Proteasome inhibition-enhanced fracture repair is associated with increased mesenchymal progenitor cells in mice.

The ubiquitin/proteasome system controls the stability of Runx2 and JunB, proteins essential for differentiation of mesenchymal progenitor/stem cells (MPCs) to osteoblasts. Local administration of proteasome inhibitor enhances bone fracture healing by accelerating endochondral ossification. However, if a short-term administration of proteasome inhibitor enhances fracture repair and potential mechanisms involved have yet to be exploited. We hypothesize that injury activates the ubiquitin/proteasome system in callus, leading to elevated protein ubiquitination and degradation, decreased MPCs, and impaired fracture healing, which can be prevented by a short-term of proteasome inhibition. We used a tibial fracture model in Nestin-GFP reporter mice, in which a subgroup of MPCs are labeled by Nestin-GFP, to test our hypothesis. We found increased expression of ubiquitin E3 ligases and ubiquitinated proteins in callus tissues at the early phase of fracture repair. Proteasome inhibitor Bortezomib, given soon after fracture, enhanced fracture repair, which is accompanied by increased callus Nestin-GFP+ cells and their proliferation, and the expression of osteoblast-associated genes and Runx2 and JunB proteins. Thus, early treatment of fractures with Bortezomib could enhance the fracture repair by increasing the number and proliferation of MPCs.

Introduction

Successful fracture repair restores broken bones to their original structural geometry and biomechanical integrity. The fracture repair process is comprised of four phases: inflammatory response; recruitment of mesenchymal progenitor/stem cells (MPCs) and their differentiation into osteoblasts (OBs); new bone formation; and remodeling of the new bone from woven to lamellar bone. Although fracture repair is rapid and efficient, about 5–10% of patients with fractures, mainly aged subjects, develop delayed union or nonunion, resulting in unsuccessful fracture repair [1, 2]. BMP7 and PTH are FDA-approved drugs for the treatment of nonunion of long-bone fractures [3, 4]. However, short half-life, side effects, and high cost limit their utilization.

The Ubiquitin/Proteasome System (UPS) is a major mechanism for regulating the ubiquitination and degradation of intracellular proteins, including transcription factors or abnormal proteins trafficking from the cytosol and nuclei [5]. Disruption of the UPS has been linked to pathophysiological conditions that provoke accumulation of aberrant proteins, such as in neurodegenerative disorders and cancers [6]. Ubiquitination is carried out via sequential enzymatic reactions involving ubiquitin activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3, which confer substrate specificity, linking the ubiquitin-protein conjugation machinery to target molecules. Ubiquitinated (Ub-) proteins often undergo degradation in proteasomes or lysosomes, resulting in decreased steady-state levels of one or a group of proteins. A number of positive regulators of OBs, including the BMP- [7] & TGFβ-Smads [8], Runx2 [9], and JunB [10-13], can be regulated via the UPS by Nedd4 sub-class of E3 ligases, which comprises 7 mammalian members, including Nedd4-1, Nedd4-2, Itch, Wwp1&2, and Smurf 1&2. We have investigated the role of Smurf1, Wwp1 and Itch in bone using Smurf1−/−, Wwp1−/−, and Itch−/− mice and demonstrated that all of them negatively regulate OB differentiation or migration by controlling the stability of Runx2, JunB, or CXCR4 proteins in OBs and MPCs [10-16]. However, the involvement of these E3s and their effects on protein ubiquitination and degradation in fracture repair has not been well studied.

Proteasome inhibitors have been reported to induce OB differentiation [17]. Local delivery of the proteasome inhibitor, PS1 (Z-Ile-Glu(OtBu)-Ala-Leu-CHO) promotes femoral closed fracture repair in rats, accompanied by increasing BMP2 expression in callus [18]. BMP2 has strong osteoinductive activity to regulate OB differentiation and has been used to accelerate fracture and wound healing in patients [19]. Bortezomib (Btz) and Carfilzomib are two FDA-approved proteasome inhibitors for treating patients with multiple myeloma. Systemic administration of Btz enhances bone formation in mice by stabilizing Runx2 protein in MPCs and promoting their differentiation into OBs [20]. However, long-term use of proteasome inhibitors also inhibits osteoclast differentiation by inhibiting NF-κB signaling [21] during the bone remodeling stage of fracture repair, which may be counteractive to their bone anabolic effects on OBs. Thus, whether proteasome inhibitors can promote fracture repair by targeting only MPCs has not been investigated. We hypothesize that fracture injury triggers the UPS in callus at the early phase of fracture repair, leading to elevated protein ubiquitination and degradation, including Runx2 and JunB, and decreased MPCs. Btz, given only at the early phase of fracture repair, could promote fracture repair by increasing MPCs, OB differentiation, and Runx2 and JunB expression.

In this study, we used a tibial fracture model in Nestin-GFP reporter mice [22] in which a subgroup of MPCs are Nestin-GFP positive [23]. We examined the expression pattern of the Nedd4 sub-class of E3s and Ub-proteins in fracture callus and the effect of Btz on fracture repair focusing on Nestin-GFP+ MPCs.

Materials and methods

Animal study

Animals and ethics statement

Nestin-GFP transgenic mice (Nestin-GFP mice) were obtained from Dr. Tatyana V Michurina (Cold Spring Harbor Laboratory) [23] and generated on a C57BL/6J background, in which the 5.8-kb fragment of the promoter region was inserted upstream of sequences encoding destabilized eGFP. Primers: Forward: 5’-ATCACATGGTCCTGCTGGAGTTC-3’, Reverse: 5’-GGAGCTGCACACAACCCATTGCC-3’ were used for genotyping Nestin-GFP mice. Three-month-old C57BL/6J wild type (WT) mice were purchased from the National Cancer Institute. Male mice were used in this study to avoid any potential influence of estrogen on bone. Mice were randomly grouped (N = 3–8 mice /group or time point). Mice were housed in conditions of specific pathogen-free and controlled temperature (22°C) and illumination (12-hour light/12-hour dark cycle). All animal procedures were conducted in accordance with approved guidelines of the University of Rochester Committee for Animal Resources (protocol number: 2001-121R).

