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Abstract: The aim of this study was to evaluate the effects of the remnants of two suture materials on osseointegration of titanium implants in a rabbit tibial model. Calibrated defects were prepared in the tibia of five Chinchilla rabbits. Filaments of nonresorbable (NR) nylon or resorbable (R) chitosan were placed at the bone to implant interface, whereas control sites had no suture material. After a healing period of 4 weeks, a pull-out test procedure was performed followed by enzymatic analyses of the wound fluid and relative quantification of mRNA levels for bone-related and cytokine markers from the peri-implant bone. A trend toward a reduced pull-out force was observed in the NR group (NR: 23.0 ± 12.8 N; R: 33.9 ± 11.3 N; control: 33.6 ± 24.0 N). Similarly, the bone resorption marker vacuolar type H+-ATPase was increased in the NR group compared with that in the control group (P = 0.041). The R group showed trends for lower alkaline phosphatase activity and osteocalcin expression and higher total protein content and RNA compared with the control group. In this submerged healing model, peri-implant bone healing was marginally affected by the two suture materials tested. However, there was a tendency toward better osseointegration and lower expression of bone resorption markers in the R group

compared with the control group. (J Oral Sci 57, 219-227, 2015)

Keywords: suture materials; chitosan; nylon; dental implants; bone; wound healing; osseointegration; biological complications.

IntroductionForeign materials, such as cement remnants from the supraconstruction, can sometimes lead to peri-implant tissue complications (1). Theoretically, suture remnants displaced toward the peri-implant bone may have a similar negative effect on the peri-implant tissues. The choice of suture material may potentially influence the outcome of periodontal and implant procedures (2,3). The ideal suture material should provide uncomplicated stable primary wound closure without affecting the treatment outcome negatively. However, suture materials often seem to worsen the inflammatory response for a variety of reasons (4); therefore, the selection of an appropriate material is important (5,6). Materials that minimize inflammatory reactions are preferred, particularly in an environment, such as the oral cavity, where microbial levels are high. Moreover, suture materials and their metabolites might affect the healing process, particularly after implant placement, and can also provide a route for communication between the fixture and the oral cavity (2). Moreover, the wicking effect of some suture types can result in harbouring and transporting bacteria to the depth of the wound; thus, evading the host defense mechanism, resulting in local infection and subsequent

Journal of Oral Science, Vol. 57, No. 3, 219-227, 2015

Original

Suture materials affect peri-implant bone healing and implant osseointegration

Oscar Villa1), Staale P. Lyngstadaas1), Marta Monjo2), Maria Satué2), Hans J. Rønold3), Christiane Petzold1), and Johan C. Wohlfahrt1)

1)Department of Biomaterials, Institute of Clinical Dentistry, University of Oslo, Oslo, Norway2)Group of Cell Therapy and Tissue Engineering, Research Institute on Health Sciences (IUNICS),

University of Balearic Islands, Palma de Mallorca, Spain3)Department of Prosthodontics, Institute of Clinical Dentistry, University of Oslo, Oslo, Norway

(Received March 25, 2015; Accepted May 19, 2015)

Correspondence to Dr. Johan Caspar Wohlfahrt, Department of Biomaterials, Institute of Clinical Dentistry, University of Oslo, P.O. Box 1109, Blindern, NO-0317 Oslo, NorwayE-mail: j.c.wohlfahrt@odont.uio.nodoi.org/10.2334/josnusd.57.219DN/JST.JSTAGE/josnusd/57.219

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acute inflammatory reaction (6,7).Suture materials are either resorbable (R) or nonresorb-

