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. Author manuscript; available in PMC: 2023 Jun 1.
Published in final edited form as: Alcohol Clin Exp Res. 2022 May 16;46(6):915–927. doi: 10.1111/acer.14836

Episodic alcohol exposure attenuates mesenchymal stem cell chondrogenic differentiation during bone fracture callus formation

Jonathan M Eby 1,2,†, Farah Sharieh 1,2,†, Jessica Azevedo 1, John J Callaci 1,2,*
PMCID: PMC9764140  NIHMSID: NIHMS1797257  PMID: 35403260

Abstract

Background:

During bone fracture repair, mesenchymal stem cells (MSC) differentiate into chondrocytes and osteoblasts to form a fracture callus. Our laboratory previously reported that alcohol exposed rodents with a surgically created tibia fracture display deficient fracture callus formation and diminished signs of endochondral ossification characterized by the absence of chondrocytes and mature hypertrophic chondrocytes, suggesting alcohol may inhibit MSC differentiation. These findings led to our hypothesis that alcohol exposure inhibits mesenchymal stem cell chondrogenic differentiation within the developing fracture callus.

Methods:

In the present study, we utilized a lineage tracing approach to determine which stage(s) of chondrogenic differentiation may be affected by alcohol exposure. We utilized lineage-specific reporter mice to determine the effects of alcohol on MSC and early and late chondrogenic cell frequencies within the fracture callus. In addition, serially sectioned slides were immunofluorescently and immunohistochemically stained and quantified to determine the effect of alcohol on cell proliferation and apoptosis within the fracture callus of alcohol administered rodents, respectively.

Results:

Alcohol-administered rodents had a reduced fracture callus area at 4, 6, and 9 days post-fracture. Alcohol had no effect on apoptosis in the fracture callus at any of the examined timepoints. Alcohol-administered rodents had significantly fewer proliferative cells in the fracture callus at 9 days post-fracture, but no effect on cell proliferation was observed in earlier fracture callus timepoints. Alcohol-administered rodents had reduced Collagen2a1- and Collagen10a1 expressing cells in the developing fracture callus, suggesting that alcohol may inhibit both early chondrogenic differentiation and later chondrocyte maturation during fracture callus development.

Conclusion:

The data suggest that alcohol might be affecting normal fracture healing through the mitigation of MSC chondrogenic differentiation at Callus site.

Keywords: Fracture healing, MSC differentiation, binge alcohol, chondrogenesis

Introduction

Bone fracture repair is a regenerative process that recapitulates primary bone formation, requiring a complex orchestration of molecular and cellular processes. While a majority of fractures heal without complication, about 5–10% of bone fractures progress to a fracture nonunion (Hak et al., 2014; Tzioupis and Giannoudis, 2007; Zimmermann and Moghaddam, 2010). Fracture nonunion is a permanent failure of fracture healing and requiring surgical intervention to achieve bone union. Fracture nonunion contributes to over a billion dollars annually in healthcare costs in the United States alone (Miclau, 2017). Risk factors associated with fracture nonunion include diabetes and age-related comorbidities such as osteoporosis (Zura et al., 2017). An important lifestyle-related risk factor for fracture nonunion is alcohol abuse (Duckworth, 2011; Zura et al., 2016)

Excessive alcohol consumption is a well-known risk factor for sustaining a traumatic injury requiring emergency medical treatment and/or hospital admission (Miller et al, 2012). Up to 40% of orthopaedic trauma patients admitted to the emergency department have elevated blood alcohol levels (Levy et al., 1996; Savola et al., 2005) suggesting that alcohol abuse may increase the risk for traumatic orthopaedic injury. Clinical studies have found that while alcohol abuse also increases the risk for a non-healing fracture injury (Chakkalakal, 2005; Elmali et al., 2002; Nyquist et al., 1997), the cellular and molecular mechanisms underlying alcohol consumption-related fracture nonunion have not been studied extensively.

During fracture healing mesenchymal stem cells (MSC) primarily of periosteal origin, migrate towards the injury site and differentiate into cartilage-producing chondrocytes and bone-forming osteoblasts (Colnot, 2009). Shortly after injury, injury-associated tissue begins to produce cytokines and chemokines to initiate the early inflammatory stage. While the inflammatory stage functions to recruit immune cells, chemokines secreted also serve to recruit local pair-related homeobox 1 positive (PRX1+) MSCs to the injury site. Early fracture callus formation is initiated by MSC proliferation and differentiation into collagen 2 positive (COL2a1+) chondrocytes to form a soft cartilaginous fracture callus. COL2a1+ chondrocytes mature into collagen 10 positive (COL10a1+) hypertrophic chondrocytes leading to the ossification of the soft cartilaginous callus into a hard, bony callus. Bone remodeling activity replaces primary bone with lamellar bone, restoring normal bone anatomy and architecture. Any disruptions to this process as a result of alcohol consumption may negatively affect fracture callus formation and bone healing. Indeed, several studies have shown that alcohol inhibits callus formation and bone union after fracture (Chakkalakal, 2005; Elmali et al., 2002; Nyquist et al., 1997). A recent rodent study found that fracture callus from rodents exposed to alcohol had less cartilaginous tissue and more immature appearing cells as determined by hematoxylin and eosin-staining of fracture callus tissue (Roper et al., 2016). These findings suggested that alcohol may inhibit fracture callus formation in part due to an inhibition of MSC lineage differentiation.

