluses between wild type (WT), PostnHET, PostnKO, and Spp1KO genotypes (see Supplementary Figure S1 for digital radiographs). Longitudinal sections from reconstructed µCT volumes are shown for each time point and genotype in Figure 1. Healing appeared to proceed normally in the WT control C57BL/6 mice (Figures 1A–C) with apparent bridging at 14 dpf, definitive bridging by 21 dpf, and callus remodeling evident at 28 dpf [37]. Healing also appeared to proceed reasonably well in the PostnHET mice with definitive bridging by 21 dpf and evident callus remodeling at 28 dpf, though at 14 dpf less calcified tissue was evident in the external callus (Figures 1D–F). The external fracture callus in the PostnKO and Spp1KO mice at 14 dpf appeared to have central areas devoid of calcified tissue (Figures 1G, J). By 21 dpf, the PostnKO and Spp1KO calluses appeared to bridge the fracture (Figures 1H, K). Callus remodeling was evident at 28 dpf in the PostnKO and Spp1KO mouse fracture calluses (Figures 1I, L). However, less bone was evident in the 28 dpf PostnKO callus and the extent of callus remodeling appeared reduced for both genotypes.

The µCT data were also used to quantify total callus volume (TV) and callus bone (calcified tissue) volume (BV). As expected, TV, BV, and BV/TV varied with time after fracture for all genotypes showing a general pattern of decreasing callus TV (means of 39, 27, and 18 mm³ at 14, 21, and 28 dpf) and BV (means of 11, 10, and 6.5 mm³ at 14, 21, and 28 dpf) but increasing BV/TV from 14 dpf (means of 32, 41, and 39% at 14, 21, and 28 dpf; Figures 1M–O). In addition, male mice also had significantly larger calluses (TV; means of 37 vs. 19 mm³) and more callus bone (BV; means of 11.5 vs. 7.4 mm³), though normalized callus bone volume (BV/TV) was in general greater in the female mice (means of 34% vs. 41%; see Supplementary Material). Across all time points and both genders, callus TV was significantly greater in the PostnHET (33.1 mm³) and Spp1KO (34.3 mm³) as compared to the WT (22.4 mm³) and PostnKO (25.1 mm³) mice. Consistent with the overall callus volume, BV was significantly greater in the Spp1KO mice (12.5 mm³) and lowest in the PostnKO mice (7.8 mm³), though differences between WT (8.6 mm³), PostnHET (9.4 mm³), and PostnKO were not significant. In contrast, BV/TV across all time points and both genders was significantly lower in the PostnHET (34.9%) and PostnKO (35.1%) mice as compared to the WT mice (40.8%), though no statistical difference was detected for the Spp1KO mice (39.2%).

Immunohistochemistry was performed on 10 dpf calluses to confirm normal expression of POSTN and SPP1 in WT mice and loss of POSTN and SPP1 expression in PostnKO and Spp1KO mice, respectively (Figure 2).

POSTN was broadly expressed throughout the WT callus (Figure 2A). POSTN levels appeared higher in osteoblast-rich areas and along the chondro-osseous junction with apparent lower levels in callus chondrocytes. POSTN expression levels appeared to be reduced in the PostnHET callus with POSTN localization limited to fibroblasts and newly differentiated chondrocytes at the center of the callus and to chondrocytes near the chondro-osseous junction (Figure 2B). As expected, POSTN was absent in the PostnKO callus (Figure 2C). POSTN expression was detected in the Spp1KO callus though at an apparent reduced level as compared to WT (Figure 2D).

SPP1 was also broadly expressed throughout the WT fracture callus with SPP1 localized in callus osteoblasts and chondrocytes (Figure 2E). As expected, SPP1 expression appeared absent in the Spp1KO callus (Figure 2H). SPP1 levels appeared reduced in the PostnHET callus with expression in osteoblasts and in a subset of chondrocytes at or near the chondro-osseous junction (Figure 2F). SPP1 expression in the PostnKO callus appeared to be even further reduced as compared to the PostnHET callus (Figure 2G).

Postn and Spp1 mRNA levels were measured in total RNA prepared from 14 dpf WT, PostnKO, and Spp1KO calluses by RTqPCR. As expected, ß-2-microglobulin (B2m) mRNA levels were similar between calluses from all genotypes (Figure 2I), while Postn and Spp1 mRNA levels were significantly reduced in the PostnKO and Spp1KO calluses, respectively (Figures 2K, L). Similar to the IHC results, Spp1 mRNA and Postn mRNA levels were significantly lower than WT levels in the PostnKO and Spp1KO calluses, respectively. We also noted that osteocalcin (Bglap) mRNA levels were reduced in the PostnKO mouse calluses (Figure 2J), which was consistent with the reduced callus BV/TV (Figure 1O) and reduced callus bone area (Figure 3J) in Postn deficient mice.