able (NR). They can also be classified into mono- and multifilamentous or braided on the basis of their physical configuration (8). Braided sutures provide easier handling and better knot stability compared with mono-filament types; however, they present a higher potential for transmitting infections (9). Monofilament sutures are usually NR, with few exceptions. Nylon consists of a group of materials with the same chemistry, and it is the most commonly used NR suture material (10). Nylon sutures can be either monofilamentous or braided and consist of synthetic polyamide polymer fibers (8). They present clinically acceptable biocompatibility and a low inflammatory response in infected wounds (4). However, there are few R monofilament suture materials. Chitin is a natural polysacharide (a poly-N-acetyl glucosamine) commonly found in the exoskeleton of crustaceans, cuticles of many invertebrates, and cell walls of fungi (11). Chitosan is obtained by deacetylation of chitin. Chitosan sutures are generally R and monofilamentous and present excellent properties for surgical application (12). They have been demonstrated to have antibacte-rial (13-15) and anti-inflammatory (16) properties and improve wound healing (17-19) and bone formation (20). They also reduce bacterial rates by 100% (21). This is an advantage as bacteria can penetrate along the suture track, leading to an inflammatory tissue reaction (6). In vitro studies have demonstrated that chitosan decreases macrophage production of pro-inflammatory cytokines, suppresses cyclooxygenase induction, and increases the release of anti-inflammatory cytokines (16). Moreover, clinical studies reported that it also improved wound healing (17-19) and increased wound breaking strength in a rat model (22). In a dog model, chitosan was shown to promote migration of inflammatory cells into the deeper wound area and increase formation of connective tissue through increased production of collagen (23). Okamoto (24) found fewer inflammatory cells migrating to the wound site in the chitosan group and a trend to enhance re-epithelialization. It also induced bone formation in surgically created bone defects in an experimental animal model but showed no indication of the same in the control group (20). To the best of our knowledge, the influence of suture remnants on peri-implant bone healing has not yet been investigated. The aim of the present study was to evaluate the effects of monofilaments of NR nylon and R chitosan materials on peri-implant bone healing in a validated animal model.

Materials and MethodsIn vivo studyThis study used five female chinchilla rabbits (Oryc-tolagus cuniculus), 6 months old and weighing 3.0-3.5 kg (ESF Produkter Estuna AB, Norrtälje, Sweden). The animals were kept in cages during the experimental period. Room temperature was maintained at 19 ± 1°C, and humidity at 55 ± 10%.

The experiment was approved by the Norwegian Animal Research Authority. The procedures were conducted in accordance with the Animal Welfare Act of January 1, 2010, Section 13 and the Regulation on Animal Experimentation of January 15, 1996.

The rabbits were sedated with 0.05-0.1 mL/kg subcu-taneous (s.c.) fluanisone/fentanyl (Hypnorm, Janssen, Belgium) and anesthetized with 2 mg/kg intravenously (i.v.). Midazolam (Dormicum, Roche, Switzerland) 10 min before surgery. In case of signs of waking up, diluted Hypnorm was injected i.v. slowly until adequate sedation effect was achieved. Lidocaine/adrenaline (Xylocaine/Adrenaline, AstraTech AB, Mölndal, Sweden) was administered locally at the operation site. The operation sites were depilated and washed with soft soap before surgery. Animals were then placed on their backs on the operating table and covered with sterile cloths, and the operating sites were disinfected with chlorhexidine gluconate 5 mg/mL (Klorhexidin, Galderma Nordic AB, Uppsala, Sweden).

An incision was made on the proximal-anterior part of the tibia, penetrating all soft tissue layers. The periosteum was elevated and retained by a self-retaining retractor. Four guide holes were made with a twist drill (Medicon CMS, Tuttlingen, Germany), using a drill guide to ensure standardized and correct positioning of the implants. Implant beds were prepared using a custom-made stainless steel bur (diameter 6.25 mm) mounted on a slow-speed dental implant drill. Copious irrigation with physiological saline solution was carried out during bone preparation. Four sites were prepared in each animal and then randomized to one of the three groups. Control sites were left empty. In the two test groups, one of the following suture materials were placed in the prepared implant sites prior to implant placement (Fig. 1):・NR monofilament nylon suture (Nylon 6/12, Dupont, Wilmington, DE, USA) USP size 6-0.・R monofilament chitosan suture (Medovant GmbH, Mainz, Germany), USP size 6-0.

The suture filaments were carefully placed on the prepared bone surface, perpendicular to the implant direc-tion, using thin tweezers. Four 5-mm fibers were placed at each implant site, avoiding fiber overlapping and over-

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filling. A coin-shaped, machined, titanium implant (6.25 mm in diameter and 1.95 mm in thickness), covered with a Teflon cap to avoid bone overgrowth, and a preshaped titanium maxillofacial bone plate (Medicon CMS) were placed over the test and control sites and retained with two titanium mini screws (1.2 × 3 mm, Medicon CMS). The soft tissue was repositioned and sutured in place with DexonII (Sherwood-Davis & Geck, Ballymoney, UK). Following surgery, each animal received an s.c. injection of 20 mL NaCl, warmed to body temperature, and 0.02-0.05 mg/kg buprenorphin (Temgesic, Reckitt & Colman, Hull, UK). A second s.c. injection of 0.05 mg/kg Temgesic was administered at least 3 h after the first/previous Temgesic injection. Health status was monitored throughout the study period, and the operation sites were examined daily until wound healing was complete. After a healing period of 4 weeks, the rabbits were euthanized using fluanison/fentanyl (Hypnorm) 1.0 mL i.v., followed by pentobarbital (Mebumal, Rikshospitalets Apotek, Norway) 1 mL/kg body weight i.v. An incision was made through the soft tissue up to the tibial bone immediately after euthanization. The titanium plate covering the implants was exposed and removed. A hole was made in the center of the PTFE cap with a hypodermic syringe, and pressurized air was applied to remove the caps and expose the reverse part of the titanium implant.