Alcohol has also been shown to affect chondrocyte marker expression. One study found that chondrocytes isolated from patients with femoral head osteonecrosis had decreased COL2a1 and SOX9 expression, an early chondrogenic transcription factor, when treated with alcohol (Qin et al., 2018). A recent in vitro study showed that alcohol inhibited MSC early osteochondral commitment and differentiation (Sharieh et al., 2020). However, the effect of alcohol on MSC differentiation during early fracture callus formation has yet to be described. In this study, we hypothesized that alcohol’s deleterious effects on fracture callus formation might be caused by a perturbation of MSC osteochondral differentiation in the early fracture callus. Lineage tracing, utilizing transgenic, tissue specific reporter mice is a technique which can be utilized to explore the origins and lineage differentiation of cells in the fracture callus (He et al.,2017). In the current study, MSC and chondrocyte-specific lineage reporter transgenic mice were utilized to determine if episodic alcohol exposure results in changes in MSC and chondrocyte cell frequencies in the developing fracture callus, in an attempt to further elucidate a cellular mechanism responsible for alcohol-induced deficit fracture healing.

Materials and Methods

Animals

All animal experiments described herein were approved by the Institutional Animal Care and Use Committee (IACUC) at Loyola University Medical Center. Transgenic mouse strains Col2a1-CreERT2 (Zhu et al., 2008) and Col10a1-Cre (Gebhard et al., 2008) were generously gifted by Matthew Hilton at Duke University School of Medicine. Prx1-CreERT2 (stock #029211) and Ai9 tdTomato reporter (stock #007909) mice were purchased from Jackson Laboratory. The Prx1-CreERT2, Col10a1-Cre and Ai9 tdTomato reporter mice were developed on a C57BL/6J background, while the Col2a1-CreERT2 animals were developed on a mixed FVB/J/C57BL/6J background. Prx1-CreERT2;Ai9, Col2a1-CreERT2;Ai9, and Col10a1-Cre;Ai9 crosses were confirmed by genotyping. Male and female animals, aged 8–9 week at the start of each treatment regimen, were used for all experiments.

Alcohol Administration

Transgenic animals were randomly assigned to either a saline-treated or alcohol treated group. Treatment Group-specific alcohol/saline administrations and surgeries were staggered by one day and animals within a group were housed together with cages placed on different rack levels. Our episodic model of alcohol administration has been described previously (Roper et al., 2016). Briefly, animals were given a daily intraperitoneal (i.p.) injection of either sterile isotonic saline (saline) or 20% v/v of 100% molecular grade absolute ethanol (EtOH) in saline, at a dose of 2g/kg for 3 consecutive days. Alcohol or saline treatments were then withheld for 4 days before animals were given a second 3-day alcohol or saline treatment regimen. One hour following EtOH administration, mice exhibit a blood alcohol concentration (BAC) of approximately 200mg/dL. This blood alcohol concentration mirrors blood alcohol concentrations common in trauma patients at the time of admission to the emergency department (Savola et al., 2005; Stoduto, 1993). One-hour following the last pre-injury ethanol dose administered, animals were subjected to a modification of a previously described mid-shaft stabilized tibia fracture, described below. Daily post-injury saline or EtOH i.p. injections were administered to mice to mimic a clinically relevant problem of post-injury drinking in patients (Savola et al., 2005). Animals had ad libitum access to food and water during the protocol. No significant effects of alcohol on mouse body weight were observed. Animals were humanely euthanized at 4, 6, and 9 days post-fracture.

Stabilized Tibia Fracture Model

Our tibia fracture model is a modification of a previously described stabilized mid-shaft mouse tibia fracture protocol (Hiltunen et al., 1993). Briefly, mice were anesthetized using 3% isoflurane and the left hind leg was prepped using a chemical depilatory cream to remove fur from the distal femur down to the ankle. Povidone-iodine solution was applied to the chemically shaved hind leg followed by 70% ethanol. A small incision was created proximal of the proximal head of the tibia. The incision site was moved to expose the patellar tendon, where a 27-gauge needle was guided behind the patellar tendon to ream a hole into the head of the tibia and into the medullary cavity. A 0.25mm “000” stainless steel pin was inserted into the medullary cavity to internally stabilize the fracture. A complete mid-shaft tibia fracture was administered using angled bone scissors and the stainless-steel pin was cut flush to the head of the tibia. The incision was closed with sutures and animals received a subcutaneous injection of 1mg/kg sustained relief buprenorphine (Simbadol, Patterson, MN, USA), and ip administration of 1mL isotonic saline and 5mg/kg gentamicin. Animals were kept on 37° C heating pads until regaining full consciousness.

