Cisplatin Inhibits Bone Healing during Distraction Osteogenesis Kimo C. Stine,1 Elizabeth C. Wahl,2 Lichu Liu,2 Robert A. Skinner,3 Jacquelyn VanderSchilden,3 Robert C. Bunn,1 Corey O. Montgomery,3 Larry J. Suva,3 James Aronson,1,2,3 David L. Becton,1 Richard W. Nicholas,3 Christopher J. Swearingen,1,4 Charles K. Lumpkin Jr.1,2 1

Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, Arkansas, 2Laboratory for Limb Regeneration Research, Arkansas Children’s Hospital Research Institute, Little Rock, Arkansas, 3Department of Orthopaedic Surgery, Center for Orthopaedic Research, University of Arkansas for Medical Sciences, Little Rock, Arkansas, 4Pediatric Biostatistics, Arkansas Children’s Hospital Research Institute, Little Rock, Arkansas Received 16 July 2013; accepted 1 November 2013 Published online 20 November 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jor.22527

ABSTRACT: Osteosarcoma (OS) is the most common malignant bone tumor affecting children and adolescents. Many patients are treated with a combination of chemotherapy, resection, and limb salvage protocols. Surgical reconstructions after tumor resection include structural allografts, non-cemented endoprostheses, and distraction osteogenesis (DO), which require direct bone formation. Although cisplatin (CDP) is extensively used for OS chemotherapy, the effects on bone regeneration are not well studied. The effects of CDP on direct bone formation in DO were compared using two dosing regimens and both C57BL/6 (B6) and tumor necrosis factor receptor 1 knockout (TNFR1KO) mice, as CDP toxicity is associated with elevated TNF levels. Detailed evaluation of the five-dose CDP regimen (2 mg/kg/day), demonstrated significant decreases in new bone formation in the DO gaps of CDP treated versus vehicle treated mice (p < 0.001). Further, no significant inhibitory effects from the five-dose CDP regimen were observed in TNFR1KO mice. The twodose regimen significantly inhibited new bone formation in B6 mice. These results demonstrate that CDP has profound short term negative effects on the process of bone repair in DO. These data provide the mechanistic basis for modeling peri-operative chemotherapy doses and schedules and may provide new opportunities to identify molecules that spare normal cells from the inhibitory effects of CDP. ß 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:464–470, 2014. Keywords: cisplatin; distraction osteogenesis; chemotherapy; mouse; limb salvage

Osteosarcoma (OS) is the most common malignant bone tumor affecting children and adolescents.1 Most patients are treated with a combination of chemotherapy and surgery with limb salvage being possible in many cases.2 Surgical reconstruction utilizing limb salvage protocols are now the standard of care in most tertiary care centers, yet these patients continue to be at risk for post-operative complications related to poor bone regeneration.1–4 Poor direct bone regeneration often leads to failure of the implanted endoprothesis, the engrafted bone, or of the lengthened bone.1–4 Currently, anti RANKL therapy (Prolia1, Amgen, Thousand Oaks, CA) and zoledronic acid (Zometa1, Novartis, Basel, Switzerland) are the only FDAapproved therapies for cancer treatment induced bone loss (CTIBL).5 However, few therapies are currently available for the treatment and/or repair of bone repair deficits.4 Chemotherapeutic agents that are used widely and accepted as efficacious in patients with OS include doxorubicin, high-dose methotrexate and cisplatin (CDP, cis-diamminedichloroplatinum (II), cis-platinum).6–9 Neoadjuvant chemotherapy followed by surgery with

Grant sponsor: Children’s University Medical Group Fund Grant Program, the Arkansas Children’s Hospital Research Institute; Grant sponsor: Arkansas Tobacco Settlement Plan; Grant sponsor: UAMS Pediatric Hematology/Oncology Division; Grant sponsor: Carl L. Nelson Endowed Chair in Orthopaedic Creativity; National Institutes of Health National Center for Research Resources; Grant number: UL1 RR029884; Grant sponsor: NIH National Center for Research Resources; Grant number: #1CORR16517-01. Correspondence to: Charles K. Lumpkin (T: 501-364-2797; F: 501-364-5880; E-mail: [email protected]) # 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

