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Simulated microgravity alters the expression of key genes involved in fracture healing
Acta Astronautica 92 (2013) 65–72
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Simulated microgravity alters the expression of key genes involved in fracture healing N. Patrick McCabe a, Caroline Androjna a, Esther Hill b, Ruth K. Globus c, Ronald J. Midura a,n a b c
Department of Biomedical Engineering, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA Lockheed Martin Exploration & Science, NASA Ames Research Center, Moffett Field, CA 94035, USA Biosciences Division, NASA Ames Research Center, Moffett Field, CA 94035, USA
a r t i c l e i n f o
abstract
Article history: Received 22 November 2011 Received in revised form 4 April 2012 Accepted 9 April 2012 Available online 26 April 2012
Fracture healing in animal models has been shown to be altered in both ground based analogs of spaceflight and in those exposed to actual spaceflight. The molecular mechanisms behind altered fracture healing as a result of chronic exposure to microgravity remain to be elucidated. This study investigates temporal gene expression of multiple factors involved in secondary fracture healing, specifically those integral to the development of a soft tissue callus and the transition to that of hard tissue. Skeletally mature female rats were subjected to a 4 week period of simulated microgravity and then underwent a closed femoral fracture procedure. Thereafter, they were reintroduced to the microgravity and allowed to heal for a 1 or 2 week period. A synchronous group of weight bearing rats was used as a normal fracture healing control. Utilizing Real-Time quantitative PCR on mRNA from fracture callus tissue, we found significant reductions in the levels of transcripts associated with angiogenesis, chondrogenesis, and osteogenesis. These data suggest an altered fracture healing process in a simulated microgravity environment, and these alterations begin early in the healing process. These findings may provide mechanistic insight towards developing countermeasure protocols to mitigate these adaptations. & 2012 IAA. Published by Elsevier Ltd. All rights reserved.
Keywords: Simulated microgravity Fracture healing Gene expression Angiogenesis Chondrogenesis Osteogenesis
1. Introduction Astronauts on spaceflight missions are faced with progressive loss of bone mass and mineral density mainly due to the reduced mechanical forces that are necessary for normal bone maintenance [1]. It has been suggested that
Abbreviations: PCR, polymerase chain reaction; VEGF, vascular endothelial growth factor; FGF2, basic fibroblast growth factor; IGF-1, insulin like growth factor-1; PDGF, platelet derived growth factor; TGF-b, transforming growth factor-b; BMPs, bone morphogenetic proteins; HLU, hind limb unloaded; WB, weight bearing; micro-CT, micro-computed tomography; MSCs, mesenchymal stem cells; ROI, region of interest n Correspondence to: Department of Biomedical Engineeering/ND20, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195, USA. Tel.: þ1 216 445 3212 ; fax: þ 1 216 444 9198. E-mail address: [email protected] (R.J. Midura).
microgravity may have a negative impact on the conversion of cartilage into cancellous bone via endochondral ossification due to alterations in the extracellular matrix composition [2]. This impaired state of bone quality can increase the risk that a trauma-induced long bone fracture might occur while in space or after return to normal gravity. In addition to an increased fracture risk, healing after fracture is impaired in both spaceflight and earth based microgravity analog animal fracture models [3]. The purpose of this study was to determine whether simulated microgravity affects ultimate fracture healing by altering gene expression of multiple factors within the first 2 weeks of bone fracture healing that is necessary for proper healing. Fracture healing is a complex process consisting of several temporally overlapping, and histologically discernable phases involving proliferation and differentiation of cells and tissues. Upon fracture, a hematoma forms at the fracture site resulting in a large clot that hinders further accumulation of blood
0094-5765/$ - see front matter & 2012 IAA. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.actaastro.2012.04.016
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at the fracture site [4]. As the hematoma becomes hypoxic, a new vasculature is recruited to facilitate nutrient delivery and waste disposal. Damaged tissue and cellular debris are then removed through the efforts of inflammatory cells that have migrated into the wound area [5]. Next, a cytokine cascade leads to the influx of reparative mesenchymal stem cells (MSCs) that differentiate into cell types that produce new tissues. First, chondrocytes deposit a new cartilage to form a firm tissue callus that bridges the disjoined bone ends and provides initial fracture site stability. The chondrocytes mature, undergo hypertrophy and the cartilaginous tissue is replaced with new woven bone through the efforts of bone depositing osteoblasts [5]. This woven bone is subsequently remodeled into a more compact bone tissue through the combined efforts of osteoclasts (bone resorbing cells) and osteoblasts [6]. While the stages of fracture healing are well characterized for bones subjected to constant gravitational and requisite ground reaction forces, alterations that occur as a result of exposure to simulated microgravity and loss of ground reaction forces are incompletely characterized. Each of these phases of bone fracture healing is driven by augmented expression of specific genes that are transcribed at basal maintenance levels in an uncompromised mature skeleton. Neovascularization occurs, at least in part, as a result of the combined efforts of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF2) [7]. An initial wave of MSCs is recruited to the fracture site and undergoes differentiation into chondrocytes. The mRNA of protein products aggrecan and Type II collagen peak within the first 2 weeks following rat bone fracture [8] in order to facilitate the matrix deposition and the formation of a firmer callus tissue. A second wave of MSCs is recruited to the fracture site [9] and undergoes differentiation into bone-forming osteoblasts. This occurs via the actions of osteoinductive molecules such as insulin like growth factor-1 (IGF-1), platelet derived growth factor (PDGF), transforming growth factorb (TGF-b) and the bone morphogenetic proteins (BMPs) [10]. Osteoblast maturation can be tracked by monitoring levels of osteogenic markers such as alkaline phosphatase, bone sialoprotein, and osteocalcin [11]. Using a NASA approved hind limb unloading (HLU) model to simulate exposure to microgravity, we found that the femora of rats subjected to microgravity exhibit reduced expression of key factors involved in the early reparative stages of fracture healing compared to full weight bearing (WB) counterparts. 2. Materials and methods 2.1. Animals Seven to eight months old, skeletally mature female Sprague–Dawley rats (Harlan Laboratories, Inc., Indianapolis, IN) were utilized in this study. Food and water were available ad libitum. Animals were group housed until time of tail suspension where all animals, regardless of study group, were singly housed until study termination. The Institutional Care and Use Committees of the Cleveland Clinic and the Biosciences Division, NASA Ames Research Center approved all procedures.
2.2. Ground based analog of microgravity The hind limb unloaded rat model developed by Morey-Holten et al. [12] is the NASA accepted ground based analog of spaceflight as it leads to physiological adaptations similar to those experienced in true spaceflight. Unloading in rats leads to a non-weight bearing condition in the hind limbs and a cephalic shift in body fluids [3]. Rats were randomly assigned to be either hind limb unloaded (HLU) or full weight bearing (WB) controls. Body weights (BW) were monitored on a weekly basis throughout the study period. 2.3. Fracture model Following a 4 week period of unloading or normal cage activity, rats were subjected to femoral fracture via a modified Bonnarens–Einhorn femoral fracture model [13]. In brief, a threaded titanium pin (Grade 2, diameter 1.30 mm, McMaster-Carr, Aurora, OH) was inserted into the medullary cavity of the femur in a retrograde fashion starting from the femoral condylar notch, seating the pin in the cancellous bone region of the greater trochanter. This pin serves as an intramedullary support to stabilize the fracture and provide a load bearing support for the injured limb. Following pin insertion and surgical closure, the stabilized femur is subjected to a 3-point bending moment in a closed configuration to generate a mid-shaft fracture of the femur. Rats underwent bilateral, middiaphyseal femoral fractures. Fractures were visualized by planar X-ray or micro-computed tomographic (microCT) imaging (Fig. 1). Rats were acclimated to Tylenol analgesia (3 mg/mL in drinking water) for a period of 3 days prior to surgery and remained on Tylenol analgesia for a period of 7 days post-fracture. Buprenorphine (2.5 mg/kg BW) was administered immediately prior to surgery and for 2 successive doses at 12 h intervals following fracture for all animals. Bupivicaine (0.5% solution) was given at the fracture site immediately postfracture as a local anesthetic. All rats remained unsuspended for a period of 24 h following fracture to avoid any potential anesthesia and analgesia related complications due to simultaneous head down tilt. Rats were then subjected to a further 1 or 2 week state of HLU or WB depending upon prior group designation. Animals/fractures were excluded from analysis if they met one of the following exclusionary criteria: (1) failure to detect a fracture following a single 3-point bending displacement; (2) fractures occurring outside the mid-diaphyseal region and too close to the proximal or distal metaphyses; (3) fracture patterns that were spiral extending beyond the 1.5 cm region of interest; (4) body weight losses greater than 20% of initial values; and (5) anesthesia complications leading to premature death. 2.4. RNA extraction and Real-Time PCR Following euthanasia by CO2 inhalation, rat hind limbs were removed and muscle stripped with care taken not to disturb the fracture callus tissue. Whole femurs were snap frozen in liquid nitrogen and stored at 80 1C until use.