Tamoxifen Administration

For animal strains Prx1-CreERT2;Ai9 and Col2a1-CreERT2;Ai9, 100mg/kg tamoxifen was intraperitoneally administrated for 5 consecutive days. Tamoxifen administration for fractures isolated at post-operative (Post-Op) day 4 began Post-Op day −1. Tamoxifen administration for fractures isolated at Post-Op day 6 and 9 began on Post-Op day 1 and Post-Op day 4, respectively.

Tissue Processing

All liquid reagents used for tissue processing were kept ice-cold and each step was performed rocking at 4° C. Injured tibias were harvested from euthanized rodents on the respective post-fracture days Cutaneous and sub-cutaneous tissue was removed from each tibia before placing specimens in 1X PBS (Corning, VA, USA); The PBS buffer was replaced with 4% paraformaldehyde in 1X PBS pH 7.1–7.4, and specimens were fixed while rocking overnight. Tibias were then washed in 1X PBS for 30 minutes, then replaced with 14% EDTA (Sigma ED4SS, GA, USA), pH 7.0 decalcification solution and replace with fresh decalcification solution every 4 days. Tibias were incubated in decalcification solution until tibias were flexible, approximately 7–10 days. After decalcification, tibias were washed with 1X PBS for 30 minutes before replacing with 30% sucrose (Sigma, GA, USA) in 1X PBS. Tibias were incubated in 30% sucrose for 2 days. Tibias were removed from the sucrose solution, placed in tissue cassettes, and covered in optimal cutting temperature compound (Fisher, IL, USA) and snap frozen. Tissue cassettes (Fisher, IL, USA) were stored at −80° C. Before sectioning, tissue blocks were placed in the cryostat to acclimate to cryostat temperature and serially sectioned at 10μm. Sections were then dried and stored at −20° C until needed.

Immunofluorescence, Immunohistochemistry, and Histology

Tissue sections used in immunofluorescent experiments were rehydrated in 1X PBS for 10 minutes, then treated with 2mg/mL hyaluronidase (Sigma, GA, USA) in 1X PBS pH 5.0 for 30 minutes at 37° C. Following hyaluronidase treatment, tissue sections were wash 2 times in 1X PBS before blocking tissues in 5% bovine serum albumin (Fisher, IL, USA) in 1X PBS for 30 minutes. Blocking solution was replaced with rabbit anti-human/rat/mouse Ki-67 (clone D3B5, Alexa Fluor® 647 Conjugate (Cell Signaling, MA, USA)) to detect proliferative cells, or rabbit anti-RFP (Abcam, ab62341, MA, USA) or rabbit anti-human/rat/mouse collagen 2 (Abcam, ab34712, MA, USA) followed by goat anti-rabbit Alexa Fluor 647 (Southern Biotech, AL, USA) to detect PRX1-, COL2a1-, and COL10a1-tdTomato positive cells or collagen 2 within fracture tissue, respectively. Tissue slides were washed 2 times with 1x PBS and ProLong™ Gold Antifade Mountant with DAPI (ThermoFisher, IL, USA) applied and cover-slipped. Mounted slides were cured overnight in the dark at room temperature. Tissue slides were panel-imaged on a DeltaVision widefield fluorescence microscope (Applied Precision, GE Healthcare) equipped with a digital camera (CoolSNAP HQ; Photometrics) and a 0.45-numerical aperture 20× objective lens. Panels were stitched together using SoftWoRx software v.7.0.0 (Applied Precision, GE Healthcare). As we have previously demonstrated that episodic alcohol exposure in fracture injured mice affects their total fracture callus area measurements (Roper et al., 2016), quantification of fluorescent tdTomato-positive cells in fracture callus sections was normalized to total cell density using of the callus using DAPI-stained cells in each callus section. TUNEL staining was performed utilizing an HRP-DAB TUNEL Assay Kit (Abcam, MA, USA) according to manufacturer instructions. For hematoxylin and eosin (H&E) staining, tissue slides were stained with 0.1% Mayer’s hematoxylin (Sigma, GA, USA) for 10 minutes, blued in tap water for 5 minutes, and dipped into 0.5% eosin Y (Sigma, GA, USA) in 95% EtOH solution before progressively dehydrating tissue slides in increasing EtOH/tap water solutions. TUNEL and H&E slides were cleared in xylene before applying Permount (Fisher Scientific, IL, USA) and cover-slipped. TUNEL and H&E slides were then scanned and digitized (HistoWiz, Brooklyn, NY). Callus size and cell frequencies were measured with the investigator blinded to treatment group using ImageJ software (Public Domain, courtesy of the National Institutes of Health, Bethesda, MD).