Tensile force test procedureThe excised tibial bone was fixed and leveled for the tensile force test. The pull-out test was performed with a tensile test machine (Zwicki; Zwick, Ulm, Germany) equipped with a 200 N load cell. A pull-out jig was attached to the screw hole on the backside of the implants, perpendicular to the implant test surface, and the load was applied until the implant detached from the bone.

The force applied was recorded on a load versus displacement plot. The implant-bone attachment was evaluated immediately after euthanasia, so that the results were not influenced by drying. It was possible to analyze

whether shear forces skewed the test from the load versus displacement plot recorded. An immediate drop in the curve indicated little influence of shear forces, whereas a tapering curve indicated a greater influence.

Wound fluid analyses: lactate dehydrogenase activity, alkaline phosphatase activity, and total proteinAfter implant detachment, two filter chromatography paper discs (same size as the coins) were placed in each drilled hole (proximal and distal) for 1 min to absorb wound fluid. Samples were then transferred to microcen-trifuge tubes containing 200 µL of phosphate-buffered saline (PBS) and placed on ice until analysis was carried out later the same day.

Lactate dehydrogenase (LDH) activity in the wound fluid was used as a marker for tissue necrosis (25). Spec-trophotometric analysis of LDH levels was carried out by first incubating 50 µL of wound fluid and 50 µL of the reaction mixture at 25°C for 30 min and then measuring the oxidation of NADH at 490 nm in the presence of pyruvate, according to the manufacturer’s kit instruc-tions (Cytotoxicity Detection kit, Roche Diagnostics, Manheim, Germany).

Alkaline phosphatase (ALP) activity was used as an indicator of mineralization around the titanium implants (26). An aliquot of 25 µL of wound fluid in duplicate was assayed for alkaline phosphatase activity by measuring the cleavage of p-nitrophenyl phosphate (Sigma, St. Louis, MO, USA) in a soluble, yellow end product that absorbs at 405 nm. A volume of 100 µL of this substrate was used. The reaction was stopped after 30 min in the dark by adding 50 µL of 3 M sodium hydroxide. The absorbance of the ceased reaction was read at 405 nm. Similar to the samples, a standard curve with calf intes-tinal alkaline phosphatase (CIAP, 1 U/µL); Promega, Madison, WI, USA) was constructed by mixing 1 µL of stock CIAP with 5 mL of alkaline phosphatase buffer (1:5,000 dilution) and then preparing 1:5 serial dilu-tions. Total protein in the wound fluid for each sample was determined using a BCA protein assay kit (Pierce, Rockford, IL, USA) according to the manufacturer’s kit instructions.

RNA isolation and real-time RT-PCR analysesA monophasic solution of phenol and guanidine thio-cyanate (Trizol, Life Technologies, Carlsbad, CA, USA) was used to isolate total RNA from the peri-implant bone tissues attached to the extracted implants, which was then quantified using a Nanodrop spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA) at 260 nm. The same amount of RNA (280 ng of the total RNA

Fig. 1 Intrasurgical pictures showing the suture filaments placed over the bone defect (a). The implant with a Teflon cap placed over the test site (b).

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isolated) was reverse transcribed to cDNA at 42°C for 60 min, using a High Capacity RNA-to-cDNA kit (Applied Biosystems, Foster City, CA, USA) that contains oligo and random hexamers. Each cDNA was diluted 1/4, and the aliquots were frozen (-20°C) until the PCR reactions were carried out.

