The Effects of Systemic Therapy of PEGylated NEL-Like Protein 1 (NELL-1) on Fracture Healing in Mice
2017; Elsevier BV; Volume: 188; Issue: 3 Linguagem: Inglês
10.1016/j.ajpath.2017.11.018
ISSN1525-2191
AutoresJustine Tanjaya, Elizabeth L. Lord, Chenchao Wang, Yulong Zhang, Jong K. Kim, Alan Nguyen, Llyod Baik, Hsin Chuan Pan, Eric Chen, Jin Hee Kwak, Xinli Zhang, Benjamin M. Wu, Chia Soo, Kang Ting,
Tópico(s)Connective tissue disorders research
ResumoFractures are common, with an incidence of 13.7 per 1000 adults annually. Systemic agents have been widely used for enhancing bone regeneration; however, the efficacy of these therapeutics for the management and prevention of fracture remains unclear. NEL-like protein 1 (NELL-1) is a potent pro-osteogenic cytokine that has been modified with polyethylene glycol (PEG)ylation [PEGylated NELL-1 (NELL-PEG)] to enhance its pharmacokinetics for systemic therapy. Our aim was to investigate the effects of systemic administration of NELL-PEG on fracture healing in mice and on overall bone properties in uninjured bones. Ten-week–old CD-1 mice were subjected to an open osteotomy of bilateral radii and treated with weekly injections of NELL-PEG or PEG phosphate-buffered saline as control. Systemic injection of NELL-PEG resulted in improved bone mineral density of the fracture site and accelerated callus union. After 4 weeks of treatment, mice treated with NELL-PEG exhibited substantially enhanced callus volume, callus mineralization, and biomechanical properties. NELL-PEG injection significantly augmented bone regeneration, as confirmed by high expression of bone turnover rate, bone formation rate, and mineral apposition rate. Consistently, the immunohistochemistry results also confirmed a high bone remodeling activity in the NELL-PEG–treated group. Our findings suggest that weekly injection of NELL-PEG may have the clinical potential to accelerate fracture union and enhance overall bone properties, which may help prevent subsequent fractures. Fractures are common, with an incidence of 13.7 per 1000 adults annually. Systemic agents have been widely used for enhancing bone regeneration; however, the efficacy of these therapeutics for the management and prevention of fracture remains unclear. NEL-like protein 1 (NELL-1) is a potent pro-osteogenic cytokine that has been modified with polyethylene glycol (PEG)ylation [PEGylated NELL-1 (NELL-PEG)] to enhance its pharmacokinetics for systemic therapy. Our aim was to investigate the effects of systemic administration of NELL-PEG on fracture healing in mice and on overall bone properties in uninjured bones. Ten-week–old CD-1 mice were subjected to an open osteotomy of bilateral radii and treated with weekly injections of NELL-PEG or PEG phosphate-buffered saline as control. Systemic injection of NELL-PEG resulted in improved bone mineral density of the fracture site and accelerated callus union. After 4 weeks of treatment, mice treated with NELL-PEG exhibited substantially enhanced callus volume, callus mineralization, and biomechanical properties. NELL-PEG injection significantly augmented bone regeneration, as confirmed by high expression of bone turnover rate, bone formation rate, and mineral apposition rate. Consistently, the immunohistochemistry results also confirmed a high bone remodeling activity in the NELL-PEG–treated group. Our findings suggest that weekly injection of NELL-PEG may have the clinical potential to accelerate fracture union and enhance overall bone properties, which may help prevent subsequent fractures. Fracture healing is an intricate process that is heavily influenced by various systemic and local factors.1Einhorn T.A. Gerstenfeld L.C. Fracture healing: mechanisms and interventions.Nat Rev Rheumatol. 2015; 11: 45-54Crossref PubMed Scopus (871) Google Scholar, 2Marsell R. Einhorn T.A. The biology of fracture healing.Injury. 2011; 42: 551-555Abstract Full Text Full Text PDF PubMed Scopus (1008) Google Scholar The goal of fracture healing is to regain function and return to weight bearing in a timely manner. Improvements in overall skeletal strength may help prevent fractures, which are estimated to reach an incidence of 13.7 per 1000 adults annually.3Court-Brown C.M. 