Tibial fracture model, Bortezomib treatment, and callus preparations

For fracture model, open tibial fracture procedure is a standard operating procedure (SOP) used in the Center for Musculoskeletal Research (CMSR) [24-27]. Briefly, a 6 mm long incision is made in the skin over the anterior tibia after anesthesia (Ketamine 100mg/kg and Xylazine 10 mg/kg, intraperitoneal injection). A sterile 27G needle is inserted via the proximal tibial articular surface into the bone marrow (BM) cavity, temporarily withdrawn to facilitate midshaft transection of the tibia using a scalpel, and reinserted to stabilize the fracture, which is confirmed by X-ray. The incision is closed with sutures. Mice are given Buprenorphine SR (ZooPharm) 0.5 mg/kg subcutaneously 4 hours before the surgery to control pain, and are monitored every 24 hours for 72 hours post-fracture to ensure that mice are under a pain-free condition. For Btz treatment, Btz is purchased from Cambridge, MA, USA, and dissolved in saline at the concentration of 0.1mg/ml. Mice are given Btz (0.6 mg/kg body weight) or vehicle by intraperitoneal (i.p.) injection on 1-, 3- and 5- day post fracture (dpf). Mice are euthanized by CO2 inhalation and secondary cervical dislocation at different time points. For callus cell preparation, callus is harvested at 10 dpf and dissected free of soft tissue and cut into pieces. Callus pieces are washed thoroughly with cold PBS and then digested with ACCUMAX cell detachment solution (Stem cell Tech) for 30 minutes at room temperature. 8×105−1.5×106 cells are obtained from each mouse (two fractures).

Micro-CT and Bio-mechanical testing

For microCT, fractured tibiae at 10 dpf were dissected free of soft tissue, fixed overnight in 70% ethanol and removed the stabilizing needle before scanning at high resolution (10.5 μm) on a VivaCT40 micro-CT scanner (Scanco Medical, Basserdorf, Switzerland) using 300 ms integration time, 55kVp energy, and 145 uA intensity. 3-D images were generated using a constant threshold of 275 for all samples. For Bio-mechanical testing, fractured tibiae at 28 dpf are stored at −80°C after removing the stabilizing needle carefully to avoid any damage to the architecture of the callus. The tibial ends are embedded in polymethylmethacrylate and placed on an EnduraTec system (Bose Corporation). A rotation rate of 1°/s is used to twist the samples to failure or up to 80°. Maximum torque, maximum rotation, and torsion rigidity are analyzed using a CMSR SOP [28].

Cell analysis

Flow cytometry and cell sorting

APC-anti-CD45, PE-anti-CD105 and PE-cy7-anti-Sca1 antibodies (Abs) are purchased from eBioscience. Callus and BM cells are stained with various fluorescein-labeled Abs and subjected to flow cytometric analysis using a Becton-Dickinson FACSCanto II Cytometer, according to the manufacturer’s instructions. Results are analyzed by Flowjo7 data analysis software (FLOWJO, LLC Ashland, OR). For cell sorting, BM cells from Nestin-GFP mice are sorted using a Becton-Dickinson FACSAria III sorter, according to the manufacture’s instructions [29].

Cell proliferation and apoptosis assays

For proliferation assays, Nestin-GFP mice are given two i.p. injections of BrdU (1 mg/mouse/injection) spaced 16 hours apart, and are euthanized 2 hours after the second injection. Callus cells are harvested and stained with APC-conjugated anti-BrdU Ab for flow cytometry using a BrdU Flow Kit (BD Pharmingen, cat#: 51-2354AK), according to the manufacturer’s instructions. The percentage of BrdU+ cells in the Nestin-GFP+ cells is assessed. For apoptosis assays, cells are stained with APC-anti-AnnexinV using an Apoptosis Detection Kit (eBioscience, cat#: 88–8007,). After being stained with Propidium iodide (PI), the percentage of PI-/AnnexinV+ apoptotic cells is assessed by flow cytometry. Of notes, some of terminally differentiated OBs may be Annexin V positive, which cannot be excluded by this method [30].

Histological analysis

Histology and histomorphometric analysis

Tibiae are fixed in 10% buffered formalin, decalcified in 10% EDTA, and paraffin sections are prepared for histology based on SOP of CMSR [31, 32]. In brief, bones are embedded in paraffin and 4 μm thick sections are cut at 3 levels. Sections are then stained with Alcian Blue/Hematoxylin (ABH) and counterstained with Eosin/Orange G. Sections are also stained for Tartrate-resistant acid phosphatase (TRAP) activity for osteoclast identification. Stained sections are scanned with an Olympus VS-120 whole-slide imaging system. Histomorphometric measurement of cartilage, woven bone area and osteoclast numbers is performed on 2–3 sections/bone using Visiopharm software (Version 2018.4) blindly, as we previously described [31, 32].

Immunostaining

Bones are embedded in Tissue-Tek and sectioned at 8 μm using a Leica CM1850 cryostat (Leica, German). Unstained sections are directly observed (below) or subjected to immunofluorescence staining after blocking in PBS with 10% normal goat serum and 0.2% Triton X-100 for 1 hour and staining overnight with rat anti-mouse Endomucin Ab (1:50, Santa Curz, cat#: sc-53941), mouse anti-Ubiquitin Ab (1:50, Santa Curz, cat#: sc-166553) or mouse anti-BrdU Ab (1:100, Santa Curz, cat#: sc-32323) at 4°C. After rinsing with PBS for 15 minutes, sections are incubated with goat anti-rat Alexa Fluor 568 (1:400, Invitrogen, cat#: A-11011) or goat anti-mouse Alexa Fluor 568 (1:400, Invitrogen, cat#: A-11004) at room temperature. Unstained and stained slides are mounted with mounting medium containing DAPI (Vector). Stained sections are scanned with an Olympus VS-120 whole-slide imaging system. Scanned images are numbered and the external callus area is outlined. The percentage of positively stained cells over the total cells within the external callus is quantified by Image J software (Version 1.53).