The three housekeeping genes used were 18S ribosomal RNA (18S rRNA), glyceraldehyde-3-phosphate dehy-drogenase (GAPDH), and β-actin. The target genes were vacuolar type proton ATPase (H+-ATPase), insulin-like growth factor-1 (IGF-1), bone morphogenetic protein-2 (BMP-2), collagen-type 1 (Coll-1), interleukin-10 (IL-10), IL-6, osteocalcin (OC), calcitonin receptor (CalcR), tartrate-resistant acid phosphatase (TRAP), and tumor necrosis factor-α (TNF-α). Primer sequences have been published elsewhere (26,27). Real-time PCR was performed in the Lightcycler 480 (Roche Diagnostics). Each reaction contained 7 µL of LightCycler-FastStart DNA Master SYBR Green I dye, 0.25 µL of sense and antisense specific primers, and 3 µL of cDNA dilution resulting in a final volume of 10 µL. The amplification program consisted of a preincubation step for denatur-ation of the template cDNA (10 min, 95°C), 45 cycles of the denaturation step (10 s, 95°C), an annealing step (10 s, 60°C), and an extension step (10 s, 95°C). After each cycle, fluorescence was measured at 72°C. A negative control without cDNA template was used in each assay.

Real-time efficiencies were calculated from the slopes provided in the LightCycler 480 software (Roche Diag-nostics) using serial dilutions showing all the investigated transcripts, high real-time PCR efficiency rates, and high linearity when different concentrations are used. PCR products were subjected to a melting curve analysis on the LightCycler, and subsequently 2% agarose/TAE gel electrophoresis to confirm amplification specificity, Tm, and amplicon size, respectively.

All samples were normalized by the geometric mean of the expression levels of 18S rRNA, GAPDH, and β-actin, and fold changes were related to 4 weeks of implant placement using the mathematical model described by Pfaffl (28) as follows: ratio = Etarget ∆Cp target (mean control 4

weeks−sample)/Ereference ∆Cp target (mean control 4 weeks-sample), where Cp is the crossing point of the reaction amplification curve as determined by the LightCycler 480 software. Stability of reference genes was calculated using the BestKeeper tool (29). Thus, values were expressed as a percentage of the control defects at 4 weeks, which were set to 100.

Statistical analysisAll data are presented as mean values ± SEM. Differ-ences between the test and control groups were examined

using the Student t-test. All analysis was performed on SPSS program for Windows (Chicago, IL, USA) version 17.0, and the overall significance threshold (α) was set at 0.05.

ResultsTensile testBiomechanical evaluation of implants using the tensile test showed no significant differences between the groups (Fig. 2). However, a trend toward a lower pull-out

Fig. 2 Tensile test measurements of implants. Box plots show the median value and distribution of the measurements for each group (n = 5). CTRL, control; NR, nonresorbable monofilaments; R, resorbable monofilaments.

Fig. 3 Picture of the bone defects after implant removal. Resorbable (a) and nonresorbable monofilaments (b). Arrows indicate filament location.

Fig. 4 LDH activity measured in the wound fluid collected from the implant site. LDH activity was expressed as percentage of the control operated site, which was set to 100%. Values represent mean ± SEM (n = 5). CTRL, control; LDH, lactate dehydrogenase; NR, nonresorbable monofilaments; R, resorbable monofilaments.

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force was observed in the NR group (approximately 30% decrease).

Macroscopic observation of the implant site and wound fluid analysesAll implant sites showed uneventful healing, and no adverse effects were observed. The implant was visu-ally inspected after removal. All sites showed minimal bleeding, regardless of the treatment group, after 4 weeks (Fig. 3). R sutures were still present in all defects but one (Fig. 3). There were no differences in the total LDH activity, ALP activity, and protein content of peri-implant wound fluid between the test and the control groups (Figs. 4, 5). However, on calculating specific ALP activity by correcting ALP activity with total protein present in the wound fluid, a trend toward lower activity was found in the R group (54% decrease) and NR group (43% decrease) when compared with the control group (Fig. 5). The R group also showed a trend for increased total protein content (21-30%), when compared with the NR and control groups (Fig. 5).

Gene expression analyses from peri-implant bone tissueNo statistically significant differences in total RNA content of bone tissue attached to the extracted implants were found between the groups, although the R group showed a trend toward higher total RNA content (Fig. 6).

There were no statistically significant differences in gene expression markers related to bone formation, i.e. BMP-2, Coll-1, OC, and IGF-1 (Fig. 7), between the test and the control groups, although there was a trend toward decreased OC expression in the R group compared with the control group (P = 0.086).

The three markers related to differentiated osteoclasts and bone resorption, i.e., H+-ATPase, TRAP, and CalcR, were also investigated: Gene expression of H+-ATPase was increased in the NR group compared with the control group, and this was statistically significant (P = 0.041). No such differences were found with regard to the expression of TRAP and CalcR (Fig. 8).