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Pharmacokinetics and osteogenic potential of PEGylated NELL-1 in vivo after systemic administration.Biomaterials. 2015; 57: 73-83Crossref PubMed Scopus (13) Google Scholar Despite the substantial evidence of NELL-1 in promoting osteogenesis, there has been little understanding of the effects of systemic delivery of NELL-1 on fracture healing. In the present study, we explored the therapeutic effects of systemically delivered NELL-PEG on fracture repair in a radial defect model. We hypothesized that systemically delivered NELL-PEG improves fracture healing by enhancing bone regeneration and remodeling processes. The current study may present a new modality for the management and prevention of bone fracture, particularly in conditions of inherently weak bone, such as osteoporosis. A total of 10 female CD-1 mice, aged 10 weeks, were obtained from Charles River Laboratories (Wilmington, MA) and treated according to the animal protocol that was approved by the University of California, Los Angeles, Office of Animal Research Oversight and Chancellor's Animal Research Committee of the Office for Protection of Research Subjects. Animals were weighed and randomly assigned into control and treatment groups, then subjected to weekly tail vein injections of either PEGylated phosphate-buffered saline (PBS) or NELL-PEG (n = 5 animals per group). All surgical procedures were performed under anesthesia. The bilateral forelimbs were shaved and draped in a sterile manner before the surgery. An incision was made over the dorsum of the forearm, and the soft tissue was dissected bluntly to expose the radial shaft. The tip of an 11-blade scalpel was inserted into the interosseous membrane between the radius and ulna to protect the ulna, which was left intact.39Ning B. Zhao Y. Buza Iii J.A. Li W. Wang W. Jia T. Surgically induced mouse models in the study of bone regeneration: current models and future directions (Review).Mol Med Rep. 2017; 15: 1017-1023Crossref PubMed Scopus (9) Google Scholar A 0.15-mm Orthodisc (Ortho Technology, Tampa, FL) was used to generate an open osteotomy in the middle third of the radial shaft (Supplemental Figure S1A). The periosteum was removed and the skin incision was closed with 5-0 absorbable polyglactin sutures (Vicryl; Ethicon, Johnson & Johnson, Somerville, NJ). Postoperatively, mice were housed individually with ad libitum access to food and water and allowed to weight bear as tolerated. Mice were weekly administered with 100 μL of NELL-PEG solution (1.25 mg/kg) or PBS solution via the lateral vein, starting from the postsurgery (week 0) until the fourth injection (week 3), with the optimal dose determined according to our previous studies.37Zhang Y. Velasco O. Zhang X. Ting K. Soo C. Wu B.M. Bioactivity and circulation time of PEGylated NELL-1 in mice and the potential for osteoporosis therapy.Biomaterials. 2014; 35: 6614-6621Crossref PubMed Scopus (14) Google Scholar, 38Kwak J.H. Zhang Y. Park J. Chen E. Shen J. Chawan C. Tanjaya J. Lee S. Zhang X. Wu B.M. Ting K. Soo C. Pharmacokinetics and osteogenic potential of PEGylated NELL-1 in vivo after systemic administration.Biomaterials. 2015; 57: 73-83Crossref PubMed Scopus (13) Google Scholar Mice were sacrificed at week 4 postoperatively via carbon dioxide overdose and harvested for radii (Supplemental Figure S1B). Samples were fixed in 4% paraformaldehyde for 48 hours and stored in 70% ethanol at room temperature. After the surgery, radiographs of the fracture site were taken immediately to confirm the transverse fracture. Subsequently, high-resolution radiographs were consecutively taken every week to monitor fracture healing. Three blinded examiners (J.T., J.K.K., and H.C.P.) assessed the healing process by using a 4-point radiograph-based scoring system from the radiographic union score.40Kooistra B.W. Dijkman B.G. Busse J.W. Sprague S. Schemitsch E.H. Bhandari M. The radiographic union scale in tibial fractures: reliability and validity.J Orthop Trauma. 