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Total RNA from 1- and 2-week fracture callus tissue (1.5 cm ROI) was isolated by tissue pulverization in a FastPrep-24 device (MP Biomedicals, Solon, OH) in the presence of Trizol (Invitrogen, Carlsbad, CA) and cleaned up by passing through RNeasy columns (Qiagen Inc, Valencia, CA). Reverse transcription reactions were performed with
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1 mcg of total RNA using the Masterscript RT cDNA synthesis kit (5Prime, Inc., Gaithersburg, MA) followed by PCR in an ABI 7500 Real-Time PCR instrument (Invitrogen) with the primers listed in Table 1. PCR was carried out at 50 1C for 2 min, 95 1C for 10 min; 40–45 cycles of 95 1C for 15 s and 60 1C for 1 min. Expression levels were determined using SYBR Green chemistry (Qiagen) and the DDCT relative quantification method described by Livak and Schmittgen [14]. For each gene, relative expression was calculated following normalization to ribosomal 18S mRNA (r18S). Each sample was quantified from the average of triplicate repeats. Dissociation curves indicated amplification specificity by illustrating single reaction products. To verify amplicon identity, an amplicon from each gene was cloned into pGEM-T easy (Promega, Madison, WI) and sequenced. All amplicons aligned 100% with the expected mRNA reference sequences. 2.5. Micro-CT image acquisition and analysis Micro-CT imaging and analyses were performed on bone specimens that were processed for histological evaluation. Ex-vivo micro-CT images were acquired at an isotropic voxel resolution of 45 mm (GE Explore Locus, GE Healthcare). The volume of mineralizing hard callus was determined from the generated 3-D (16-bit) image volumes, for a 1 cm ROI, utilizing custom designed software (Image IQ, Inc., Cleveland, OH). Specifically, original cortical bone was manually masked from each volume and grayscale segmentation values of 600–6000 for hard callus and 46000 for the titanium rod were applied for the volumetric analysis. 2.6. Histology
Fig. 1. Depiction of the ex-vivo micro-CT imaging and analyses of the rat femoral fractures, over the 14 day healing period. (A, left panel) The box around a femur, one day post surgery, denotes the 1.5 cm region of interest (ROI) surrounding the fracture site that was utilized in all analyses. (A, right panels) Representative magnified views of the femoral ROI surrounding the fracture site, at designated time points, over the 14 day healing period. (B) Representative segmented volumes for WB and HLU groups that were computer generated based on cortical mask and threshold values applied for implant volume, cortical bone and mineralizing bone; yellow depicts the titanium rod, blue depicts the original cortical bone and red depicts the volume of mineralizing callus formed. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Histological evaluation was performed on sagittal sections from 10–13 fractures per group at each time point, representing a range of fracture types including diaphyseal wedge, simple and complex fractures. Fractured femora were fixed in 10% neutral buffered formalin and decalcified in a 5% trichloroacetic acid solution at 4 1C. Tissue was embedded in paraffin and sectioned at 7 mm. Hyaline cartilage tissue areas were determined by staining with Safranin O/Fast Green followed by static histological evaluation. Measurements of Safranin O-stained cartilage
Table 1 List of primers used in Real-Time PCR gene expression analysis. Gene
Product
Refseq/rRNA
Primer sequences
Acan Alpl Bmp2 Bmp7 Ibsp Col2a1 Fgf2 Bglap
Aggrecan Alkaline phoshpatase, Liver/bone/kidney Bone morphogenetic protein 2 Bone morphogenetic protein 7 Bone sialoprotein Type II collagen Fibroblast growth factor 2 Bone gamma-carboxyglutamate (gla) protein, osteocalcin Vascular endothelial growth factor 18S ribosomal subunit
NM_022190.1 NM_013059.1 NM_017178.1 NM_001191856.1 NM_012587.2 NM_012929.1 NM_019305.2 NM_013414.1
50 -TCTATCTGCACGCCAACC-30 , 50 -ACAAAGTCTTCACCTGTGTAGC-30 50 -CAACCTGACTGACCCTTCC-30 , 50 -TAACCTCTGTGACCCTTGAC-30 50 -AGAAGCCATCGAGGAACTTT-30 , 50 -CCTGGTGTCCAATAGTCTGG-30 50 -TATGCTGCCTACTACTGTGAG-30 , 50 -GTTGATGAAGTGAACCAGTGTC-30 50 -AACGGTTTCCAGTCCAGG-30 , 50 -TTTGAAGTCTCCTCTTCCTC-30 50 -GTGATGATGGTGAAGCTGGA-30 , 50 -TAACCTCTGTGACCCTTGAC-30 50 -ACCCACACGTCAAACTACAG-30 , 50 -GTCCATCTTCCTTCATAGCCAG-30 50 -CTGACAAAGCCTTCATGTCCA-30 , 50 -GCTCCAAGTCCATTGTTGAAG-30
NM_031836 M11188.1
50 -CAGCTATTGCCGTCCAATTGA-30 , 50 -CCAGGGCTTCATCATTGCA-30 50 -CGCGGTTCATTTTGTTGGT-30 , 50 -AGTCGGCATCGTTTATGGTC-30
Vegf r18S
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areas were made using Bioquants software (Bioquant Image Analysis Corporation, Nashville, TN). 2.7. Statistical analysis Comparisons at each time point were made between the WB and HLU groups by the parametric t test analysis (GraphPad Prism v5, GraphPad Software, Inc., La Jolla, CA). Real-Time PCR statistical analysis was conducted on Log2 transformed relative expression data. Data are plotted in box and whisker format, indicative of 5–95 percentiles (whiskers), 25–75 percentiles (box), and median values (line inside each box). Differences were considered significant at p r0.05. 3. Results and discussion 3.1. Angiogenesis While it is known that both VEGF [7,15,16] and FGF2 [17,18] have multiple roles in the healing of bone fractures, their roles in the growth of new blood vessels are crucial as vasculature plays a key role in each phase of the healing process. The hematoma formed immediately after fracture rapidly becomes hypoxic which drives the expression and accumulation of inflammatory mediators and growth factors [4]. This promotes the influx of endothelial cells and the formation of the new vasculature facilitating delivery of nutrients, MSCs, and eventually the hypertrophic differentiation of chondrocytes [15]. Two genes with protein products known to be key players in promoting vascularization of fracture callus tissue, Vegf and Fgf2, were measured. VEGF mRNA levels (Fig. 2A) in HLU callus tissue 7 days post-fracture was significantly less than that of WB fracture callus tissue (po0.05). Reduced VEGF mRNA levels were also present in the callus tissue of HLU rats at 2 weeks post-fracture (po0.05). Levels of FGF2 mRNA (Fig. 2B) were similarly reduced in HLU rat callus tissue at both 7 and 14 days post-fracture (each, po0.05). Endothelial cells exposed to the simulated microgravity in vitro exhibited reductions in responsiveness to VEGF and FGF2 treatments [19], introducing the possibility that a similar phenomenon might occur in vivo