Biochemical analysis

Quantitative real time RT-PCR

Fracture callus tissues are homogenized under liquid nitrogen. Total RNA is extracted using TRIzol reagent. cDNAs are synthesized using an iSCRIPT cDNA Synthesis Kit (Bio-Rad). Quantitative RT-PCR amplification is performed in an iCycler (Bio-Rad) real time PCR machine using an iQ SYBR Green supermix (Bio-Rad), according to the manufacturer’s instructions. β-actin is amplified on the same plates and used to normalize the data. Each sample is prepared in triplicate and each experiment is repeated at least three times. The relative abundance of each gene is calculated by subtracting the CT value of each sample for an individual gene from the corresponding CT value of β-actin (ΔCT). ΔΔCT are obtained by subtracting the ΔCT of the reference point. These values are then raised to the power 2 (2ΔΔCT) to yield fold-expression relative to the reference point. Representative data are presented as means ± SD of the triplicates or individual samples. The sequences of primers and qPCR conditions in the current study are shown in S1 Table.

Western blotting analysis

Fracture calluses are homogenized under liquid nitrogen. Proteins are extracted with RIPA lysis buffer. Proteins are quantitated using a kit from Bio-Rad and loaded in 10% SDS-PAGE gels and blotted with anti-Ubiquitin, JunB, Runx2, NF-κB RelA and β-actin (Santa Cruz), IκBα and β-catenin (Cell Signaling), Osteocalcin (Enzo ALX) Abs as we described previously [11]. Bands are visualized using ECL chemiluminescence (Bio-rad).

Statistical analysis

All results are given as mean ± SD. Statistical analysis is performed using GraphPad Prism 5 software (GraphPad Software Inc., San Diego, CA, USA). Comparisons between 2 groups are analyzed using a 2-tailed unpaired Student’s t test. One-way ANOVA and Tukey’s post-hoc multiple comparisons are used for comparisons among 3 or more groups. p values <0.05 are considered statistically significant. The sample size for biomechanical testing is determined based on our previous published data using mice with tibial fracture, in which callus samples were harvested at 28 dpf following drug treatments [33]. The sample size n> = 3 is calculated given expected means of torsional rigidity 1491 ± 58 [N.mm/(rad/mm)] in vehicle-treated mice, 639 ± 133 [N.mm/(rad/mm)] in drug 1-treated mice, and 1141 ± 197 [N.mm/(rad/mm)] in drug 2-treated mice, with an alpha level of 0.05, and power of 80%, for One-way ANOVA with Dunnett’s test.

Results

Increased expression of Nedd4 sub-class E3 ligases and ubiquitinated proteins in fracture callus

We and others have reported that deletion of Nedd4 sub-class of E3 ligases promotes OB differentiation by preventing degradation of OB positive regulators, Runx2 [9] and JunB [13]. To determine if E3-mediated ubiquitination and degradation of these proteins play a role in fracture repair, we examined the expression levels of Nedd4 sub-class of E3s, Wwp1, Itch, Smurf1 and Smurf2, in callus samples at different time points after fracture by qPCR. Compared to samples isolated from time 0, the expression of Wwp1, Itch, and Smurf2 increased significantly, starting from 4 dpf, peaked at 7 dpf, and returned to baseline at 14 dpf and 21 dpf. Smurf1 expression increased only mildly during the whole fracture repair process, while Itch expression increased more than the others (Fig 1A). We examined levels of total Ub-proteins at 1 hour, 4 and 7 dpf when E3 ligase levels were increased. We found that the expression levels of Ub-proteins in callus were markedly increased compared to the time 0 sample and to non-fractured bones at all time points. In contract, total Ub-proteins in non-fractured bones were no change (Fig 1B).

Fig 1

Increased expression of E3 ligases and ubiquitinated proteins in fracture callus.

3-month-old C57BL/6J male mice received an open tibial fracture surgery. (A) Expression levels of E3 ligases in callus tissues determined by qPCR at different time points. N = 3 mice. Values are means ± SD. Relative mRNA expression is the fold-change vs. the time 0 samples as 1. *p<0.05 vs. time 0 samples. (B) Total Ub-proteins in callus tissues determined by Western blot analysis using anti-Ubiquitin Ab at different time points. N = 3 mice. Experiments were repeated independently at least 2 times.

Bortezomib increases fracture repair

Because E3 ligases cause ubiquitination of target proteins that subsequently undergo proteasomal degradation and Ub-proteins are increased in the early phase of fracture repair (Fig 1), we want to know if short-term of proteasome inhibition at the early phase of fracture repair could enhance fracture repair. We treated mice with Btz, a proteasome inhibitor used clinically, at 1-, 3- and 5-dpf and sacrificed them at different time points. We used Btz at 0.6 mg/kg body weight, a dose for treating multiple myeloma and increasing bone volume in mice [34]. ABH-stained sections revealed that Btz significantly increased cartilage and woven bone area at 10 and 14 dpf (Fig 2A & 2B), which was confirmed by micro-CT analysis (Fig 3A). Btz increased the mRNA expression of genes related to OB differentiation (Osx, Runx2) and maturation (Bsp, Ocn, Opn), and chondrocyte differentiation (Sox9, Col2) in callus (Fig 3B). Increased OB function was further demonstrated by increased Osteocalcin protein levels in callus samples from Btz-treated mice (Fig 3C). TRAP-stained sections showed that Btz slightly decreased osteoclast numbers at 10 dpf, but not in other time points (Fig 4A & 4B). Bio-mechanical testing showed increased torsional rigidity and maximum Torque in Btz-treated mic at 28 dpf (Fig 4C). Osteoclastogenesis is controlled by NF-κB/NFATc1 signal pathway in osteoclast precursors [35]. We examined the expression levels of nf-κb and nfatc1 mRNA in callus at 10 and 21 dpf and found that Btz significantly reduced nf-κb, and nfatc1 expression at 10, but not at 21 dpf (Fig 4D). Of note, Btz did not reduce the expression of Rankl in the same samples, suggesting that the inhibitory effect of Btz in osteoclasts is likely due to reduced expression of osteoclast transcription factors, nf-κb and nfatc1.