2010; 24 Suppl 1: S81-S86Crossref PubMed Scopus (116) Google Scholar Fracture union was identified by the presence of bridging callus on two cortical edges. All animals were subjected to dual X-ray absorptiometry scan (PIXImus2 GE Lunar Corp., Madison, WI) with an interval of every 2 weeks to monitor the changes in BMD. Scanning was performed under anesthesia. A square region of interest of 10 × 10 mm2 was drawn at the fracture site. Longitudinal assessment of the BMD (g/cm2) was performed starting at the baseline until the final time point of the study. At the end of the study, all animals were subjected to live micro–positron emission tomography–computed tomography (microPET-CT) scans at the University of California, Los Angeles, California NanoSystem Institute before harvest. Mice were preheated on a heating pad for 15 minutes before injection of 100 μCi of 18F-NaF ion via tail vein injection and maintained under anesthesia on a heated induction chamber during the 1-hour tracer uptake, as stated previously.30James A.W. Shen J. Zhang X. Asatrian G. Goyal R. Kwak J.H. Jiang L. Bengs B. Culiat C.T. Turner A.S. Seim Iii H.B. Wu B.M. Lyons K. Adams J.S. Ting K. Soo C. NELL-1 in the treatment of osteoporotic bone loss.Nat Commun. 2015; 6: 7362Crossref PubMed Scopus (84) Google Scholar, 38Kwak J.H. Zhang Y. Park J. Chen E. Shen J. Chawan C. Tanjaya J. Lee S. Zhang X. Wu B.M. Ting K. Soo C. Pharmacokinetics and osteogenic potential of PEGylated NELL-1 in vivo after systemic administration.Biomaterials. 2015; 57: 73-83Crossref PubMed Scopus (13) Google Scholar, 41Tanjaya J. Zhang Y. Lee S. Shi J. Chen E. Ang P. Zhang X. Tetradis S. Ting K. Wu B. Soo C. Kwak J.H. Efficacy of intraperitoneal administration of PEGylated NELL-1 for bone formation.Biores Open Access. 2016; 5: 159-170Crossref PubMed Scopus (7) Google Scholar Briefly, all mice underwent 10 minutes of microPET scanning (Siemens Medical Solutions Inc., Knoxville, TN), followed by microCT scanning (Siemens Inveon CT). The filtered back projection of superimposed microCT radiographs on the reconstructed microPET images was used for analysis with A Medical Image Data Examiner software version 1.0.5 (https://en.freedownloadmanager.org/Mac-OS/AMIDE-FREE.html). A standardized region of interest was drawn in a box of 10 × 10 × 10 mm3 to encompass the fracture site at the radii, whereas a box of 6 × 6 × 6 mm3 was drawn to cover the lower lumbar vertebrae (L6). Mean signal intensity (% injected dose/g) of the tracer uptake was calculated, and values were quantified with decay correction and normalized according to the actual tracer-injected dose. All specimens were scanned with SkyScan 1172 (Bruker microCT N.V., Kontich, Belgium), with settings of 55 kVp, 181 mA, and 0.5-mm Al filter at the resolution of 16 μm. Scanned images were then reconstructed with NRecon version 2.0.4 (Bruker microCT N.V.) for attenuation correction, ring artifact reduction, and beam hardening. Subsequently, samples were aligned in a three-dimensional view according to the long axis of the ulna with Data Viewer software version 1.5.4.0 (Bruker microCT N.V.). A total volume of interest of 200 slices was used for analysis, with the middle gap of the fracture as the midpoint reference. MicroCT images were analyzed with CTAn software version 1.13 (Bruker microCT N.V.) and segmented manually by comparing the binarized image with the unsegmented image. Threshold within the range of 60 to 225 was determined on the basis of subjective observation. The region of interest was manually drawn for improved accuracy by encompassing the callus and excluding the radius and ulna bones. Bone parameters of BMD (g/cm3), tissue mineral density, and trabecular thickness were used for analysis. CTVox software version 3.3 (Bruker microCT N.V.) was used to generate three-dimensional representative images and videos. The linear measurement was performed with CTAn software version 1.17.7.2 by using a modified fracture displacement method.42Grauer A.L. Roberts C.A. Paleoepidemiology, healing, and possible treatment of trauma in the medieval cemetery population of St. Helen-on-the-Walls, York, England.Am J Phys Anthropol. 