leading to potential neovascularization deficiencies when compared to what is Earth normal. As the recruitment of vasculature is necessary for proper fracture healing [20], the chondrogenic and osteogenic phases of fracture healing also may be adversely affected. 3.2. Chondrogenesis Initial fracture site stabilization occurs through the production of cartilage matrix proteins that form a firmer callus tissue bridging the fractured bone ends. Expression levels of Acan and Col2a1 (genes encoding cartilage matrix proteins aggrecan and Type II collagen, respectively) were analyzed in the 7 and 14 days healing callus of fractured femora from WB rats and from rats exposed to 4 weeks of HLU prior to fracture as well as post-fracture. Expression of both of these genes are known to peak around 7–14 days after fracture and drops rapidly after 2 weeks in a rat diaphyseal fracture model [8]. As seen in Fig. 3A, Acan levels at both 1 and 2 weeks were similar in callus tissue from HLU and WB rat femora (p¼ 0.1591 and p¼0.1123, respectively). However, at 7 and 14 days post-fracture, Col2a1 levels were significantly less (each, po0.05) in HLU healing callus tissue compared to that of WB rats (Fig. 3B). Safranin O detection of sulfated proteoglycans revealed comparable levels of cartilage tissue proteoglycan area in the callus tissue of HLU and WB fractured femora (Fig. 4(A–C)). Given the histological appearance of hyaline cartilage tissue in these calluses (Fig. 4D), and that aggrecan comprises the bulk of proteoglycan in cartilage, these histochemistry results imply similar aggrecan distributions in HLU and WB fracture calluses. Under normal circumstances both Acan and Col2a1 expression patterns are tightly coordinated during cartilage tissue formation, yet we observed discordant expression patterns of Acan versus Col2a1 during fracture healing in HLU rats. The relevance of this observation is not known presently though disrupted patterns of Acan and Col2a1 have been reported in cartilage pathologies [21]. Interestingly, spaceflight and HLU exposure lead to declines in the steady state levels of Acan and Col2a1 [2]. In the case of traumatic injury, such as that described herein, Col2a1 levels in fracture callus are similarly reduced in HLU rats compared to WB rats while a proportional difference in Acan levels are not discernible, possibly
Fig. 2. Angiogenic factors have reduced expression levels in the callus tissue of fractured WB and HLU rat femora. Log2 transformed data are presented (whiskers: 5–95 percentile) (A) Vegf; WB: 7 days (n¼ 16), 14 days (n ¼18); HLU: 1 week (n¼ 19), 2 weeks (n¼ 20). (B) Fgf2; WB: 7 day (n¼ 15), 14 day (n¼ 16); HLU: 1 week (n ¼19), 2 weeks (n¼19). *po 0.05 versus WB at same time point.
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Fig. 3. Chondrogenic factor levels in the callus tissue of fractured WB and HLU rat femora. (A) Acan levels are similar in both WB and HLU callus tissue 7 and 14 days post-fracture (WB: 1 week (n¼ 16), 2 weeks (n¼ 18); HLU: 1 week (n¼19), 2 weeks (n¼19)). (B) Decrease in Col2a1 gene expression in HLU callus tissue 7 and 14 days post-fracture (WB: 1 week (n¼ 16), 2 weeks (n ¼18); HLU: 1 week (n ¼19), 2 weeks (n ¼19)). Log2 transformed data are presented (whiskers: 5–95 percentile). *po 0.05 versus WB at same time point.
Fig. 4. Cartilage tissue histology of callus tissue sections from fractured WB and HLU rat femora at (A) 7- and (B) 14-day post-fracture. The 0.5 mm scale black scale bar applies to panels A and B. (C) Percent cartilage area per healing callus area was measured from Safranin O stained proteoglycan deposits (arrows in panels A and B). (D) Higher magnification views of both WB and HLU callus tissues at 14 days of healing stained with Safranin O. The white scale bars in each image represents 0.1 mm.
due to the large variation in Acan expression in WB fracture callus. 3.3. Osteogenesis and related biomarkers Along with the formation of cartilage tissue occurring within the healing fracture callus, continual MSC recruitment from local or systemic sites to the fracture site leads to the differentiation of bone-forming osteoblasts. Numerous growth factors with variable osteogenic inductive capacity
can promote, either alone or in concert, the differentiation of MSCs into osteoblasts. The bone morphogenetic proteins (BMPs) are among the most potent for stimulating osteogenic differentiation and mineral deposition. Rat calvarial osteoblast cells treated with BMP2 expressed elevated levels of osteoblast early differentiation markers such as alkaline phosphatase (Alpl), intermediate differentiation markers such as bone sialoprotein (Ibsp), and late differentiation markers such as osteocalcin (Bglap) [22]. To a lesser extent, BMP7 induces an osteogenic differentiation in vitro [23] but
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appears to be a more potent stimulator of bone growth in vivo [24]. We determined the relative expression levels of Bmp2 and Bmp7 in fracture callus tissue of HLU and WB rats to gage the relative levels of osteogenic induction potential of their respective callus tissue. At 7 days, BMP2 mRNA levels (Fig. 5A) were decreased in callus tissue of HLU rats (po0.05) suggesting a reduced osteogenic induction potential of fracture site tissue as a result of exposure to simulated microgravity. At 14 days, however, BMP2 mRNA levels in HLU fracture callus tissue approached but did not achieve statistically quite lower values than that of WB rats (p¼ 0.064). Interestingly, relative levels of Bmp7 (Fig. 5B) remained lower in the callus tissue of HLU rats compared to WB rats at both 7 and 14 days (po0.05, both time points). At present the data cannot discriminate whether this continued reduction in Bmp7 expression is due to its role in cartilage formation [25] as opposed to its acting as a promoter of osteogenesis. Alternatively, BMP7 may not be as critical in fracture repair as BMP2 because conditional deletion of BMP7 allows an unhindered fracture healing [26] whereas BMP2 has been shown to be necessary [27] for the initiation of fracture healing. Together, the reductions
in BMP gene transcription in HLU fracture callus tissue relative to that of WB rats suggests a reduction in osteogenic inductive capacity of reparative tissues within limbs exposed to reduced ground reaction forces. Whether this is the result of reductions in osteoprogenitor cells within callus tissue of HLU rats as a consequence of simulated microgravity or the result of upstream delays in healing remains to be determined. Alkaline phosphatase (Alpl; early marker), bone sialoprotein (Ibsp; intermediate marker), and osteocalcin (Bglap; late marker) are temporally expressed during the differentiation of preosteoblast lineage cells into mineralizing osteoblasts [11]. Spaceflight has been shown to result in decreased steady state levels of Alpl [28] and Bglap [29]. It should be mentioned that only osteocalcin is uniquely expressed by osteoblasts; in the context of fracture healing, alkaline phosphatase is expressed by adipocytes [30] and chondrocytes [31,32] and bone sialoprotein is also expressed by hypertrophic chondrocytes [33,34]. As shown in Fig. 5C, fracture callus tissue from HLU rats contained relatively lower levels of Alpl at 7 days (po0.05) but levels at 14 days were comparable to callus tissue from WB rats (p¼0.116).
Fig. 5. Fracture callus tissue from WB and HLU rats has reduced levels of osteo-inductive factors and osteogenic biomarkers. (A) Bmp2 levels were lower at 7 days but similar to WB fracture callus tissue at 14 days. WB: 1 week (n ¼13), 2 weeks (n ¼11); HLU: 1 week (n ¼15), 2 weeks (n ¼29). (B) Bmp7 levels were lower at both 7 and 14 day after fracture in the callus tissue of HLU rats. (WB: 1 week (n¼ 15), 2 weeks (n¼16); HLU: 1 week (n¼ 18), 2 weeks (n¼ 20)). (C) Alpl levels in HLU fracture callus tissue was lower at 7 days but not at 14 days compared to WB tissue. (WB: 1 week (n ¼16), 2 weeks (n¼ 18); HLU: 1 week (n¼18), 2 weeks (n ¼20)). (D) Ibsp levels were also only lower the 7 day time point compared to WB fracture callus tissue. (WB: 1 week (n¼ 16), 2 weeks (n ¼18); HLU: 1 week (n¼ 19), 2 weeks (n¼20)). (E) Osteocalcin mRNA (Bglap) levels in HLU callus tissue was lower at 7 and 14 day compared to that of WB rats. (WB: 1 week (n¼ 16), 2 weeks (n¼18); HLU: 1 week (n¼ 19), 2 weeks (n¼ 20)). *p o0.05 versus WB at same time point.