Fig 2

Bortezomib increases cartilage and woven bone areas in fracture callus.

3-month-old C57BL/6J male mice received fracture surgery as in Fig 1. Mice were treated with Btz (0.6 mg/kg body weight, i.p.) on 1-, 3- and 5-dpf, and were sacrificed at different times. (A) Representative ABH-stained sections showing higher woven bone and callus areas in Btz-treated mice. Scale bar = 1 mm. (B) Woven bone and cartilage areas analyzed by Image J software. N = 8 mice. Values are means ± SD. *p< 0.05 vs. vehicle-treated mice.

Fig 3

Bortezomib increases callus volume and the expression of genes related to osteoblast and chondrocyte differentiation in fracture callus.

3-month-old C57BL/6J male mice received fracture surgery and treatment as in Fig 2. (A) Callus volume measured at 10 dpf by micro-CT. N = 8 mice. Values are means ± SD. *p< 0.05 vs. vehicle-treated mice. (B) Expression levels of genes related to OB differentiation and maturation, and chondrocyte differentiation in callus tissues examined by qPCR at 10 dpf. N = 3 mice. Values are means ± SD. Relative mRNA expression is the fold-change vs. vehicle-treated mice as 1. *p< 0.05 vs. vehicle-treated mice. (C) Expression levels of Osteocalcin in callus tissues following Btz treatment at 10 dpf examined by Western blot analysis. Data is normalized to callus samples from vehicle-treated mice. N = 3 mice. *p< 0.05 vs. vehicle-treated mice.

Fig 4

Bortezomib decreases osteoclasts and improves fracture repair in mice.

3-month-old C57BL/6J male mice received fracture surgery were treated with Btz as in Fig 2. (A) Representative TRAP-stained sections showing fewer TRAP+ osteoclasts at 10 dpf in Btz-treated mice. Scale bar = 0.25 mm. (B) TRAP+ osteoclasts in callus analyzed by Image J software. N = 8 mice. Values are means ± SD. *p< 0.05 vs. vehicle-treated mice. (C) Bone stiffness and strength assessed by biomechanical testing at 28 dpf. N = 5 mice. Values are means ± SD. *p< 0.05 vs. vehicle-treated mice. (D) Expression of osteoclast transcription factors, nf-kb and nfatc1 examined by qPCR at 10 dpf. N = 3. Values are means ± SD. Relative mRNA expression is the fold-change vs. samples from the vehicle-treated mice as 1. Unpaired t-test is used to compare Veh and Btz treated day 10 and day 21 callus, respectively. *p< 0.05 vs. vehicle-treated mice.

Increased Nestin-GFP+ mesenchymal progenitor/stem cells in callus during bone fracture repair

In WT mice, Btz enhances bone formation in mice by preventing Runx2 degradation in BM MPCs [20]. To study if Btz-enhanced fracture repair involves the similar cell population in callus, we utilized Nestin-GFP reporter mice, which allows us to detect Nestin-GFP+ cells in callus by flow cytometry and fluorescent microscope. We used CD45-CD105+Sca1+ as surface markers for MPCs and demonstrated that in BM of non-fractured mice, less than 0.1% of cells were Nestin-GFP+ cells. Among them 80% of Nestin-GFP+ cells were CD105+Sca1+ and 74% of CD105+Sca1+ cells were Nestin-GFP+ (Fig 5A). Correlation analysis revealed a significant association between Nestin-GFP+ cells and CD45-CD105+Sca1+ cells (R2 = 0.884) (Fig 5B). In fracture callus, the percentage of Nestin-GFP+ cells increased at the early phase of fracture repair. Compared to time 0 samples, the time point when less than 0.1% of cells were Nestin-GFP+ cells, Nestin-GFP+ cells markedly increased at 7 dpf, peaked at 10 dpf, and returned to very low levels at 21 dpf (Fig 5C). Nestin-GFP+ cells can give rise to pericytes during the bone repair process in a calvarial defect model [36]. We examined the association between Nestin-GFP+ cells and blood vessels in callus sections by immunofluorescence staining with anti-Endomucin Ab (Fig 5D) and found that 34% of Nestin-GFP+ cells were located adjacent to Endomucin+ blood vessels (Fig 5E).

Fig 5

Increased Nestin-GFP+ mesenchymal stem/progenitor cells in callus at the early phase of fracture repair.

3-month-old Nestin-GFP male mice were used. (A) cells were stained with anti-CD105 and Sca1 Abs and subjected to flow cytometry. GFP+ (upper) or CD105+Sca1+ cells were gated for analysis. N = 5 mice. SSC: side-scattered light. (B) Correlation of Nestin-GFP+ and CD45-CD105+Sca1+ cells. (C) The percentage of Nestin-GFP+ cells in callus tissues analyzed by flow cytometry. N = 3. Values are means ± SD. *p< 0.05 vs. time 0 samples. (D) Frozen sections subjected to immunofluorescence stained with anti-Endomucin Ab for blood vessels. The insert indicates enlarged immunofluorescence stained image showing Nestin-GFP+ cells adjacent to Endomucin+ blood vessels (arrows). Scale bar = 1 mm. (E) The Nestin-GFP+ cells adjacent to Endomucin+ blood vessels (distance < 2 cell diameter to blood vessel) counted. The percentage of Nestin-GFP+ cells adjacent to blood vessels over the total number Nestin-GFP+ cells calculated. N = 3 mice. Values are means ± SD.