Statistical Analysis

Normally distributed data are expressed as the mean ± standard error of the mean of four or more animals per group. Sample size was estimated from prior in vivo work from the laboratory. Statistical differences between saline and EtOH groups were calculated using Student’s T-test. A p-value below 0.05 was considered statistically significant. Data were analyzed using GraphPad Prism 7.

Results

General Observations

Animals returned to weight bearing on all four limbs immediately following recovery from anesthesia. No differences in animal weights were observed during the experimental period between any of the treatment groups. No evidence of infection was noted at the fracture site in any of the experimental animals. No animals were euthanized during the experimental period for complications resulting from fracture surgery or treatments and no animals were excluded from the experiment or resulting data analysis. Fracture callus analysis of both female and male mice revealed no sex-related differences in fracture callus area or effects of alcohol on Col2a1 or Col10a1 expression in reporter animals. Thus, animals of both sexes were combined in each treatment group for further analysis.

Effects of Alcohol Administration on Early Fracture Callus Formation

Previous rodent studies have demonstrated that MSC collect at the injury site and begin to form cartilaginous callus tissue as early as 3 days post-fracture (Einhorn, 1998). To investigate the effect of alcohol on early fracture callus formation, we harvested injured tibias at 4 days post-fracture. Figure 1a shows representative images of H&E histological stained sections of early fracture callus from saline- and alcohol-administered rodents 4 days post-fracture. Alcohol-administered rodents had less fracture callus area when compared to saline-administered rodents (Figure 1b, p=0.01). Histological examination of fracture callus from alcohol-administered rodents shows densely packed cells and/or more immature cells when compared to saline-administered rodents (Figure 1a, green arrows). Hematoxylin positive cell quantification revealed similar number of total cells within the fracture callus tissue (Figure 1c). To determine whether alcohol-related decreases in callus size were driven by alterations in fracture cell proliferation, Ki-67 was detected by immunofluorescence and quantified. We observed similar numbers of Ki-67+ cells between the saline- and alcohol-administered rodents (Figure 1d–e).

Figure 1. Effects of alcohol on fracture callus area at day 4 post-fracture.

Figure 1.

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(a) Representative hematoxylin and eosin stained histological samples from each treatment group 4 days after fracture at 20X magnification. Solid and dotted black outlines indicate fracture callus. Red dotted line and red asterisks indicates fracture site. Green arrows indicate dense, immature cells. (b) Quantification of the total area of the early fracture callus 4 days post-fracture. (c) Quantification of hematoxylin positive cells in the early fracture callus 4 days post-fracture. (d) Representative Ki-67 fluorescent stained samples from each treatment group 4 days after fracture at 20X magnification. Solid and dotted white outlines indicate fracture callus. Green dotted line indicates fracture site. (e) Quantification of Ki-67 positive (Ki-67+) DAPI positive (DAPI+) cells in the early fracture callus 4 days post-fracture. (f) Representative terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) stained histology samples from each treatment group 4 days after fracture at 20X magnification. Black dotted outlines indicate fracture callus. Red dotted line and red asterisk indicates fracture site. Black arrows indicated TUNEL positive (TUNEL+) cells. (g) Quantification of the TUNEL+ cells in the early fracture callus 4 days post-fracture. n=7–9 per group. Data displayed as mean ± standard error of the mean, two-tailed Student’s T-test.

Figure 1b: Saline: N=7 EtOH: N=9: Figure 1c: Saline: N=8 EtOH: N=8: Figure 1e:

Saline N=7 EtOH: N=9: Figure 1g: Saline: N=7 EtOH: N=8

To determine whether alcohol-related decreases in callus size were driven by alterations in fracture callus cell apoptosis, TUNEL staining was performed and apoptotic cells were quantified. Though there were some apoptotic cells in both groups, there were no significant differences between the groups. (Figure 1f–g).

Effects of Alcohol Administration on PRX1 Cell Frequency in Early Fracture Callus Stages

To test whether alcohol-administration affected MSC density in early fracture callus formation, we utilized Prx1-CreERT2;Ai9 reporter mice to quantify the frequency of PRX1-tdTomato+ cells in early (4 days post-fracture) and later (6 days post-fracture) fracture callus development. We found no significant differences in the frequency of PRX1-tdTomato+ cells between the saline- and alcohol-administered rodents at either post-fracture timepoints (Figure 2a–b and c-d, respectively).

Figure 2. Effects of alcohol on PRX1-tdTomato+ cells within the fracture callus 4- and 6-days post-fracture.

Figure 2.