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Fig. 6. Box and whisker (whiskers: 5–95 percentile) plot of the amount of mineralizing callus tissue at 7 and 14 days, determined in a 1 cm ROI surrounding the femoral fracture site. The differences in mineralizing tissue content were found to be statistically significant between WB and HLU groups at both time points (*p o 0.05).
This temporal pattern is comparable to those reported by Herrmann et al. [35] and Oni et al. [36] where slight lags in the levels of serum ALP were observed in patients with delayed fracture healing. Similarly, relative BSP mRNA levels were lower at 7 days (po0.05) but were not significantly different (p¼0.488) after 14 days (Fig. 5D) in the fracture callus tissue of HLU rats. These temporal differences may be explained by changes in both chondrogenesis and osteogenesis induced by HLU during these early phases of fracture healing. Alternatively, since Ibsp and Alpl expression levels act as indicators of early and intermediate stages of osteoblast differentiation, reduced expression of each of these genes may also suggest delayed osteoblast maturation in HLU fracture callus formation. In a more definitive indication that osteogenic maturation seems to be delayed, relative osteocalcin mRNA levels (Fig. 5E) were found to be reduced at both 7 and 14 days (po0.05, both time points) in the callus tissue of HLU fractures as compared to WB counterparts. In fact, spaceflight leads to an impaired osteoblast maturation even in the presence of dihydroxyvitamin D3 [1,25(OH)2D3] [37]. In the case of fracture healing, our findings are in accord with Herrmann et al. [35] and Oni et al. [36], where serum osteocalcin levels were higher in patients with normal fracture healing for a period of up to a month than that of patients with delays in fracture healing. 3.4. Hard callus tissue volumes Overall, the HLU groups exhibited a bone healing response that results in a significantly smaller sized mineralizing callus volume than that of the WB groups at both the 7-day and 14-day time points (Fig. 6). The HLU callus volumes were on average 50% smaller at 7 days (4.872.0 mm3 and 9.172.0 mm3, HLU and WB respectively) and 30% smaller at 14 days (30.4712.1 mm3 and 43.279.2 mm3, HLU and WB respectively). Statistical analyses indicate that the mineralizing tissue content is significantly different between groups, at each time point, over the 14-day period (po0.05). This is in agreement with our previous work illustrating smaller callus size and callus formation rates in HLU rats with fibular osteotomies
7 and 14 days following fracture [38], although the alterations were more severe in the fibular fracture model. These data support the observed differences in the gene expression of the angiogenic, chondrogenic and osteoinductive proteins as well as the osteogenic biomarkers that were investigated. Specifically, at both time points, the decreased expression of the chondrogenic gene, Col2a1, and the various osteogenic genes within the HLU groups correlate with decreased mineralization of the fracture callus. Therefore it is possible that both differentiation and maturation of MSCs into osteoblasts is delayed in the HLU group and/or requisite protein content required within the fracture callus site for an appropriate matrix tissue deposition to occur is deficient, thereby leading to an overall initial delay in the fracture healing process. 4. Conclusion Although histological analyses of fracture callus tissue indicates that exposure to simulated microgravity (HLU) leads to relatively normal cartilage proteoglycan deposition responses during secondary fracture healing, reductions in Col2a1 gene expression would likely lead to reductions in Type II collagen deposition in the healing callus tissue of HLU versus WB rats. Specifically, while levels of Acan appear in accord with that of Safranin O stained callus tissue, an uncoupling of Col2a1 and Acan expression at the molecular level is observed. These molecular alterations during early fracture healing stages suggest that the extracellular matrix composition of HLU callus tissue may be different than that of WB counterparts. If true, then this might contribute to a delay in the acquisition of a cartilaginous tissue connection across the fracture site. Simulated microgravity exposure results in a reduction in the expression of pro-angiogenic genes. If this decreased transcription leads to a reduction in pro-angiogenic protein levels at the fracture callus, then these alterations would likely delay a hypertrophic cartilage response and/or hinder callus ossification. Indeed, reductions in the levels of osteo-inductive and osteogenic gene
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expression characteristic of a normal secondary or endochondral healing response were detected and are consistent with this possibility. In addition, the decreased amount of mineralizing callus tissue present at the HLU fracture site supports the contention of a delayed healing response at early healing times. However, transient reductions in gene expression at early healing time points do not necessarily indicate compromised healing outcomes at later healing time points. While this study supplies data concerning alterations in fracture healing at early time points following extended periods of exposure to HLU, further investigations are necessary to determine the effects that long term exposure to microgravity has on functional fracture healing outcomes.
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[17] W. Chen, S. Jingushi, I. Aoyama, J. Anzai, G. Hirata, M. Tamura, et al., Effects of FGF-2 on metaphyseal fracture repair in rabbit tibiae, J. Bone Miner. Metab. 22 (2004) 303–309. [18] H. Tokuda, K. Hirade, X. Wang, Y. Oiso, O. Kozawa, Involvement of SAPK/JNK in basic fibroblast growth factor-induced vascular endothelial growth factor release in osteoblasts, J. Endocrinol. 177 (2003) 101–107. [19] D. Grimm, J. Bauer, C. Ulbrich, K. Westphal, M. Wehland, et al., Different responsiveness of endothelial cells to vascular endothelial growth factor and basic fibroblast and simulated microgravity, Tissue Eng. A 16 (2010) 1559–1573. [20] M.R. Hausman, M.B. Schaffler, R.J. Majeska, Prevention of fracture healing in rats by an inhibitor of angiogenesis, Bone 29 (2001) 560–564. [21] J.R. Matyas, M.E. Adams, D. Huang, L.J. Sandell, Discoordinate gene expression of aggrecan and type II collagen in experimental osteoarthritis, Arthritis Rheum. 38 (1995) 420–425. [22] D. Chen, M. Harris, G. Rossini, C.R. Dunstan, S.L. Dallas, J.Q. Feng, et al., Bone morphogenetic protein 2 (BMP-2) enhances BMP-3, BMP-4, and bone cell differentiation marker gene expression during the induction of mineralized bone matrix formation in cultures of fetal rat calvarial osteoblasts, Calcif. Tissue Int. 60 (1997) 283–290. [23] T.K. Sampath, J.C. Maliakal, P.V. Hauschka, W.K. Jones, H. Sasak, R.F. Tucker, et al., Recombinant human osteogenic protein-1 (hOP-1) induces new bone formation in vivo with a specific activity comparable with natural bovine osteogenic protein and stimulates osteoblast proliferation and differentiation in vitro, J. Biol. Chem. 267 (1992) 20352–20362. [24] T. Barr, A.J.A. McNamara, G.K.B. Sa´ndor, C.M.L. Clokie, S.A.F. Peel, Comparison of the osteoinductivity of bioimplants containing recombinant human bone morphogenetic proteins 2 (Infuse) and 7 (OP-1), Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 109 (2010) 531–540. [25] S. Chubinskaya, M. Hurtig, D.C. Rueger, OP-1/BMP-7 in cartilage repair, Int. Orthop. 31 (2007) 773–781. [26] K. Tsuji, K. Cox, L. Gamer, D. Graf, A. Economides, V. Rosen, Conditional deletion of BMP7 from the limb skeleton does not affect bone formation or fracture repair, J. Orthop. Res. 28 (2010) 384–389. [27] K. Tsuji, A. Bandyopadhyay, B.D. Harfe, K. Cox, S. Kakar, L. Gerstenfeld, et al., BMP2 activity, although dispensable for bone formation, is required for the initiation of fracture healing, Nat. Genet. 38 (2006) 1424–1429. [28] D.D. Bikle, J. Harris, B.P. Halloran, E. Morey-Holton, Altered skeletal pattern of gene expression in response to spaceflight and hindlimb elevation, Am. J. Physiol. 267 (1994) E822–E827. [29] P. Backup, K. Westerlind, S. Harris, T. Spelsberg, B. Kline, R. Turner, Spaceflight results in reduced mRNA levels for tissue-specific proteins in the musculoskeletal system, Am. J. Physiol. 