Bortezomib increases Nestin-GFP+ cells, ubiquitinated proteins, and proliferation of Nestin-GFP+ cells in fracture callus

To determine if Btz increases MPCs in fracture callus, we treated Nestin-GFP mice with Btz and examined the frequency of GFP+, CD105+ or Sca1+ cells by flow cytometry using callus samples harvested at 10 dpf. Compared to vehicle-treated mice, Btz-treated mice had significantly more Nestin-GFP+ cells (1.2±0.9% vs. 9.6±0.2%), CD105+ cells (4.1±0.8% vs. 8.9±0.4%), Sca1+ cells (4±1.5% vs. 9±0.5%), and CD45-CD105+Sca1+ cells (0.3±1.1% vs. 1.4±0.1%) (Fig 6A), but not B220+ B cells, CD3+ T cells and CD11b+ monocytes (Fig 6B). Western blotting analysis showed more Ub-proteins and higher expression levels of Runx2 and JunB proteins in callus of Btz-treated mice (Fig 6C).

Fig 6

Bortezomib increases the number of Nestin-GFP+ cells and expression of osteogenic proteins in fracture callus.

3-month-old Nestin-GFP male mice received fracture surgery were treated with Btz as in Fig 2. Callus tissues were harvested at 10 dpf. The percentage of mesenchymal (A) and hematopoietic (B) lineage cell populations analyzed by flow cytometry. N = 4 mice. Values are means ± SD. *p< 0.05 vs. vehicle-treated mice. (C) Expression levels of total Ub-proteins, Runx2, and JunB examined by Western blot analysis. Numbers are the fold-change by densitometry analysis of Western blot images using callus samples harvested at 10 dpf from vehicle-treated mice as 1. N = 3 mice.

To determine the distribution of Ub-proteins, we performed immunofluorescence staining with an anti-Ubiquitin Ab on callus sections. Btz markedly increased the Ubiquitin-positive stained areas and cells that were both Nestin-GFP and Ubiquitin positive, which were located mainly in the callus (Fig 7A & 7B). To study the potential mechanisms that mediate Btz-induced increase in Nestin-GFP+ cells, we examined cell proliferation by BrdU incorporation assays at 10 dpf, a time when increased Nestin-GFP+ cells in callus peaked. Immunofluorescence staining with an anti-BrdU Ab revealed more Nestin-GFP+BrdU+ cells in callus sections from Btz-treated mice than those from vehicle-treated mice (Fig 7C & 7D). Flow cytometric analysis indicated that Btz increased the percentage of Nestin-GFP+BrdU+ cells by 2-fold (21.7±4.8% in Btz- vs. 11.2±2.1% in vhecile-treated mice, Fig 7E). In contrast, Btz did not affect AnnexinV+ apoptotic cell numbers (Fig 7F). These data indicate that Btz increases MPCs and protein ubiquitination in fracture callus. Btz increases proliferation of Nestin-GFP+, which may contribute to increasing their numbers in fracture callus.

Fig 7

Bortezomib increases the proliferation of Nestin-GFP+ cells in fracture callus.

3-month-old Nestin-GFP male mice received fracture surgery were treated with Btz as in Fig 2. Callus tissues were harvested at 10 dpf. (A) Frozen sections subjected to immunofluorescence staining with anti-Ubiquitin Ab and examined under a fluorescent microscopy. Nestin-GFP+ cells are green and Ubiquitin+ cells are stained for red. Nestin-GFP+Ubiquitin+ cells are stained for yellow (indicated by yellow arrows). Scale bar = 1 mm. The adjacent sections stained for ABH. Inserts indicate enlarged immunofluorescence stained images. (B) The percentage of Nestin-GFP+, Ubiquitin+, or Nestin-GFP+Ubiquitin+ cells were counted. N = 3 mice. Values are means ± SD. *p< 0.05 vs. vehicle-treated mice. (C) Mice were given two injections of BrdU (1 mg/i.p. injection) spaced 16 hours apart, starting at 9 dpf. Frozen sections subjected to immunofluorescence staining with anti-BrdU Ab and analyzed under a fluorescent microscopy. Nestin-GFP+ cells are green and BrdU+ cells are stained for red. Nestin-GFP+BrdU+ cells are stained for yellow (indicated yellow arrows). Scale bar = 1 mm. (F) The percentage of Nestin-GFP+BrdU+ cells was counted. N = 3 mice. Values are means ± SD. *p< 0.05 vs. vehicle-treated mice. (D) The percentage of BrdU+ cells in callus Nestin-GFP+ cells analyzed by flow cytometry. N = 3 mice. Values are means ± SD. *p< 0.05 vs. vehicle-treated mice. (E) Cell apoptosis assessed by flow cytometry as the percentage of Annexin V+ cells in Nestin-GFP+ cells. N = 3 mice. Values are means ± SD.

Bortezomib increases β-catenin and inhibits NF-κB protein expression in fracture callus

β-catenin and NF-κB signal pathways play critical roles in controlling osteoblastogenesis and osteoclastogenesis, respectively, and both of them are regulated by the UPS [21]. To examine if Btz promotes fracture repair by affecting β-catenin and NF-κB signal proteins, we examined the expression levels of β-catenin, NF-κB inhibitor IκBα, and NF-κB RelA proteins in callus tissues by Western blot analysis. We found that Btz increased the expression of IκBα and β-catenin on 10 dpf, but not 21 dpf, the time when Btz’s effect has disappeared. Accordingly, NF-κB-RelA levels were decreased at 10 dpf in Btz-treated mice (Fig 8).

Fig 8

Bortezomib increases IκBα, β-catenin and decreases RelA protein levels in fracture callus at 10-day post-fracture.