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(a) Representative PRX1-tdTomato+ fluorescent stained samples from each treatment group 4 days after fracture at 20X magnification. Solid and dotted white outlines indicate fracture callus. White dotted line indicates fracture site. (b) Quantification of the PRX1-tdTomato+ DAPI positive (DAPI+) cells in the fracture callus 4 days post-fracture. (c) Representative PRX1-tdTomato+ fluorescent stained samples from each treatment group 4 days after fracture at 20X magnification. Solid and dotted white outlines indicate fracture callus. Green dotted line indicates fracture site. (d) Quantification of the PRX1-tdTomato+ DAPI positive (DAPI+) cells in the fracture callus 6 days post-fracture. Data displayed as mean ± standard error of the mean, two-tailed Student’s T-test. Figure 2b: Saline: N=7 EtOH: N=9: Figure 2d: Saline: N=6 EtOH: N=5

Effects of Alcohol Administration on Fracture Callus Formation and COL2a1- Cell Frequency at 6 Days Post-Fracture

Next, we examined the effect of alcohol-administration on fracture callus formation at 6-days post-fracture. Figure 3a shows representative H&E histological stains from saline- and alcohol-administered rodents 6 days post-fracture. Consistent with previous findings from our laboratory, we found that alcohol-administration significantly reduced total fracture callus area when compared to callus area from saline-administered rodents (Figure 3b, p=0.046). As observed in 4 days post-fracture fracture callus tissue, fracture callus tissue from alcohol-administered rodent appears denser with less extracellular matrix deposition at 6 days post-fracture when compared to saline-administered rodents (Figure 3a, green arrows). No significant differences were observed in proliferative and apoptotic cell frequencies between saline- and alcohol-administered rodents at the 6 days post-fracture timepoint (Supplemental Figure 1a–b and c–d, respectively).

Figure 3. Effects of alcohol on callus area and callus COL2a1-tdTomato+ cells at day 6 post-fracture.

Figure 3.

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(a) Representative hematoxylin and eosin stained histological samples from each treatment group 6 days after fracture at 20X magnification. Solid and dotted black outlines indicate fracture callus. Red dotted line and red asterisks indicates fracture site. Green arrows indicate dense, immature cells. (b) Quantification of the total area of the early fracture callus 6 days post-fracture. (c) Representative COL2a1-tdTomato+ fluorescent stained samples from each treatment group 6 days after fracture at 20X magnification. Solid and dotted white outlines indicate fracture callus. Green dotted line indicates fracture site. (d) Quantification of the COL2a1-tdTomato+ DAPI positive (DAPI+) cells in the fracture callus 6 days post-fracture. n=5–8 per group, Data displayed as mean ± standard error of the mean, two-tailed Student’s T-test. Figure 3b: Saline: N= 5 EtOH: N=6: Figure 3d: Saline: N= 8 EtOH: N=8

Collagen 2a1 is an early chondrocyte cell marker and previous studies have determined that COL2a1 mRNA is detectable at a mouse bone fracture site as early as 5 days post-fracture (Einhorn, 1998). We next examined the effects of alcohol-administration on COL2a1 expression in the fracture callus 6 days post-fracture as early MSC osteochondral commitment is marked by COL2a1 expression. Utilizing Col2a1-CreERT2;Ai9 reporter mice, we observed a significant decrease in COL2a1-tdTomato+ cell frequency within the fracture callus of alcohol-administered rodents when compared to saline-administered rodents (Figure 3c–d, p=0.0245).

Effects of Alcohol Administration on Fracture Callus Formation, Cell Proliferation and COL2a1- Cell Frequency at 9 Days Post-Fracture

Previous findings from our laboratory and others showed peak cartilaginous callus development in a mouse tibia fracture to be at 9 days post-fracture with histological evidence of numerous hypertrophic chondrocytes (Roper et al., 2016; Einhorn, 1998). Figure 4a shows representative H&E histological images of fracture callus tissue 9 days post-fracture. Fracture callus area quantification revealed alcohol-administered rodents had significantly less fracture callus area when compared to saline-administered rodents (Figure 4b, p=0.0463) with no differences in apoptotic cell frequencies (Supplemental Figure 2). However, Ki-67+ cell frequencies were significantly reduced in Alcohol-administered rodents when compared to saline-administered rodents (Figure 4c–d, p=0.027). Utilizing Col2a1-CreERT2;Ai9 reporter mice, we observed significantly fewer COL2a1-tdTomato+ cells within the fracture callus of alcohol-administered rodents when compared to saline-administered rodents (Figure 5a–b, p=0.0389).

Figure 4. Effect of alcohol on callus area and callus proliferative cells at day 9 post-fracture.

Figure 4.