266 (1994) E567–E573. [30] A. Herbertson, J.E. Aubin, Cell sorting enriches osteogenic populations in rat bone marrow stromal cell cultures, Bone 21 (1997) 491–500. [31] R. Majeska, R. Wuthier, Studies on matrix vesicles isolated from chick epiphyseal cartilage. Association of pyrophosphatase and ATPase activities with alkaline phosphatase, Biochim. Biophys. Acta 391 (1975) 51–60. [32] B. de Bernard, P. Bianco, E. Bonucci, M. Costantini, G. Lunazzi, P. Martinuzzi, et al., Biochemical and immunohistochemical evidence that in cartilage an alkaline phosphatase is a Ca2 þ -binding glycoprotein, J. Cell Biol. 103 (1986) 1615–1623. [33] J. Chen, Q. Zhang, C.A. McCulloch, J. Sodek, Immunohistochemical localization of bone sialoprotein in foetal porcine bone tissues: comparisons with secreted phosphoprotein 1 (SPP-1, osteopontin) and SPARC (osteonectin), Histochem. J. 23 (1991) 281–289. [34] J. Chen, H. Shapiro, J. Sodek, Development expression of bone sialoprotein mRNA in rat mineralized connective tissues, J. Bone Miner. Res. 7 (1992) 987–997. [35] M. Herrmann, D. Klitscher, T. Georg, J. Frank, I. Marzi, W. Herrmann, Different kinetics of bone markers in normal and delayed fracture healing of long bones, Clin. Chem. 48 (2002) 2263–2266. [36] O.O.A. Oni, J.P. Mahabir, S.J. Iqbal, P.J. Gregg, Serum osteocalcin and total alkaline phosphatase levels as prognostic indicators in tibial shaft fractures, Injury 20 (1989) 37–38. [37] G. Carmeliet, G. Nys, R. Bouillon, Microgravity reduces the differentiation of human osteoblastic MG-63 cells, J. Bone Miner. Res. 12 (1997) 786–794. [38] C. Androjna, X. Su, P.R. Cavanagh, R.J. Midura, Fracture healing may be impaired during spaceflight, in: P.R. Cavanagh, A.J. Rice (Eds.), Bone Loss During Spaceflight, Cleveland Clinic Press, 2007, pp. 93–101 (Chapter 10).
Contents lists available at ScienceDirect
Acta Astronautica journal homepage: www.elsevier.com/locate/actaastro
Simulated microgravity alters the expression of key genes involved in fracture healing N. Patrick McCabe a, Caroline Androjna a, Esther Hill b, Ruth K. Globus c, Ronald J. Midura a,n a b c
Department of Biomedical Engineering, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA Lockheed Martin Exploration & Science, NASA Ames Research Center, Moffett Field, CA 94035, USA Biosciences Division, NASA Ames Research Center, Moffett Field, CA 94035, USA
a r t i c l e i n f o
abstract
Article history: Received 22 November 2011 Received in revised form 4 April 2012 Accepted 9 April 2012 Available online 26 April 2012
Fracture healing in animal models has been shown to be altered in both ground based analogs of spaceflight and in those exposed to actual spaceflight. The molecular mechanisms behind altered fracture healing as a result of chronic exposure to microgravity remain to be elucidated. This study investigates temporal gene expression of multiple factors involved in secondary fracture healing, specifically those integral to the development of a soft tissue callus and the transition to that of hard tissue. Skeletally mature female rats were subjected to a 4 week period of simulated microgravity and then underwent a closed femoral fracture procedure. Thereafter, they were reintroduced to the microgravity and allowed to heal for a 1 or 2 week period. A synchronous group of weight bearing rats was used as a normal fracture healing control. Utilizing Real-Time quantitative PCR on mRNA from fracture callus tissue, we found significant reductions in the levels of transcripts associated with angiogenesis, chondrogenesis, and osteogenesis. These data suggest an altered fracture healing process in a simulated microgravity environment, and these alterations begin early in the healing process. These findings may provide mechanistic insight towards developing countermeasure protocols to mitigate these adaptations. & 2012 IAA. Published by Elsevier Ltd. All rights reserved.
Keywords: Simulated microgravity Fracture healing Gene expression Angiogenesis Chondrogenesis Osteogenesis
1. Introduction Astronauts on spaceflight missions are faced with progressive loss of bone mass and mineral density mainly due to the reduced mechanical forces that are necessary for normal bone maintenance [1]. It has been suggested that
Abbreviations: PCR, polymerase chain reaction; VEGF, vascular endothelial growth factor; FGF2, basic fibroblast growth factor; IGF-1, insulin like growth factor-1; PDGF, platelet derived growth factor; TGF-b, transforming growth factor-b; BMPs, bone morphogenetic proteins; HLU, hind limb unloaded; WB, weight bearing; micro-CT, micro-computed tomography; MSCs, mesenchymal stem cells; ROI, region of interest n Correspondence to: Department of Biomedical Engineeering/ND20, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195, USA. Tel.: þ1 216 445 3212 ; fax: þ 1 216 444 9198. E-mail address: [email protected] (R.J. Midura).
microgravity may have a negative impact on the conversion of cartilage into cancellous bone via endochondral ossification due to alterations in the extracellular matrix composition [2]. This impaired state of bone quality can increase the risk that a trauma-induced long bone fracture might occur while in space or after return to normal gravity. In addition to an increased fracture risk, healing after fracture is impaired in both spaceflight and earth based microgravity analog animal fracture models [3]. The purpose of this study was to determine whether simulated microgravity affects ultimate fracture healing by altering gene expression of multiple factors within the first 2 weeks of bone fracture healing that is necessary for proper healing. Fracture healing is a complex process consisting of several temporally overlapping, and histologically discernable phases involving proliferation and differentiation of cells and tissues. Upon fracture, a hematoma forms at the fracture site resulting in a large clot that hinders further accumulation of blood
0094-5765/$ - see front matter & 2012 IAA. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.actaastro.2012.04.016
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at the fracture site [4]. As the hematoma becomes hypoxic, a new vasculature is recruited to facilitate nutrient delivery and waste disposal. Damaged tissue and cellular debris are then removed through the efforts of inflammatory cells that have migrated into the wound area [5]. Next, a cytokine cascade leads to the influx of reparative mesenchymal stem cells (MSCs) that differentiate into cell types that produce new tissues. First, chondrocytes deposit a new cartilage to form a firm tissue callus that bridges the disjoined bone ends and provides initial fracture site stability. The chondrocytes mature, undergo hypertrophy and the cartilaginous tissue is replaced with new woven bone through the efforts of bone depositing osteoblasts [5]. This woven bone is subsequently remodeled into a more compact bone tissue through the combined efforts of osteoclasts (bone resorbing cells) and osteoblasts [6]. While the stages of fracture healing are well characterized for bones subjected to constant gravitational and requisite ground reaction forces, alterations that occur as a result of exposure to simulated microgravity and loss of ground reaction forces are incompletely characterized. Each of these phases of bone fracture healing is driven by augmented expression of specific genes that are transcribed at basal maintenance levels in an uncompromised mature skeleton. Neovascularization occurs, at least in part, as a result of the combined efforts of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF2) [7]. An initial wave of MSCs is recruited to the fracture site and undergoes differentiation into chondrocytes. The mRNA of protein products aggrecan and Type II collagen peak within the first 2 weeks following rat bone fracture [8] in order to facilitate the matrix deposition and the formation of a firmer callus tissue. A second wave of MSCs is recruited to the fracture site [9] and undergoes differentiation into bone-forming osteoblasts. This occurs via the actions of osteoinductive molecules such as insulin like growth factor-1 (IGF-1), platelet derived growth factor (PDGF), transforming growth factorb (TGF-b) and the bone morphogenetic proteins (BMPs) [10]. Osteoblast maturation can be tracked by monitoring levels of osteogenic markers such as alkaline phosphatase, bone sialoprotein, and osteocalcin [11]. Using a NASA approved hind limb unloading (HLU) model to simulate exposure to microgravity, we found that the femora of rats subjected to microgravity exhibit reduced expression of key factors involved in the early reparative stages of fracture healing compared to full weight bearing (WB) counterparts. 2. Materials and methods 2.1. Animals Seven to eight months old, skeletally mature female Sprague–Dawley rats (Harlan Laboratories, Inc., Indianapolis, IN) were utilized in this study. Food and water were available ad libitum. Animals were group housed until time of tail suspension where all animals, regardless of study group, were singly housed until study termination. The Institutional Care and Use Committees of the Cleveland Clinic and the Biosciences Division, NASA Ames Research Center approved all procedures.