464

JOURNAL OF ORTHOPAEDIC RESEARCH MARCH 2014

additional adjuvant chemotherapy has become a common treatment approach (Cure Search/COG). However, since CDP is extensively used for the treatment of OS, we believe the effects of CDP treatment on direct bone regeneration need further study.10–12 Therefore, we tested the effects of two CDP dosing regimens on bone regeneration in a mouse model of distraction osteogenesis (DO).13–16 During DO, bone formation process spatially well organized and temporally distinct from resorptive processes induced later. In addition, recent literature suggests that DO is emerging as an appropriate protocol for osteosarcoma patients and does provide significant benefit to patients compared with other critical size defect repair procedures.17–20 As such, the studies described herein provide important new information regarding potential clinical applications.20 Based on previous studies, we observed that exposure to chemotherapy can occur preoperatively, postoperatively, and even simultaneously with bone repair/regeneration.21 Therefore, to develop and standardize a mouse model for CDP toxicity we chose to perform the DO surgery simultaneously with the first of five daily CDP injections. The dosage and delivery was also chosen based on previous experimental and clinical protocols (Cure Search: Children’s Oncology Group/COG).21 The CDP dose was 2 mg/kg/day which is approximately equivalent to the pediatric dose of 60 mg/m2/day. CDP administered during this protocol produced significant damage to the regenerating bone. Based on previous studies that demonstrated (1) excess levels of tumor necrosis factor alpha (TNFa) are inhibitory to bone regeneration in DO and act through TNF receptor 1 (TNFR1),22 and (2) that CDP toxicity is associated with elevated TNFa levels,23,24 we tested

CISPLATIN AND DISTRACTION OSTEOGENESIS

the hypothesis that the TNFR1 axis was mediating the negative effects of this regimen of CDP dosing using TNFR1 knockout mice.22 Finally, to model more closely the current human therapeutic CDP dose common in clinical practice and to examine the persistence of the CDP effects, the same dose of CDP was administered for two consecutive days followed a week later by the initiation of the standard DO protocol. In sum, both the five dose and two dose regimens produced significant inhibitory effects on new bone formation, suggesting that the inhibition of bone repair in CDP-treated OS patients may be associated with CDP-mediated dysregulation of TNFa signaling.

MATERIALS AND METHODS Animals Adult male C57BL/6 (B6, Jax #000664) and TNFR1 KO (Jax #002818) mice were purchased from Jackson Industries (Bar Harbor, ME). Animals were housed in individual cages in temperature (22˚C) and humidity (50%) controlled rooms having a 12 h light/12 h dark cycle. All mice were acclimated by animal care personnel for 5–7 days prior to surgery. In all studies, the mice were assigned to respective experimental groups with mean body weights equal to that of the control group (4 g) for the study. All research protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Arkansas for Medical Sciences. Study Designs

Study 1: Effects of CDP Treatment on DO in B6 Mice: Regimen 1 To study the effects of CDP administration on direct bone formation in the mouse DO model, 9-week old C57BL/6 male mice (n ¼ 20) underwent the standard DO protocol (described below). All mice received an IP injection (under isoflurane anesthesia) of either vehicle (saline) or CDP (2 mg/kg/day in saline) daily for 5 consecutive days beginning the day of surgery. The CDP was made fresh daily and at harvest distracted tibiae were collected.

Study 2: Effects of CDP Treatment on DO in TNFR1 Knockout Mice Based on previous studies which suggest that TNFR1 mediates the negative effects of exogenous TNF, we performed a study identical to that above except we used 9-week old male TNFR1 deficient mice (n ¼ 6: saline & n ¼ 6 CDP).22 Serum TNF levels were measured.

Study 3: Effects of CDP Treatment on DO in B6 Mice: Regimen 2 To better model clinical practice and to look at the persistence of the CDP effects, we administered the same dose of CDP (2 mg/kg/day) for 2 consecutive days followed 1-week later by DO. The selected dose is equivalent of the clinical dose but may underestimate overall dose due to the higher metabolism of the mouse. Male 12-week old B6 mice were used (n ¼ 6: saline vs. n ¼ 6 CDP). Distraction Protocol Distraction was performed as we have previously described.14,22,25 Following acclimation and under Nembutal anesthesia, each mouse underwent placement of an external fixator and osteotomy to the left tibia.22 Four 27-gauge, 1.25 in needles were manually drilled through the tibia (two

465

proximally, two distally). The titanium external fixator was then secured to the pins. A small incision was made in the skin distal to the tibia crest and the soft tissue was carefully retracted to visualize the bone. A single hole was manually drilled through both cortices of the mid-diaphysis, and surgical scissors were used to fracture the cortex on either side of the hole. The fibula was fractured by direct lateral pressure. The periosteum and dermal tissues were closed with a single suture. Finally, buprenex (1.0 mg/kg) was given by intramuscular injection post-surgery for analgesia. Distraction began 3 days after surgery (3-day latency) at a rate of 0.075 mm b.i.d. (0.15 mm/day) and continued for 11 days at which time the tibias were harvested. MicroCT (mCT) Analysis Following analytical radiography, bone formation was analyzed using a mCT40 (Scanco Medical AG, Bassersdorf, Switzerland) and the manufacturer’s software. The distracted tibia including the entire distraction gap was scanned in cross section with an isotropic voxel size of 12 mm at 55 kV, 114 mA, and 1,000 projections per rotation with threshold and noise filter the same as that used for the trabecular analysis of intact mouse bones (sigma 0.8, support 1, threshold 245). The DO gaps and new bone formation were analyzed as we have described.25