1996; 100: 531-544Crossref PubMed Scopus (101) Google Scholar Most fractures were analyzed on the basis of the coronal or sagittal views of radiographs. Depending on the rotation of the object to the X-ray source, fracture displacement measurement was proved to be inconsistent unless a specific standardized protocol was established.43Owen J.L. Stephens D. Wright J.G. Reliability of radiographic measurement of fracture angulation in children with femoral shaft fractures.Can J Surg. 2007; 50: 115-118PubMed Google Scholar Herein, we established a standardized protocol for measuring fracture displacement by calculating the shortest distance between fractured bones and the uninjured ulna on the two-dimensional transaxial images. The ulna was selected as a stable reference point. Finite element models were generated by first converting microCT images into DICOM files using SkyScan Dicom Converter software version 2.5 (DicomCT application, SkyScan 1172F; Bruker microCT N.V.). Next, tetrahedral three-dimensional mesh models were generated by using Mimics software version 20.0 (Materialise, Leuven, Belgium) to isolate the volume of interest by drawing directly near the fracture site. The 3-matic version 12.0 (Materialise) was then used to remesh the models at an average of approximately 197,000 elements for each. Three-point bending finite element analysis simulation44Shefelbine S.J. Simon U. Claes L. Gold A. Gabet Y. Bab I. Muller R. Augat P. Prediction of fracture callus mechanical properties using micro-CT images and voxel-based finite element analysis.Bone. 2005; 36: 480-488Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar was then performed using the ABAQUS software version 6.6 (Dassault Systèmes, Velizy-Villacoublay, France), with boundary conditions set as encastre at the most distal and proximal points. A Young's modulus of 3.5 GPa and a Poisson's ratio of 0.33 were assigned to the radius and ulna.45Chung C.G. James A.W. Asatrian G. Chang L. Nguyen A. Le K. Bayani G. Lee R. Stoker D. Zhang X. Ting K. Peault B. Soo C. Human perivascular stem cell-based bone graft substitute induces rat spinal fusion.Stem Cells Transl Med. 2014; 3: 1231-1241Crossref PubMed Scopus (53) Google Scholar Subsequently, a uniform and uniaxial pressure of 0.5 MPa was applied at the fracture site, to test the biomechanical strength of the healed bone. The average von Mises stress experienced by the samples (n = 7 limbs per group) was analyzed. For the analysis of the sixth lumbar vertebrate (L6), the same workflow was used to obtain the meshed models, except for the volume of interest, which was now selected to be a 0.6 × 0.6 × 0.6-mm cube of trabeculae in the inferior portion of the vertebrae. On average, each meshed model is approximately 623,000 elements. In ABAQUS, a Young's modulus of 10 GPa and a Poisson's ratio of 0.33 were assigned to simulate the material properties of the trabeculae bone. Similarly, a uniaxial compressive load of 0.5 MPa was applied to the superior surface of the cube, whereas the inferior surface was set to encastre. The resulting average von Mises stress was then analyzed (n = 5 animals per group). For bone fluorescent labeling, mice were injected intraperitoneally with Calcein (20 mg/kg) and Alizarin Red Complexon (30 mg/kg; Sigma Aldrich, St. Louis, MO) at weeks 1 and 3, respectively. After microCT scanning, samples were embedded in methylmethacrylate and processed for a longitudinal cut with a microtome (n = 3 limbs per group). Bone double-labeling analysis was performed using the Osteomeasure Morphometric System version 3.0 (Osteometrics, Atlanta, GA), and parameters of mineral apposition rate (μm per day) and bone formation rate (μm3 μm−2 per day) were used. The mineral apposition rate is the distance between the labels/the interval of injection time, whereas bone formation rate multiplies mineral apposition rate by the fraction of bone surface that is labeled. The analysis was performed at the fracture site as selected volume of interest. For each section, three consecutive images were taken at the fracture sit
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