2.2. Ground based analog of microgravity The hind limb unloaded rat model developed by Morey-Holten et al. [12] is the NASA accepted ground based analog of spaceflight as it leads to physiological adaptations similar to those experienced in true spaceflight. Unloading in rats leads to a non-weight bearing condition in the hind limbs and a cephalic shift in body fluids [3]. Rats were randomly assigned to be either hind limb unloaded (HLU) or full weight bearing (WB) controls. Body weights (BW) were monitored on a weekly basis throughout the study period. 2.3. Fracture model Following a 4 week period of unloading or normal cage activity, rats were subjected to femoral fracture via a modified Bonnarens–Einhorn femoral fracture model [13]. In brief, a threaded titanium pin (Grade 2, diameter 1.30 mm, McMaster-Carr, Aurora, OH) was inserted into the medullary cavity of the femur in a retrograde fashion starting from the femoral condylar notch, seating the pin in the cancellous bone region of the greater trochanter. This pin serves as an intramedullary support to stabilize the fracture and provide a load bearing support for the injured limb. Following pin insertion and surgical closure, the stabilized femur is subjected to a 3-point bending moment in a closed configuration to generate a mid-shaft fracture of the femur. Rats underwent bilateral, middiaphyseal femoral fractures. Fractures were visualized by planar X-ray or micro-computed tomographic (microCT) imaging (Fig. 1). Rats were acclimated to Tylenol analgesia (3 mg/mL in drinking water) for a period of 3 days prior to surgery and remained on Tylenol analgesia for a period of 7 days post-fracture. Buprenorphine (2.5 mg/kg BW) was administered immediately prior to surgery and for 2 successive doses at 12 h intervals following fracture for all animals. Bupivicaine (0.5% solution) was given at the fracture site immediately postfracture as a local anesthetic. All rats remained unsuspended for a period of 24 h following fracture to avoid any potential anesthesia and analgesia related complications due to simultaneous head down tilt. Rats were then subjected to a further 1 or 2 week state of HLU or WB depending upon prior group designation. Animals/fractures were excluded from analysis if they met one of the following exclusionary criteria: (1) failure to detect a fracture following a single 3-point bending displacement; (2) fractures occurring outside the mid-diaphyseal region and too close to the proximal or distal metaphyses; (3) fracture patterns that were spiral extending beyond the 1.5 cm region of interest; (4) body weight losses greater than 20% of initial values; and (5) anesthesia complications leading to premature death. 2.4. RNA extraction and Real-Time PCR Following euthanasia by CO2 inhalation, rat hind limbs were removed and muscle stripped with care taken not to disturb the fracture callus tissue. Whole femurs were snap frozen in liquid nitrogen and stored at 80 1C until use.
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Total RNA from 1- and 2-week fracture callus tissue (1.5 cm ROI) was isolated by tissue pulverization in a FastPrep-24 device (MP Biomedicals, Solon, OH) in the presence of Trizol (Invitrogen, Carlsbad, CA) and cleaned up by passing through RNeasy columns (Qiagen Inc, Valencia, CA). Reverse transcription reactions were performed with
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1 mcg of total RNA using the Masterscript RT cDNA synthesis kit (5Prime, Inc., Gaithersburg, MA) followed by PCR in an ABI 7500 Real-Time PCR instrument (Invitrogen) with the primers listed in Table 1. PCR was carried out at 50 1C for 2 min, 95 1C for 10 min; 40–45 cycles of 95 1C for 15 s and 60 1C for 1 min. Expression levels were determined using SYBR Green chemistry (Qiagen) and the DDCT relative quantification method described by Livak and Schmittgen [14]. For each gene, relative expression was calculated following normalization to ribosomal 18S mRNA (r18S). Each sample was quantified from the average of triplicate repeats. Dissociation curves indicated amplification specificity by illustrating single reaction products. To verify amplicon identity, an amplicon from each gene was cloned into pGEM-T easy (Promega, Madison, WI) and sequenced. All amplicons aligned 100% with the expected mRNA reference sequences. 2.5. Micro-CT image acquisition and analysis Micro-CT imaging and analyses were performed on bone specimens that were processed for histological evaluation. Ex-vivo micro-CT images were acquired at an isotropic voxel resolution of 45 mm (GE Explore Locus, GE Healthcare). The volume of mineralizing hard callus was determined from the generated 3-D (16-bit) image volumes, for a 1 cm ROI, utilizing custom designed software (Image IQ, Inc., Cleveland, OH). Specifically, original cortical bone was manually masked from each volume and grayscale segmentation values of 600–6000 for hard callus and 46000 for the titanium rod were applied for the volumetric analysis. 2.6. Histology
Fig. 1. Depiction of the ex-vivo micro-CT imaging and analyses of the rat femoral fractures, over the 14 day healing period. (A, left panel) The box around a femur, one day post surgery, denotes the 1.5 cm region of interest (ROI) surrounding the fracture site that was utilized in all analyses. (A, right panels) Representative magnified views of the femoral ROI surrounding the fracture site, at designated time points, over the 14 day healing period. (B) Representative segmented volumes for WB and HLU groups that were computer generated based on cortical mask and threshold values applied for implant volume, cortical bone and mineralizing bone; yellow depicts the titanium rod, blue depicts the original cortical bone and red depicts the volume of mineralizing callus formed. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Histological evaluation was performed on sagittal sections from 10–13 fractures per group at each time point, representing a range of fracture types including diaphyseal wedge, simple and complex fractures. Fractured femora were fixed in 10% neutral buffered formalin and decalcified in a 5% trichloroacetic acid solution at 4 1C. Tissue was embedded in paraffin and sectioned at 7 mm. Hyaline cartilage tissue areas were determined by staining with Safranin O/Fast Green followed by static histological evaluation. Measurements of Safranin O-stained cartilage
Table 1 List of primers used in Real-Time PCR gene expression analysis. Gene
Product
Refseq/rRNA
Primer sequences
Acan Alpl Bmp2 Bmp7 Ibsp Col2a1 Fgf2 Bglap