Masson’s trichrome staining was performed on tissue sections of fracture calluses collected at 7, 10, 14, and 21 dpf to visualize morphological differences between mouse genotypes during the healing process. Images from one quadrant of the external fracture callus of a WT, PostnHET, PostnKO, and Spp1KO mouse at 14 dpf are shown in Figures 3A, C, E, G, respectively. External callus bone tissue and cartilage were identified based on staining and morphology and quantified at each time point as shown in Figures 3I–K. Similarly, TRAP staining was performed on serial sections to identify osteoclasts in the fracture calluses (Figures 3B, D, F, H). At 14 dpf, the external callus of the WT mouse has apparent newly formed bone, is highly cellularized, and has a clearly demarcated chondro-osseous junction that is lined with osteoclasts (TRAP⁺ cells; Figures 3A, B). While the 14 dpf PostnHET callus also appears to have a considerable amount of new bone and is highly cellularized, the chondro-osseous junction is neither distinct nor abundantly populated with osteoclasts (Figures 3C, D). The PostnKO mouse callus appears to have less new bone, more cartilage, and a poorly defined chondro-osseous junction (Figure 3E, F). The Spp1KO mouse callus appears to have amounts of new bone and cartilage that are comparable to the WT callus (Figures 3G, H).

Similar to the PostnHET and PostnKO chondro-osseous junctions though, the Spp1KO chondro-osseous junction also appears to have fewer osteoclasts and displays an abnormal morphology.

Callus area, percent bone area, percent cartilage area, and density of TRAP⁺ cells were quantified at each time point and for each genotype as shown in Figures 3I–L. As expected, callus area was dependent upon time after fracture and gender but was not dependent upon genotype.

For all genotypes, callus percent bone area increased with time after fracture (Figure 3J) while callus percent cartilage area peaked at 10 dpf before declining (Figure 3K). Callus percent bone area was dependent upon time after fracture, gender, and genotype with WT significantly different from all other genotypes and Spp1KO different from PostnHET and PostnKO. In contrast, callus percent cartilage area was only dependent upon on time after fracture and not gender or genotype. The density of osteoclasts (TRAP⁺ cells/mm²) was dependent upon time after fracture and genotype, but not gender (Figure 3L). Osteoclast density rapidly increased between 10 and 14 dpf for all genotypes but overall osteoclast density was greater in the WT mouse fracture calluses than in the PostnHET, PostnKO, or Spp1KO mouse calluses.

The effects of Postn and Spp1 null mutations on callus chondrocyte hypertrophy were assessed by immunohistochemical (IHC) detection and quantification of Collagen X (COL10A1) and Matrix Metallopeptidase-13 (MMP13). Serial sections from mouse fracture calluses collected at 7, 10, or 14 dpf were stained with Safranin-O to detect cartilage (Figures 4A, D, G, J, see also Figure 3K) or used for IHC detection of collagen X (Figures 4B, E, H, K) or MMP-13 (Figures 4C, F, I, L). Safranin-O stained areas appeared to roughly correlate with callus areas expressing collagen X. In contrast, callus cells expressing MMP-13 were generally not evident in callus cartilage areas stained with Safanin-O. Instead, cells expressing MMP-13 also appeared to express collagen X and were localized in areas of newly forming bone.

The percentage of callus cartilage area that expressed collagen X was determined at 7, 10, and 14 dpf (Figure 4M). Callus area expressing collagen X was dependent upon time after fracture and mouse genotype, but not gender. Across all time points, the percent of callus area that stained for COL10A1 by IHC was significantly lower for the PostnKO (21%) and Spp1KO (21%) mouse calluses as compared to WT (29%; p < 0.009).

Callus chondrocytes were counted at 10 and 14 dpf (Figure 4N) and compared to the number of callus chondrocytes expressing MMP-13, and thus presumably undergoing hypertrophy (Figure 4O). The number of callus chondrocytes was significantly reduced in the PostnKO and Spp1KO mouse calluses as compared to WT (p = 0.039). The number of callus chondrocytes also decreased between 10 dpf to 14 dpf by 33% in WT, 34% in PostnHET, 23% in PostnKO, and 29% in Spp1KO calluses. Conversely, the percentage of callus chondrocytes expressing MMP-13 increased between 10 and 14 dpf for all genotypes (p < 0.0001), even though the percentage of MMP-13 expressing chondrocytes was significantly lower in the PostnHET, PostnKO, and Spp1KO mouse calluses as compared to WT (p < 0.0001).

Neovascularization of fracture calluses collected at 7, 10, and 14 dpf was assessed in WT, PostnHET, PostnKO, and Spp1KO mice by immunohistochemical (IHC) detection of CD31 (Figure 5). CD31 is encoded by Pecam1 and is expressed by vascular endothelial cells [40]. At 10 and 14 dpf, blood vessels expressing CD31 were abundant in areas of the WT fracture callus that had or were undergoing endochondral ossification but were absent in the cartilage (Figures 5A, B). Blood vessels were similarly abundant in the PostnHET fracture calluses at 10 and 14 dpf (Figures 5C, D). However, fewer blood vessels were apparent in the 10 and 14 dpf fracture calluses of the PostnKO and Spp1KO mice (Figures 5E–H). CD31⁺ lumens in the fracture callus, and therefore presumptive blood vessels, were counted at 7, 10, and 14 dpf for all mouse genotypes and normalized to callus area (Figure 5I). Blood vessel density was dependent upon time after fracture, gender, and genotype. For all genotypes, the number of callus CD31⁺ lumens increased with time after fracture. However, the rate of increase appeared greater in the WT and PostnHET calluses, leading to a relative paucity of new blood vessels in the PostnKO and Spp1KO fracture calluses. The reduced density of CD31⁺ lumens in fracture calluses of male mice was consistent with previously observed sexual dimorphism in vasculogenesis associated with wound healing [41–43]. Quantification of the new blood vessels lumen area found an apparent time-dependent increase in lumen area but no effect of gender or genotype was detected (Figure 5J).