Radiographic and Histologic Analysis After 48 h of fixation in 10% neutral buffered formalin the left tibiae were removed from the fixators for high-resolution single beam radiography and subsequent histological processing. Radiographs were quantified as we have described.13,15,22 Following radiography, the distracted tibiae were decalcified in 5% formic acid, dehydrated, and embedded in paraffin for histologic evaluation.26 Five to seven micron longitudinal sections were cut on a microtome (Leitz 1512, Wetzlar, Germany) for hematoxylin and eosin staining (H&E). Sections were selected to represent a central or near central gap location as we have described.14,22,25 To be included in both radiographic and histologic analyses the DO samples had to (1) be well aligned, (2) have no broken pin sites, (3) have few bone chips within the DO gap, (4) have an intact ankle, and (5) have had no significant weight loss or health problems during the distraction period as we have described previously.14,22,25 Serum Mouse TNFa Bead Array Murine serum samples were analyzed on a Luminex1 100TM Bioanalyzer in the Pediatric Endocrinology Core Facility, using the Linco mouse adipokine kits (MPXMCYTO-70K-1). Statistics Differences between individual group means were estimated by the Student’s t-test. All data are reported as mean  standard error of the mean (SEM). When assumptions of equal variances between groups failed, differences between group means were estimated using a generalized linear model assuming the underlying distribution of the outcome.27 Differences were considered significant when p < 0.05 and are reported as such.

RESULTS Study 1: CDP Exposure during the Early Stages of DO Inhibits New Bone Formation: Regimen 1 The study was designed to test the hypothesis that a five-dose administration of CDP during the early JOURNAL OF ORTHOPAEDIC RESEARCH MARCH 2014

466

STINE ET AL.

stages of the distraction protocol would result in osteoinhibition in B6 mice. Comparison of the distracted tibial radiographs demonstrated a significant decrease in the mineralized area of distraction gaps of CDP treated mice (3.6%  2.1) versus vehicle treated mice (63.3%  5.8) (p < 0.001) (Fig. 1). Histological analysis of the DO gaps confirmed the significant decrease in cellular bone formation between the CDP treated mice (7.6%  7.0) versus vehicle treated mice (75.4%  5.4) (p < 0.001) (Fig. 1). Representative radiographs, and histograms are shown in Fig. 2A,B. Analysis of the same distracted tibiae by microCT (Fig. 3A) demonstrated significant decreases in both new endosteal and periosteal bone formation in CDPtreated mice compared with vehicle-treated controls (Fig. 3B,C). One mouse was excluded from both analyses. Study 2: Effects of CDP Treatment on DO in TNFR1 Knockout Mice We next examined the role of TNF in this process using TNFR1-deficient mice. Comparison of the distracted tibial radiographs demonstrated no significant decrease in mineralized area in the distraction gaps of CDP treated TNFR1 knockout mice (20.6%  8.7) versus vehicle treated knockout mice (44.1%  7.5) (p ¼ 0.07). Similarly, histological analysis of the DO gaps showed no significant decrease in cellular bone formation between the CDP treated mice (40.5%  12.7) versus vehicle treated mice (64.1%  4.0) (p ¼ 0.09) (Fig. 4). However, standard deviations between CDP treated mice and vehicle treated were statistically different (p ¼ 0.04). Therefore a generalized linear model assuming a beta distribution (i.e., beta regression) was used.27 Accounting for differences in variability between treatment groups indicates that the decrease in cellular bone formation between the CDP treated mice

Figure 1. Five-day CDP Regimen 1: Left: Comparison of the distracted tibial radiographs. A significant decrease in the mineralized area of distraction gaps of CDP treated mice (3.6%  2.1) versus vehicle treated mice (63.3%  5.8) (p < 0.001). Right: Histology of the DO gaps. A significant decrease in cellular parameters of bone formation between the CDP treated mice (7.6%  7.0) versus vehicle treated mice (75.4%  5.4) (p < 0.001). JOURNAL OF ORTHOPAEDIC RESEARCH MARCH 2014

Figure 2. Representative radiographs (A) and H&E histology (B) for B6 mice in Regimen 1 are shown. In 2B new bone is outlined by dashes.

(37.6%  11.1) versus vehicle treated mice (64.0%  3.5) is statistically significant (p ¼ 0.024). As previously reported,22 serum TNF analyses demonstrated relatively higher levels of TNF in both groups of the TNFR1 knockout mice (32.6  7.7 pg/ml vs. wild type below sensitivity at

Cisplatin inhibits bone healing during distraction osteogenesis.

Osteosarcoma (OS) is the most common malignant bone tumor affecting children and adolescents. Many patients are treated with a combination of chemothe...
760KB Sizes 0 Downloads 0 Views