3-month-old C57BL/6J mice received fracture surgery were treated with Btz as in Fig 2. Callus tissues were harvested at 10 and 21 dpf. (A) Expression levels of IκBα, β-catenin and RelA examined by Western blot analysis. (B) Densitometry analysis of Western blot images in A. Data is normalized to 10 dpf callus samples from vehicle-treated mice. N = 3 mice. Unpaired t-test is used to compare vehicle- and Btz-treated 10 dpf and 21 dpf callus, respectively. *p< 0.05 vs. vehicle-treated mice.

Discussion

Protein ubiquitination and degradation mediated by the UPS plays an important role in bone cell regulation under normal and pathological conditions. The expression levels of a number of positive regulators in MPCs are tightly controlled by their ubiquitination status. Proteasome inhibitors promote OB differentiation and bone formation by reducing the degradation of ubiquitinated OB positive regulators [7, 8, 20, 37–41]. Bone fracture repair requires proliferation and OB differentiation of MPCs. Proteasome inhibitors enhanced fracture repair in mouse models of bone defects [42]. Here, we demonstrated that total ubiquitinated protein levels in fracture callus increases during the early stages of fracture repair. Short-term administration of the proteasome inhibitor, Btz in fractured Nestin-GFP mice enhanced fracture repair, which is associated with increased proliferation of Nestin-GFP+ cells, CD45-CD105+Sca1+ MPCs and expression of ubiquitinated proteins and positive OB regulators, Runx2 and JunB in callus. Thus, UPS-mediated cellular processes may play a role in fracture repair, which involves in regulating proliferation of MPCs.

Our data indicate that the majority of protein ubiquitination and degradation happens early during fracture repair, associated with an increased expression of the Nedd4 sub-class of E3 ligases. We and others have reported that pro-inflammatory cytokines, such as TNF, increase transcription of E3 ligases via NF-κB, and that NF-κB binding elements are present in the promoter regions of Smurf1 [14] and Itch [43]. Cytokines, including IL-1, IL-6 and TNF, are highly expressed during the early inflammatory stage of fracture repair to initiate the repair cascade following injury [44, 45]. Thus, these pro-inflammatory cytokines may activate the UPS by up-regulating E3 ligases in fracture callus. Although depletion of a single E3, such as Smurf1 [16] or Itch [11], leads to enhanced bone formation by preventing the degradation of positive osteoblast regulatory proteins, we chose to use Btz in our present study because 1) Btz is an FDA-approved drug and has known bone anabolic effects, and 2) E3 ligases often have overlapping substrate proteins and vice versa. 3) We have successfully developed a bone targeted Btz (Bisphophonate conjugated Btz, BP-Btz) [33], which can facilitate the clinic use of Btz in bone fracture without the systemic side effect. Thus, targeting a single E3 ligase may not be efficient, especially since we have found levels of multiple E3 ligases elevated in fracture callus and Itch deficient mice have similar fracture repair as WT littermates (S1 Fig).

In this study, we used a short-term regimen for Btz, e.g. 3 administrations at early phase of fracture repair. The rationale why Btz was administered at day 1, 3, and 5 include 1) we hypothesize that Btz will increase MPCs at the early phase of fracture repair and day 1–5 is the time when MPCs are rapidly expanded [27, 46]; 2) a major mechanism of action of Btz is to block the degradation of Ub-proteins while we found increased Ub-proteins at the early phase of fracture repair (Fig 1B); and 3) our pharmacology study reported recently that blood Btz levels return to un-detectable levels at 12 hours [47] and that the inhibitory effect of Btz on Ub-protein degradation is abolished at 24 hours following a single Btz administration (manuscript in press). Treatment of mice with Btz on 1-, 3-, and 5-dpf enhanced fracture repair and bone strength (Figs 2 and 3), indicating that this short-term regimen is beneficial. At the cell level, Btz mainly increased cells in the mesenchymal lineage, but not other cell populations, such as B, T and monocytes at 10-dpf (Fig 3). Furthermore, BrdU incorporation assays revealed more BrdU+Nestin-GFP+ cells in Btz-treated mice, suggesting that Btz may increase proliferation of this cell population. Because BrdU is able to participate the DNA repair as well [48], it is possible that Btz may prevent cells from entering apoptosis. This possibility can be studied in the future.

Nestin-GFP+ cells in Nestin-GFP mice have been used in stem cell studies with controversial results. Nestin was initially and widely used as the marker of neural stem cells [22]. Nestin+ cells in BM include pericytes [36, 49], neural crest stem cells and mesenchymal stem cells [50]. Despite these controversial findings, our study for the first time using young adult mice indicated markedly increased Nestin-GFP+ cells in long bone fracture callus. We demonstrated that about 70% of Nestin-GFP+ cells are CD45-CD105+Sca1+, indicating that during the fracture healing, these Nestin-GFP+ cells are likely MPCs because they are rapidly expanded in response to injury as a recent study pointed out [46]. In addition, about 30% of Nestin-GFP+ cells are located adjacent to blood vessels, suggesting they are likely pericytes that to contribute to angiogenesis, another critical component of fracture repair.

One concern is the non-specificity of proteasome inhibitors because many proteins in various cell types can undergo ubiquitination in fracture callus. We believe that proteasome inhibitor promotes the fracture repair by increasing osteoblast differentiation through BMP2 [18]. Apart from this, our study strongly suggests that Btz increasing mesenchymal stem/progenitor cell is critical for fracture repair as well, particularly with a short-term regimen at early phase of fracture repair, and provides a strong rationale and evidence for the beneficial effects of short-term administration of proteasome inhibitors when they are given early during fracture repair when expansion of MPCs takes place. Short-term administration of proteasome inhibitors is unlikely to affect the OB differentiation in the whole repair process. Another issue is that fractures in young subjects typically heal well by themselves. Thus, it will be very important to determine if proteasome inhibitors could promote healing of delayed or nonunion fractures in aged or diabetic mice.