Aggrecan Alkaline phoshpatase, Liver/bone/kidney Bone morphogenetic protein 2 Bone morphogenetic protein 7 Bone sialoprotein Type II collagen Fibroblast growth factor 2 Bone gamma-carboxyglutamate (gla) protein, osteocalcin Vascular endothelial growth factor 18S ribosomal subunit
NM_022190.1 NM_013059.1 NM_017178.1 NM_001191856.1 NM_012587.2 NM_012929.1 NM_019305.2 NM_013414.1
50 -TCTATCTGCACGCCAACC-30 , 50 -ACAAAGTCTTCACCTGTGTAGC-30 50 -CAACCTGACTGACCCTTCC-30 , 50 -TAACCTCTGTGACCCTTGAC-30 50 -AGAAGCCATCGAGGAACTTT-30 , 50 -CCTGGTGTCCAATAGTCTGG-30 50 -TATGCTGCCTACTACTGTGAG-30 , 50 -GTTGATGAAGTGAACCAGTGTC-30 50 -AACGGTTTCCAGTCCAGG-30 , 50 -TTTGAAGTCTCCTCTTCCTC-30 50 -GTGATGATGGTGAAGCTGGA-30 , 50 -TAACCTCTGTGACCCTTGAC-30 50 -ACCCACACGTCAAACTACAG-30 , 50 -GTCCATCTTCCTTCATAGCCAG-30 50 -CTGACAAAGCCTTCATGTCCA-30 , 50 -GCTCCAAGTCCATTGTTGAAG-30
NM_031836 M11188.1
50 -CAGCTATTGCCGTCCAATTGA-30 , 50 -CCAGGGCTTCATCATTGCA-30 50 -CGCGGTTCATTTTGTTGGT-30 , 50 -AGTCGGCATCGTTTATGGTC-30
Vegf r18S
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areas were made using Bioquants software (Bioquant Image Analysis Corporation, Nashville, TN). 2.7. Statistical analysis Comparisons at each time point were made between the WB and HLU groups by the parametric t test analysis (GraphPad Prism v5, GraphPad Software, Inc., La Jolla, CA). Real-Time PCR statistical analysis was conducted on Log2 transformed relative expression data. Data are plotted in box and whisker format, indicative of 5–95 percentiles (whiskers), 25–75 percentiles (box), and median values (line inside each box). Differences were considered significant at p r0.05. 3. Results and discussion 3.1. Angiogenesis While it is known that both VEGF [7,15,16] and FGF2 [17,18] have multiple roles in the healing of bone fractures, their roles in the growth of new blood vessels are crucial as vasculature plays a key role in each phase of the healing process. The hematoma formed immediately after fracture rapidly becomes hypoxic which drives the expression and accumulation of inflammatory mediators and growth factors [4]. This promotes the influx of endothelial cells and the formation of the new vasculature facilitating delivery of nutrients, MSCs, and eventually the hypertrophic differentiation of chondrocytes [15]. Two genes with protein products known to be key players in promoting vascularization of fracture callus tissue, Vegf and Fgf2, were measured. VEGF mRNA levels (Fig. 2A) in HLU callus tissue 7 days post-fracture was significantly less than that of WB fracture callus tissue (po0.05). Reduced VEGF mRNA levels were also present in the callus tissue of HLU rats at 2 weeks post-fracture (po0.05). Levels of FGF2 mRNA (Fig. 2B) were similarly reduced in HLU rat callus tissue at both 7 and 14 days post-fracture (each, po0.05). Endothelial cells exposed to the simulated microgravity in vitro exhibited reductions in responsiveness to VEGF and FGF2 treatments [19], introducing the possibility that a similar phenomenon might occur in vivo
leading to potential neovascularization deficiencies when compared to what is Earth normal. As the recruitment of vasculature is necessary for proper fracture healing [20], the chondrogenic and osteogenic phases of fracture healing also may be adversely affected. 3.2. Chondrogenesis Initial fracture site stabilization occurs through the production of cartilage matrix proteins that form a firmer callus tissue bridging the fractured bone ends. Expression levels of Acan and Col2a1 (genes encoding cartilage matrix proteins aggrecan and Type II collagen, respectively) were analyzed in the 7 and 14 days healing callus of fractured femora from WB rats and from rats exposed to 4 weeks of HLU prior to fracture as well as post-fracture. Expression of both of these genes are known to peak around 7–14 days after fracture and drops rapidly after 2 weeks in a rat diaphyseal fracture model [8]. As seen in Fig. 3A, Acan levels at both 1 and 2 weeks were similar in callus tissue from HLU and WB rat femora (p¼ 0.1591 and p¼0.1123, respectively). However, at 7 and 14 days post-fracture, Col2a1 levels were significantly less (each, po0.05) in HLU healing callus tissue compared to that of WB rats (Fig. 3B). Safranin O detection of sulfated proteoglycans revealed comparable levels of cartilage tissue proteoglycan area in the callus tissue of HLU and WB fractured femora (Fig. 4(A–C)). Given the histological appearance of hyaline cartilage tissue in these calluses (Fig. 4D), and that aggrecan comprises the bulk of proteoglycan in cartilage, these histochemistry results imply similar aggrecan distributions in HLU and WB fracture calluses. Under normal circumstances both Acan and Col2a1 expression patterns are tightly coordinated during cartilage tissue formation, yet we observed discordant expression patterns of Acan versus Col2a1 during fracture healing in HLU rats. The relevance of this observation is not known presently though disrupted patterns of Acan and Col2a1 have been reported in cartilage pathologies [21]. Interestingly, spaceflight and HLU exposure lead to declines in the steady state levels of Acan and Col2a1 [2]. In the case of traumatic injury, such as that described herein, Col2a1 levels in fracture callus are similarly reduced in HLU rats compared to WB rats while a proportional difference in Acan levels are not discernible, possibly
Fig. 2. Angiogenic factors have reduced expression levels in the callus tissue of fractured WB and HLU rat femora. Log2 transformed data are presented (whiskers: 5–95 percentile) (A) Vegf; WB: 7 days (n¼ 16), 14 days (n ¼18); HLU: 1 week (n¼ 19), 2 weeks (n¼ 20). (B) Fgf2; WB: 7 day (n¼ 15), 14 day (n¼ 16); HLU: 1 week (n ¼19), 2 weeks (n¼19). *po 0.05 versus WB at same time point.
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Fig. 3. Chondrogenic factor levels in the callus tissue of fractured WB and HLU rat femora. (A) Acan levels are similar in both WB and HLU callus tissue 7 and 14 days post-fracture (WB: 1 week (n¼ 16), 2 weeks (n¼ 18); HLU: 1 week (n¼19), 2 weeks (n¼19)). (B) Decrease in Col2a1 gene expression in HLU callus tissue 7 and 14 days post-fracture (WB: 1 week (n¼ 16), 2 weeks (n ¼18); HLU: 1 week (n ¼19), 2 weeks (n ¼19)). Log2 transformed data are presented (whiskers: 5–95 percentile). *po 0.05 versus WB at same time point.
Fig. 4. Cartilage tissue histology of callus tissue sections from fractured WB and HLU rat femora at (A) 7- and (B) 14-day post-fracture. The 0.5 mm scale black scale bar applies to panels A and B. (C) Percent cartilage area per healing callus area was measured from Safranin O stained proteoglycan deposits (arrows in panels A and B). (D) Higher magnification views of both WB and HLU callus tissues at 14 days of healing stained with Safranin O. The white scale bars in each image represents 0.1 mm.