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(a) Representative hematoxylin and eosin stained histological samples of Col2-creERT2/Ai9 mice, from each treatment group 9 days after fracture at 20X magnification. Solid and dotted black outlines indicate fracture callus. Red dotted line and red asterisks indicates fracture site. (b) Quantification of the total area of the early fracture callus 9 days post-fracture. (c) Representative Ki-67 fluorescent stained samples from each treatment group 9 days after fracture at 20X magnification. Solid and dotted white outlines indicate fracture callus. Green dotted line indicates fracture site. (d) Quantification of Ki-67 positive (Ki-67+) DAPI positive (DAPI+) cells in the early fracture callus 9 days post-fracture. Data displayed as mean ± standard error of the mean, two-tailed Student’s T-test. Figure 4b: Saline: N=11 EtOH: N=11: Figure 4d: Saline: N=4 EtOH: N=5

Figure 5. Effects of alcohol on COL2a1-tdTomato+ cells and COL10a1-tdTomato+ cells within the fracture callus 9 days post-fracture.

Figure 5.

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(a) Representative COL2a1-tdTomato+ fluorescent stained samples from each treatment group 9 days after fracture at 20X magnification. Solid and dotted white outlines indicate fracture callus. Green dotted line indicates fracture site. (b) Quantification of the COL2a1-tdTomato+ DAPI positive (DAPI+) cells in the fracture callus 9 days post-fracture. (c) Representative COL10a1-tdTomato+ fluorescent stained samples from each treatment group 9 days after fracture at 20X magnification. Solid and dotted white outlines indicate fracture callus. Green dotted line indicates fracture site. (d) Quantification of the COL10a1-tdTomato+ DAPI positive (DAPI+) cells in the fracture callus 9 days post-fracture. per group, Data displayed as mean ± standard error of the mean, two-tailed Student’s T-test. Figure 5b: Saline: N=11 EtOH: N=11: Figure 5d: Saline: N=4 EtOH: N=5

Effects of Alcohol Administration on COL10a1-tdTomato+ Cell Frequency at 9 days Post-Fracture

To determine the frequency of cells in the fracture callus which reached a hypertrophic chondrocyte stage, we utilized Col10a1-Cre;Ai9 reporter mice as collagen 10 is only expressed by hypertrophic chondrocytes, which are precursors for endochondral bone formation. We found that rodents administered alcohol had significantly fewer COL10a1-tdTomato+ cells in the fracture callus when compared to saline-administered rodents (Figure 5c–d, p=0.0077).

Discussion

Tibia fractures have one of the highest rates of fracture nonunion of all skeletal sites (Mills et al., 2017; Tzioupis and Giannoudis, 2007; Zura et al.,2016) with two case studies finding tibia nonunion rates as high as 23% (Clifford et al., 1987; Antonova et al.,2013). Alcohol administered, either chronically or episodically in rodents, has been shown to negatively affect the mineral density, size, and biomechanical properties of the fracture callus (Elmali et al., 2002; Janicke-Lorenz and Lorenz, 1984; Volkmer et al., 2011; Lauing et al., 2008; Chakkalakal et al., 2002, 2005). Previous studies focused primarily on the effects of chronic alcohol administration at later stages of fracture callus development (Elmali et al., 2002; Nyquist et al., 1999; Perrien et al., 2002, 2004; Chakkalakal et al., 2002, 2005; Perry et al., 1998; Janicke-Lorenz and Lorenz, 1984). Our objective was to characterize the effects of acute, episodic alcohol exposure on the early stages of mouse tibia fracture callus development to test a hypothesis of whether alcohol’s deleterious effects on tibia fracture callus formation were related to a perturbation of MSC osteochondral differentiation in the developing fracture callus.

Previous rodent data from our laboratory has shown that episodic alcohol exposure prior to and following a fracture impairs tibia fracture callus formation (Lauing et al., 2012; Roper et al., 2016). Examination of H&E-stained histological sections showed a reduced cartilaginous callus area and effects on chondrocyte maturation (Roper et al., 2016; Natoli et al., 2018; Lauing et al., 2014). While similar numbers of total cells were observed in callus from control and alcohol-treated animals, less Col2a1 and Col10a1-expressing positive cells were observed in the callus. Because fracture callus area is also affected by alcohol exposure, quantification of Col2a1 and Col10a1-driven tdTomato expression was normalized to total cell number (total DAPI+ cells). Because less cartilaginous cells are present in the callus, the reduction in callus area seen in alcohol-treated rodents was likely related to a decreased amount of callus-associated cartilaginous extracellular matrix. Effects of alcohol on extracellular matrix-related gene expression have been previously observed in intact rodent bone (Callaci et al., 2009; Alund et al., 2017) suggesting that an alcohol-related perturbation of bone extracellular matrix protein expression may occur in both intact and injured bone tissue. As endochondral bone formation relies on a pre-existing cartilaginous matrix, reductions in callus cartilage matrix material could negatively affect bone deposition at the fracture site, leading to fracture malunion or nonunion. Interestingly, alcohol consumption has also been associated with radiological evidence of knee osteoarthritis (Kang et al., 2020), which may suggest that alcohol also affects the matrix integrity of pre-existing cartilaginous tissue.