Another concern is the severe systemic side effect of Btz, which limits its use in clinic. Fortunately, we developed the BP-Btz, which was synthesized by conjugating Btz to a bisphosphonate with no antiresorptive activity. BP-Btz has significantly less systemic side than Btz, including thymic cell death, sympathetic nerve damage and thrombocytopenia. BP-Btz treatment prevented the bone loss in wild-type and ovariectomy mice and promoted the fracture healing in aged mice [33]. It will be also interesting to examine if newer generation of proteasome inhibitors with fewer side effects than Btz [51, 52], such as Carfilzomib, is efficacious with reduced systemic toxicity.

Conclusion

Our study demonstrated elevated UPS in callus tissues during the early stages of fracture repair and the enhanced fracture healing by increasing MPCs via a short-term Btz administration. Our study provides the theoretical basis for treating fracture patients with proteasome inhibitors. Thus, manipulation of the UPS early after fracture may be a good strategy to promote bone fracture repair by proteasome inhibitors, particularly with our published bone targeting approach.

Primer sequences and qPCR conditions.

(PPT)

Click here for additional data file.

Itch knockout mice have comparable bone fracture healing.

C57/B6 WT mice and Itch knockout out mice received open tibial fracture surgery and were sacrificed at 10, 14, 21, and 28 days post fracture. Fractured tibiae examined by micro-CT. (A) Representative micro-CT images showing fracture callus. (B) Callus volume analyzed from micro-CT images. N = 3-5/time points.

(PPT)

Click here for additional data file.

(PPT)

Click here for additional data file.

22 Oct 2021

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This study needs to be considerably amended for English. A native speaker expert in Scientific English must revise it in deep.

Results section should must be focused ONLY to the obtained results: is this the first manuscript they write?

Therefore please move all comments to the Discussion Section.

Authors must analyze several other osteogenic markers, such as BSP, OPN, OC, both for qRT-PCR and WB.

Better images related to colony formation in figure 4B MUST be added: they are not available.

Micro-Computed Tomography (μCT) and Radiological Analysis must also be added to increase the interest to this paper.

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Reviewer #1: Partly

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Reviewer #1: In this study Authoirs examined the expression pattern of the Nedd4 sub-class of E3s and Ub proteins in fracture callus and the effect of Bortezomib on fracture repair on a tibial fracture model in Nestin-GFP reporter mice.

English should be revised. In particular, in paragraph: “Tibial fracture model, Bortezomib treatment, and callus preparations.” Authors used different time.

More references are needed in the first part of introduction.

“Bortezomib and Carfilzomib are 2 FDA-approved drugs”: please change 2 with two.

Results section should be revised. They should be reduced to results obtained. All the comments should be moved to the discussion section.

Authors should analyze other osteogenic markers, whose expression is related to that of Runx2, such as BSP, OPN, OC, both for qRT-PCR and WB.

Better images of colony formation in figure 4B should be provided.

Micro-Computed Tomography (μCT) and Radiological Analysis would add interest to the paper.

**********

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13 Jan 2022

Jan 14, 2022

Responses to the journal staff

Re: PONE-D-21-32074R1

Thank you very much for working on our submission.

1) We have included “Role of Funder statement: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript” in the cover letter.

2) In our previous submission, we have indicated in the cover letter that we have included “Supporting information files-uncut gels”. We ensured that we have uploaded this file and named it “Zhang uncut gels”.

3) In our previous submission, we have uploaded 3 Supporting Information files-1) Supporting-supplemental figure-Zhang S1 Fig; 2) Supporting-supplemental table-Zhang S1 Table; 3) supporting file-uncut gels-Zhang uncut gels. We ensured that we have uploaded these files.

We have double checked our submission to ensure that we have uploaded all files.

Please contact us ASAP if you find there are any problems.

END

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

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We have changed our manuscript to PLOS ONE's style requirements.

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We have followed the PLOS ONE submissions requirements. We have included the details on methods of anesthesia and/or analgesia (page 5)

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We have made correction.

4. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or

Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at

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We have included the original uncropped and unadjusted images for Figure 1B, 2E, 5C, 7A in Supporting Information files #1-4.

When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#locoriginal-images-for-blots-and-gels.

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permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public

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In the previous submission, we had two places used “data not shown”. One place states “Btz treatment did not alter the percentage of B220+ B cells, CD3+ T cells and CD11b+ monocytes in the same samples (data not shown)”. Another place states “targeting a single E3 ligase may not be efficient, especially since we have found levels of multiple E3 ligases elevated in fracture callus and Itch deficient mice have similar fracture repair as WT littermates (data not shown)”. In the revised manuscript, we have included these data in Figure 5B and S1 Fig.

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delete it from any other section.

In the revised manuscript, ethics statement is only in the Methods Section.

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We have included S1 Table in the revised manuscript. (page 8, line 234)

Additional Editor Comments:

This study needs to be considerably amended for English. A native speaker expert in Scientific English must revise it in deep.

We have extensive edited English in the revised manuscript.

Results section should must be focused ONLY to the obtained results: is this the first manuscript they write? Therefore please move all comments to the Discussion Section.

We have extensively revised result section and depleted or moved the comments to the Discussion Section.

Authors must analyze several other osteogenic markers, such as BSP, OPN, OC, both for qRT-PCR and WB.

We have examined the expression levels of additional osteogenic markers, bone sialoprotein, osteopontin and osteocalcin by qPCR, and osteocalcin protein levels by Western blot analysis as reviewer suggested. We have included these new data in Figure 2D and 2E and in text. (page 10, line 290-294)

Better images related to colony formation in figure 4B MUST be added: they are not available.

We decided to remove colony formation image from the figure 4B because we have eliminated Nestin-GFP mouse colony in University of Rochester Medical Center due to limited budget and Covid 19. Since more recent publication have further characterized Nestin-GFP cells as skeletal or perivascular mesenchymal progenitor cells [1-4] we believe that removal these data do not affect the overall conclusion of our current study.