due to the large variation in Acan expression in WB fracture callus. 3.3. Osteogenesis and related biomarkers Along with the formation of cartilage tissue occurring within the healing fracture callus, continual MSC recruitment from local or systemic sites to the fracture site leads to the differentiation of bone-forming osteoblasts. Numerous growth factors with variable osteogenic inductive capacity
can promote, either alone or in concert, the differentiation of MSCs into osteoblasts. The bone morphogenetic proteins (BMPs) are among the most potent for stimulating osteogenic differentiation and mineral deposition. Rat calvarial osteoblast cells treated with BMP2 expressed elevated levels of osteoblast early differentiation markers such as alkaline phosphatase (Alpl), intermediate differentiation markers such as bone sialoprotein (Ibsp), and late differentiation markers such as osteocalcin (Bglap) [22]. To a lesser extent, BMP7 induces an osteogenic differentiation in vitro [23] but
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appears to be a more potent stimulator of bone growth in vivo [24]. We determined the relative expression levels of Bmp2 and Bmp7 in fracture callus tissue of HLU and WB rats to gage the relative levels of osteogenic induction potential of their respective callus tissue. At 7 days, BMP2 mRNA levels (Fig. 5A) were decreased in callus tissue of HLU rats (po0.05) suggesting a reduced osteogenic induction potential of fracture site tissue as a result of exposure to simulated microgravity. At 14 days, however, BMP2 mRNA levels in HLU fracture callus tissue approached but did not achieve statistically quite lower values than that of WB rats (p¼ 0.064). Interestingly, relative levels of Bmp7 (Fig. 5B) remained lower in the callus tissue of HLU rats compared to WB rats at both 7 and 14 days (po0.05, both time points). At present the data cannot discriminate whether this continued reduction in Bmp7 expression is due to its role in cartilage formation [25] as opposed to its acting as a promoter of osteogenesis. Alternatively, BMP7 may not be as critical in fracture repair as BMP2 because conditional deletion of BMP7 allows an unhindered fracture healing [26] whereas BMP2 has been shown to be necessary [27] for the initiation of fracture healing. Together, the reductions
in BMP gene transcription in HLU fracture callus tissue relative to that of WB rats suggests a reduction in osteogenic inductive capacity of reparative tissues within limbs exposed to reduced ground reaction forces. Whether this is the result of reductions in osteoprogenitor cells within callus tissue of HLU rats as a consequence of simulated microgravity or the result of upstream delays in healing remains to be determined. Alkaline phosphatase (Alpl; early marker), bone sialoprotein (Ibsp; intermediate marker), and osteocalcin (Bglap; late marker) are temporally expressed during the differentiation of preosteoblast lineage cells into mineralizing osteoblasts [11]. Spaceflight has been shown to result in decreased steady state levels of Alpl [28] and Bglap [29]. It should be mentioned that only osteocalcin is uniquely expressed by osteoblasts; in the context of fracture healing, alkaline phosphatase is expressed by adipocytes [30] and chondrocytes [31,32] and bone sialoprotein is also expressed by hypertrophic chondrocytes [33,34]. As shown in Fig. 5C, fracture callus tissue from HLU rats contained relatively lower levels of Alpl at 7 days (po0.05) but levels at 14 days were comparable to callus tissue from WB rats (p¼0.116).
Fig. 5. Fracture callus tissue from WB and HLU rats has reduced levels of osteo-inductive factors and osteogenic biomarkers. (A) Bmp2 levels were lower at 7 days but similar to WB fracture callus tissue at 14 days. WB: 1 week (n ¼13), 2 weeks (n ¼11); HLU: 1 week (n ¼15), 2 weeks (n ¼29). (B) Bmp7 levels were lower at both 7 and 14 day after fracture in the callus tissue of HLU rats. (WB: 1 week (n¼ 15), 2 weeks (n¼16); HLU: 1 week (n¼ 18), 2 weeks (n¼ 20)). (C) Alpl levels in HLU fracture callus tissue was lower at 7 days but not at 14 days compared to WB tissue. (WB: 1 week (n ¼16), 2 weeks (n¼ 18); HLU: 1 week (n¼18), 2 weeks (n ¼20)). (D) Ibsp levels were also only lower the 7 day time point compared to WB fracture callus tissue. (WB: 1 week (n¼ 16), 2 weeks (n ¼18); HLU: 1 week (n¼ 19), 2 weeks (n¼20)). (E) Osteocalcin mRNA (Bglap) levels in HLU callus tissue was lower at 7 and 14 day compared to that of WB rats. (WB: 1 week (n¼ 16), 2 weeks (n¼18); HLU: 1 week (n¼ 19), 2 weeks (n¼ 20)). *p o0.05 versus WB at same time point.
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Fig. 6. Box and whisker (whiskers: 5–95 percentile) plot of the amount of mineralizing callus tissue at 7 and 14 days, determined in a 1 cm ROI surrounding the femoral fracture site. The differences in mineralizing tissue content were found to be statistically significant between WB and HLU groups at both time points (*p o 0.05).
This temporal pattern is comparable to those reported by Herrmann et al. [35] and Oni et al. [36] where slight lags in the levels of serum ALP were observed in patients with delayed fracture healing. Similarly, relative BSP mRNA levels were lower at 7 days (po0.05) but were not significantly different (p¼0.488) after 14 days (Fig. 5D) in the fracture callus tissue of HLU rats. These temporal differences may be explained by changes in both chondrogenesis and osteogenesis induced by HLU during these early phases of fracture healing. Alternatively, since Ibsp and Alpl expression levels act as indicators of early and intermediate stages of osteoblast differentiation, reduced expression of each of these genes may also suggest delayed osteoblast maturation in HLU fracture callus formation. In a more definitive indication that osteogenic maturation seems to be delayed, relative osteocalcin mRNA levels (Fig. 5E) were found to be reduced at both 7 and 14 days (po0.05, both time points) in the callus tissue of HLU fractures as compared to WB counterparts. In fact, spaceflight leads to an impaired osteoblast maturation even in the presence of dihydroxyvitamin D3 [1,25(OH)2D3] [37]. In the case of fracture healing, our findings are in accord with Herrmann et al. [35] and Oni et al. [36], where serum osteocalcin levels were higher in patients with normal fracture healing for a period of up to a month than that of patients with delays in fracture healing. 3.4. Hard callus tissue volumes Overall, the HLU groups exhibited a bone healing response that results in a significantly smaller sized mineralizing callus volume than that of the WB groups at both the 7-day and 14-day time points (Fig. 6). The HLU callus volumes were on average 50% smaller at 7 days (4.872.0 mm3 and 9.172.0 mm3, HLU and WB respectively) and 30% smaller at 14 days (30.4712.1 mm3 and 43.279.2 mm3, HLU and WB respectively). Statistical analyses indicate that the mineralizing tissue content is significantly different between groups, at each time point, over the 14-day period (po0.05). This is in agreement with our previous work illustrating smaller callus size and callus formation rates in HLU rats with fibular osteotomies
7 and 14 days following fracture [38], although the alterations were more severe in the fibular fracture model. These data support the observed differences in the gene expression of the angiogenic, chondrogenic and osteoinductive proteins as well as the osteogenic biomarkers that were investigated. Specifically, at both time points, the decreased expression of the chondrogenic gene, Col2a1, and the various osteogenic genes within the HLU groups correlate with decreased mineralization of the fracture callus. Therefore it is possible that both differentiation and maturation of MSCs into osteoblasts is delayed in the HLU group and/or requisite protein content required within the fracture callus site for an appropriate matrix tissue deposition to occur is deficient, thereby leading to an overall initial delay in the fracture healing process. 4. Conclusion Although histological analyses of fracture callus tissue indicates that exposure to simulated microgravity (HLU) leads to relatively normal cartilage proteoglycan deposition responses during secondary fracture healing, reductions in Col2a1 gene expression would likely lead to reductions in Type II collagen deposition in the healing callus tissue of HLU versus WB rats. Specifically, while levels of Acan appear in accord with that of Safranin O stained callus tissue, an uncoupling of Col2a1 and Acan expression at the molecular level is observed. These molecular alterations during early fracture healing stages suggest that the extracellular matrix composition of HLU callus tissue may be different than that of WB counterparts. If true, then this might contribute to a delay in the acquisition of a cartilaginous tissue connection across the fracture site. Simulated microgravity exposure results in a reduction in the expression of pro-angiogenic genes. If this decreased transcription leads to a reduction in pro-angiogenic protein levels at the fracture callus, then these alterations would likely delay a hypertrophic cartilage response and/or hinder callus ossification. Indeed, reductions in the levels of osteo-inductive and osteogenic gene
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expression characteristic of a normal secondary or endochondral healing response were detected and are consistent with this possibility. In addition, the decreased amount of mineralizing callus tissue present at the HLU fracture site supports the contention of a delayed healing response at early healing times. However, transient reductions in gene expression at early healing time points do not necessarily indicate compromised healing outcomes at later healing time points. While this study supplies data concerning alterations in fracture healing at early time points following extended periods of exposure to HLU, further investigations are necessary to determine the effects that long term exposure to microgravity has on functional fracture healing outcomes.
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