Prior examinations of the periosteal callus; nascent intramembranous bone secreted by periosteal osteoblasts on the periphery of the external cartilaginous callus, suggest that this callus compartment may be less affected by alcohol exposure (Roper et al., 2016; Volkmer et al., 2011). These findings suggested the effect of alcohol on fracture callus formation occurs in the external cartilaginous callus compartment formed primarily by periosteal-derived MSC differentiation into chondrocytes.

In the present study, we examined whether alcohol attenuated MSC chondrogenic lineage differentiation within the fracture callus as a cellular mechanism for alcohol-related impairment of callus formation and ultimately for fracture nonunion. As in past studies from our laboratory (Lauing et al., 2012; Roper et al., 2016), we found that alcohol significantly decreased fracture callus area at days 4, 6 and 9 post-fracture. We found that the fracture callus from alcohol-administered rodents had reduced frequencies of early (COL2a1) and late (COL10a1) chondrogenic marker positive cells when compared to saline-treated control animals. The frequency of COL2a1, an early marker of osteochondral lineage commitment and chondrogenic commitment, was significantly reduced in the fracture callus of alcohol administered rodents at day 6 and day 9 post-fracture, while COL10a1, a marker of hypertrophic chondrocytes and late stage chondrogenesis, was significantly reduced with alcohol administration in the fracture callus of rodents at day 9 post fracture. Alcohol administration did not affect the number of PRX1-tdTomato+ MSC progenitor cells in the fracture callus at either day 4 or day 6 post-fracture. This finding suggests that alcohol does not negatively impact stem cell density following fracture injury, suggesting that the reduced levels of COL2a1 and COL10a1-expressing cells in the callus results from attenuated MSC differentiation and not simply decreased numbers of MSC present at the injury site. As to the question of which stage of chondrocyte development is being affected by alcohol, our data suggests that alcohol may primarily block the early transition of MSC to chondro-osteo progenitor cells, as suggested by the significant decrease in Col2a1 positive cells in the early fracture callus from alcohol-treated mice. There may be additional effects of alcohol on chondrocyte maturation, as Col10a1+ cells also decrease in callus from alcohol-treated mice, but this effect may be primarily the result of an alcohol-related decrease in the number of early chondrocyte cells that are available to mature to hypertrophic cells.

To determine whether alcohol administration affected the frequency of apoptosis within the fracture callus, we quantified the number of TUNEL positive cells within the fracture callus at all three examined timepoints. Alcohol did not significantly alter apoptosis at any of the examined timepoints, suggesting the effect of alcohol on fracture callus size is not due to increased or decreased levels of cell death. To answer the question of whether alcohol administration could alter cell proliferation within the fracture callus, we next visualized Ki-67 by immunofluorescent staining at all three examined timepoints. While previous studies have shown alcohol alters the proliferation of in vitro cultured MSCs and osteoblasts (Chavassieux et al., 1993; Chen et al., 2009; Chen et al.,2017; Dyer et al., 1998; Huff et al., 2011; Klein et al., 1996) we did not observe alterations in Ki-67 frequency within the fracture callus at days 4 and 6 post-fracture. However, we did observe a significant decrease in Ki-67+ cells at day 9 post-fracture in rodents administered alcohol when compared to control animals. In rodent models, peak soft and peak hard fracture callus development is reached approximately 9 and 14-days post-fracture, respectively (Einhorn, 1998). While it is currently unclear which callus-associated cell type is showing evidence of alcohol-compromised proliferation, the alcohol-related decrease in cell proliferation observed at day 9 post fracture could reflect an effect on a remnant callus-associated MSC population undergoing self-renewal. Effects of alcohol on MSC proliferative activity have been previously described in cultured rodent MSC (Huff et al., 2011), it is not clear why we did not observe this effect of alcohol at earlier time points and what the actual significance of this finding is with respect to alcohol inhibition of callus formation. Given the timeline of cellular activity during rodent fracture callus formation (Einhorn, 1998), we would not expect an alcohol effect on MSC proliferative activity at post-injury day 9 to contribute significantly to defects in callus formation. Overall, this data continues to support our supposition that the effects of alcohol on cartilaginous callus formation appear to be caused primarily by an attenuation of MSC-chondrogenic differentiation.