Micro-Computed Tomography (μCT) and Radiological Analysis must also be added to increase the interest to this paper.

We have included μCT data for this study in Figure 2C and in text. (page 10, line 290)

We do not use X-ray in fracture study because X-ray is not an ideal and quantitative method to monitor the fracture healing process. The Center for Musculoskeletal Research, University of Rochester Medical Center, has an established SOP to evaluate the fracture healing process, including histology, micro-CT and bio-mechanical testing [5-7].

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted

rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception

(please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or

deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If

there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical

errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual

publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this study Authoirs examined the expression pattern of the Nedd4 sub-class of E3s and Ub proteins in fracture callus and the effect of Bortezomib on

fracture repair on a tibial fracture model in Nestin-GFP reporter mice.

English should be revised. In particular, in paragraph: “Tibial fracture model, Bortezomib treatment, and callus preparations.” Authors used different time.

We have extensive edited English in the revised manuscript.

More references are needed in the first part of introduction.

We have added more references in the first part of introduction.

“Bortezomib and Carfilzomib are 2 FDA-approved drugs”: please change 2 with two.

Results section should be revised. They should be reduced to results obtained. All the comments should be moved to the discussion section.

We have changed “2” to “two” on page 4. We

Authors should analyze other osteogenic markers, whose expression is related to that of Runx2, such as BSP, OPN, OC, both for qRT-PCR and WB.

We have examined the expression levels of additional osteogenic markers, bone sialoprotein, osteopontin and osteocalcin by qPCR, and osteocalcin protein levels by Western blot analysis as reviewer suggested. We have included these new data in Figure 2D and 2E and in text. (page 10, line 290-294)

Better images of colony formation in figure 4B should be provided.

We decided to remove colony formation image from the figure 4B because we have eliminated Nestin-GFP mouse colony in University of Rochester Medical Center due to limited budget and Covid 19. Since more recent publication have further characterized Nestin-GFP cells as skeletal or perivascular mesenchymal progenitor cells [1-4] we believe that removal these data do not affect the overall conclusion of our current study.

Micro-Computed Tomography (μCT) and Radiological Analysis would add interest to the paper.

We have included μCT data for this study (Figure 2C, text page 10, line 290).

We do not use X-ray in fracture study because X-ray is not an ideal and quantitative method to monitor the fracture healing process. The Center for Musculoskeletal Research, University of Rochester Medical Center, has an established SOP to evaluate the fracture healing process, including histology, micro-CT and bio-mechanical testing [5-7].

6. PLOS authors have the option to publish the peer review history of their article. If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

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Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

References

1. Wang S, Su TT, Tong H, Shi W, Ma F, Quan Z. CircPVT1 promotes gallbladder cancer growth by sponging miR-339-3p and regulates MCL-1 expression. Cell Death Discov. 2021;7(1):191. Epub 2021/07/28. doi: 10.1038/s41420-021-00577-y. PubMed PMID: 34312371; PubMed Central PMCID: PMCPMC8313687.

2. Tournaire G, Stegen S, Giacomini G, Stockmans I, Moermans K, Carmeliet G, et al. Nestin-GFP transgene labels skeletal progenitors in the periosteum. Bone. 2020;133:115259. Epub 2020/02/10. doi: 10.1016/j.bone.2020.115259. PubMed PMID: 32036051.

3. Nakahara F, Borger DK, Wei Q, Pinho S, Maryanovich M, Zahalka AH, et al. Engineering a haematopoietic stem cell niche by revitalizing mesenchymal stromal cells. Nat Cell Biol. 2019;21(5):560-7. Epub 2019/04/17. doi: 10.1038/s41556-019-0308-3. PubMed PMID: 30988422; PubMed Central PMCID: PMCPMC6499646.

4. Mendez-Ferrer S, Michurina TV, Ferraro F, Mazloom AR, Macarthur BD, Lira SA, et al. Mesenchymal and haematopoietic stem cells form a unique bone marrow niche. Nature. 2010;466(7308):829-34. Epub 2010/08/13. doi: 10.1038/nature09262. PubMed PMID: 20703299; PubMed Central PMCID: PMCPMC3146551.

5. Shares BH, Smith CO, Sheu TJ, Sautchuk R, Jr., Schilling K, Shum LC, et al. Inhibition of the mitochondrial permeability transition improves bone fracture repair. Bone. 2020;137:115391. Epub 2020/05/04. doi: 10.1016/j.bone.2020.115391. PubMed PMID: 32360587; PubMed Central PMCID: PMCPMC7354230.

6. Wang H, Zhang H, Srinivasan V, Tao J, Sun W, Lin X, et al. Targeting Bortezomib to Bone Increases Its Bone Anabolic Activity and Reduces Systemic Adverse Effects in Mice. J Bone Miner Res. 2020;35(2):343-56. Epub 2019/10/15. doi: 10.1002/jbmr.3889. PubMed PMID: 31610066.

7. Wang C, Inzana JA, Mirando AJ, Ren Y, Liu Z, Shen J, et al. NOTCH signaling in skeletal progenitors is critical for fracture repair. J Clin Invest. 2016;126(4):1471-81. Epub 2016/03/08. doi: 10.1172/JCI80672. PubMed PMID: 26950423; PubMed Central PMCID: PMCPMC4811137.

Submitted filename: Zhang Response to Reviewers.docx

Click here for additional data file.

28 Jan 2022

Proteasome Inhibition-Enhanced Fracture Repair is Associated with Increased Mesenchymal Progenitor Cells in Mice

PONE-D-21-32074R1

Dear Dr. Liu,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Reviewer #1: All comments have been addressed

**********

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Reviewer #1: (No Response)

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Reviewer #1: (No Response)

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Reviewer #1: No

14 Feb 2022

PONE-D-21-32074R1

Proteasome Inhibition-Enhanced Fracture Repair is Associated with Increased Mesenchymal Progenitor Cells in Mice

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