Fracture callus distribution of macrophages and osteoclasts was determined by immunohistochemical detection of F4/80 and cathepsin K (CTSK), respectively (Figure 6). At 14 dpf, F4/80⁺ cells were detected in calluses from all genotypes (Figures 6A, C, E, G). The F4/80⁺ cells appeared to be absent from callus cartilage but enriched in areas of newly formed bone. Osteoclasts detected by CTSK expression were evident along the callus chondro-osseous junction and within areas of newly formed bone (Figures 6B, D, F, H). Relative to WT mouse calluses, IHC detection of CTSK appeared to be reduced in the PostnHET and PostnKO mouse calluses and further reduced in the Spp1KO mouse callus. Interestingly, F4/80⁺ cells were rarely observed in the region adjacent to the chondro-osseous junction.

The F4/80⁺ and CTSK⁺ cells in fracture calluses collected at 7, 10, and 14 dpf were counted for each mouse genotype and normalized to callus area. The density of F4/80⁺ cells was dependent upon time after fracture and genotype, but not gender (Figure 6I). The density of F4/80⁺ cells increased with time after fracture for all genotypes, but the density of F4/80⁺ cells was reduced in the PostnKO and Spp1KO mouse calluses. Similarly, the density of CTSK⁺ osteoclasts was dependent upon on time after fracture and genotype but not gender (Figure 6J). The density of osteoclasts increased with time after fracture for all genotypes. Osteoclast density at all time points was highest in the WT mouse calluses and lowest in the PostnKO mouse calluses.

Fracture callus cells expressing COX-2 were detected by IHC (Figure 7). In 14 dpf WT fracture calluses, COX-2 expression was easily detectable in osteoblasts, some chondrocytes, and osteoclasts, including those at the chondro-osseous junction (Figure 7A). Strong COX-2 expression was evident in osteoblasts and some chondrocytes of the PostnHET and PostnKO mouse calluses (Figures 7B, C). COX-2 expression was evident but comparatively reduced in osteoclasts of the PostnHET and PostnKO mouse calluses as compared to the osteoclasts and osteoblasts in the WT mouse calluses and the osteoblasts in the PostnHET and PostnKO mouse calluses. In the Spp1KO mouse calluses, COX-2 expression was evident in osteoblasts and some chondrocytes but osteoclast COX-2 expression was markedly faint in comparison to callus osteoclasts from the WT, PostnHET, and PostnKO mice (Figure 7D).

Callus osteoclasts and COX-2 expressing osteoclasts were counted at 10, 14, and 21 dpf from WT, PostnHET, PostnKO, and Spp1KO mice (Figure 7E). The percentage of osteoclasts expressing COX-2 was not dependent upon time after fracture or gender. However, the percentage of osteoclasts expressing COX-2 was reduced in the PostnHET (63%), PostnKO (62%), and Spp1KO (51%) calluses as compared to calluses from WT mice (91%).

Total RNA was isolated from 14 dpf calluses of WT, PostnKO, and Spp1KO mice and target mRNA levels were measured by RT-qPCR. Chondrogenesis and chondrocyte hypertrophy were assessed by measuring Aggrecan (Acan) and MMP-13 (Mmp13) mRNA levels, respectively. Acan mRNA levels were similar between WT, PostnKO and Spp1KO mouse calluses, though Acan mRNA levels were greater in PostnKO calluses than Spp1KO calluses (Figure 8A). In contrast and similar to the IHC results (Figure 4), Mmp13 mRNA levels were significantly reduced in the PostnKO and Spp1KO mouse calluses as compared to WT (Figure 8B). Vasculogenesis was assessed by measuring CD31 (Pecam1) mRNA levels and similar to the IHC results (Figure 5), Pecam1 mRNA levels were significantly reduced in the Spp1KO mice, though not in the PostnKO mouse calluses (P = 0.13; Figure 8C). Osteoclast activity was assessed by measuring Cathepsin K (Ctsk) and Tartrate Resistant Acid Phosphatase (Acp5) mRNA levels. While IHC results showed significant reductions in TRAP⁺ cell density (Figure 3) and CTSK⁺ cell density (Figure 6), only mRNA levels for Acp5 were significantly lower in the Spp1KO mouse calluses (Figures 8D, E). COX-2 (Ptgs2) mRNA levels were also measured and were significantly reduced in both the PostnKO and Spp1KO mouse calluses as compared to WT calluses (Figure 8F).