Post-fracture Day 9 is considered to be the peak of cartilaginous callus development during fracture healing in rodents (Einhorn, 1998). The presence of COL10a1-tdTomato expressing cells in our Col10a1-Cre;Ai9 reporter mice, suggests chondrocyte maturity has been reached. In a normal healing fracture callus, the next stage is the conversion of the soft fracture callus to a hard callus. This step is marked by the mineralization and ossification of the cartilaginous compartment. When analyzing double-stained immunofluorescent tissue sections for COL10a1-tdTomato+ and Ki-67+ cells, it was revealed that the COL10a1-tdTomato+ areas were Ki-67 negative (data not shown). These findings may suggest the Ki-67+ cells are possibly proliferating MSCs, immature chondrocytes, or osteoblasts. Therefore, we hypothesize that the increased proliferation observed at day 9 post-fracture may signal proliferating MSCs or nascent osteoblasts necessary for the formation of the hard callus. Further studies are necessary to determine which cell type(s) proliferating at this stage of fracture callus development were significantly decreased with alcohol administration.

Previous studies from our laboratory showed that alcohol administration deregulates bone fracture associated Wnt/β-catenin signaling (Lauing et al., 2012, 2014) which is indispensable for fracture repair as it regulates both MSC osteochondral commitment differentiation (Hoogeboom et al., 2008; Almeida et al., 2007; Chen et al., 2008, 2010; Zhou et al., 2009; Essers et al., 2005; Iyer et al., 2013) and hypertrophic chondrocyte maturation (Guo et al., 2009).

Cellular FoxO1/3 activity, which sequesters and redirects β-catenin during cellular stress is also modulated by alcohol in fracture callus and cultured primary MSC (Roper et al., 2016; Sharieh et al., 2020), Thus alcohol-related perturbation of Wnt/FoxO signaling are likely at least partially responsible for the defects in chondrocyte cell differentiation observed in this study.

Our study did have some limitations that should be noted. The age of animals used in our study (8–9 weeks at study initiation) is somewhat younger that rodents used in other fracture studies (Chen et al., 2007), though the age of animals used herein is consistent with other studies performed in our laboratory (Roper et al., 2016; Lauing et al., 2012, 2014) and is consistent with the age of animals used in the tibia fracture paradigm that our fracture model is based on (Hiltunen et al., 1993) Our chosen analysis time points did not include those closer to the injury day, specifically days 1–3 following fracture injury. There is a possibility that alcohol administration may be affecting stem cell recruitment or proliferation in the immediate days following fracture injury. Alcohol has documented effects on early fracture site-related inflammation suggesting that the early fracture site microenvironment is disrupted by alcohol exposure (Chakkalakal et al., 2005; Sampson et al., 2011). Transgenic models were evaluated at specific time points and not over the entire range of the experiment. While the design utilized does limit direct comparisons over time of healing within a genotype, the time points analyzed for evaluation of the effects of alcohol on PRX1, Col2a1 and Col10a1 expression in fracture callus were chosen based on the literature (Einhorn, 1998) and our own experience in histological evaluation of fracture callus formation in mice, which allowed us to examine the effects of alcohol on early and later MSC-chondrogenic differentiation during fracture repair. Fracture callus area was quantified using H & E-stained histological sections; future studies should include sections stained using a cartilage-specific stain such as safranin-O (Kambrath et al., 2020) to confirm the cartilaginous area of the callus.

One question which was not addressed in the study is whether alcohol administration has effects on fracture callus-related cellular senescence. Past studies showed that alcohol-induced cellular senescence in MSCs and osteoblasts cultured ex vivo (Chavassieux et al., 1993; Chen et al., 2009; Chen et al.,2017; Dyer et al., 1998; Huff et al., 2011; Klein et al., 1996), and at least in the case of osteoblasts, inhibited normal osteoblast function. One commonly used biomarker of cellular senescence is β-galactosidase activity; however, tissue fixation has been shown to inactivate senescence-associated β-galactosidase (Ma et al., 2002). Future studies in unfixed fracture callus tissue will be required to determine the effects of alcohol on cellular senescence in the developing fracture callus.

In summary, we demonstrate here that acute, episodic alcohol exposure in mice with a tibia fracture has profound effects on the developing fracture callus, including reductions in callus area and decreases in Collagen 2 and Collagen 10 expressing cells, while overall callus number, cell proliferation and apoptosis are unaffected by alcohol. This data suggests that alcohol-related deficient fracture healing may be driven in part by an underlying perturbation of MSC differentiation towards a chondrogenic lineage. Future work will examine if previously identified alcohol-related deregulation of MSC-specific cell signaling underlies this cellular defect and if this work translates to human fracture repair complicated by alcohol abuse.

Supplementary Material

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Acknowledgements:

We thank Dr. Matthew Hilton at Duke University School of Medicine for generously gifting the Col2a1-CreERT2 and Col10a1-Cre mouse strains. Research reported in this publication was supported by the National Institute on Alcohol Abuse and Alcoholism of the National Institutes of Health under award number R21AA025551 to JJC, F31AA028147 and T32AA013257 to JME, and the National Institute of General Medical Sciences under award number T32GM008750 to FS.

Footnotes

Conflict of Interest: The authors report no conflict of interest.

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