The gene expression of pro-inflammatory (TNF-α and IL-6) and anti-inflammatory (IL-10) cytokines was also investigated, and no statistically significant differences were found between the test and control groups (Fig. 9). However, the mRNA levels of IL-10 were reduced in the NR and R groups, although these were not statistically significant (P = 0.069 and 0.125, respectively). There was also a trend toward reduced expression of IL-6 in the R group compared with the control group, and this was also not statistically significant (P = 0.098).

DiscussionThe present study shows that suture materials, such as nylon or chitosan, may have an impact on early peri-implant osseous healing. Although the results were not conclusive, they suggest that processes like bone resorp-tion and inflammation may be altered by the presence of suture materials and that different materials may act differently on these processes. Presence of NR material

Fig. 5 ALP activity, total protein, and specific ALP activity (corrected by total protein) measured in the wound fluid collected from the implant site. Total protein content (expressed in microgram) extracted from each defect. Values represent mean ± SEM (n = 5). ALP, alkaline phosphatase; CTRL, control; NR, nonresorbable monofila-ments; R, resorbable monofilaments.

Fig. 6 Quantification of total RNA content (ng/defect) isolated from the peri-implant bone tissues attached to the extracted implants. Values represent mean ± SEM (n = 5). CTRL, control; NR, nonresorbable monofilaments; R, resorbable monofilaments.

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at the bone-implant interface decreased implant retention to a greater extent than other groups and also resulted in statistically significant higher gene expression of the bone resorption marker H+-ATPase. The R group showed a trend toward lower specific ALP activity and

OC expression, but this did not influence the outcome of the biomechanical test used to assess bone-implant attachment.

Although tissue reactions around nylon monofilament sutures have been reported to be minimal (30), they can

Fig. 7 In vivo expression of bone formation related markers (BMP-2, Coll-1, and OC), and a bone repair marker (IGF-1). Data represent fold changes of target genes normalized with reference genes (18S, GAPDH, β-actin), expressed as a percentage of the control group set at 100%. Values represent the mean ± SEM (n = 5). BMP-2, bone morphogenetic protein-2; Coll-1, collagen-type 1; CTRL, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IGF, insulin-like growth factor-1; NR, nonresorbable monofilaments; OC, osteocalcin; R, resorb-able monofilaments.

Fig. 8 In vivo expression of bone resorption related markers (H+-ATPase, TRAP, and CalcR). Data represent fold changes of target genes normalized with reference genes (18S, GAPDH, β-actin), expressed as a percentage of the control group set at 100%. Values represent the mean ± SEM (n = 5). CTRL, control; GAPDH, glyceral-dehyde-3-phosphate dehydrogenase; NR, nonresorbable monofilaments; R, resorbable monofilaments; TRAP, tartrate-resistant acid phosphatase.

Fig. 9 In vivo expression of inflammatory markers (TNF-α, IL-6, and IL-10). Data represent fold changes of target genes normalized with reference genes (18S, GAPDH, β-actin), expressed as a percentage of the control group set to 100%. Values represent mean ± SEM (n = 5). CTRL, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL, interleukin; NR, nonresorbable monofilaments; R, resorbable monofilaments; TNF-α, tumor necrosis factor-α.

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elicit a foreign body tissue reaction in certain clinical situations (31,32). In our study, visual inspection of the bone defects after implant removal and measurement of LDH activity indicated that nylon sutures did not induce any adverse reactions at the bone-implant interface. However, nylon suture remnants showed a trend toward decreased implant attachment compared with chitosan monofilaments. Moreover, increased gene expression of H+-ATPase was observed in the nylon group. The vacuolar-type proton pump (H+-ATPase) is present in the ruffled membrane of osteoclasts and is involved in osteoclastic bone resorption (33). Thus, the lower implant retention observed in the NR group may be related to higher bone resorption at the bone-implant interface. This might be explained by the nature of the nylon sutures used in the present experiment. The presence of NR filaments at the bone-implant interface may interfere with the initial phase of bone healing, and histological evaluation could provide further insight into the osseointegration process taking place at the interface. In the present study, the bone-implant interface was disrupted on extraction of the implants, and the samples were processed for enzymatic and molecular analyses. Therefore, histological analysis of the intact interface could not be carried out.

The chitosan-based R suture material used in this study was still present in all defects, except for one site, after a healing period of 4 weeks. The degradation time of the chitosan material in this closed environment may be longer than in an inflamed highly vascularized soft tissue. Some R suture materials require longer periods to degrade, even in infected or acidic environments (6,34). As reviewed by Pillai et al. (35), several modifications can be made to the polymerization degree and polymer structure of chitosan such as etherification, esterifica-tion, cross-linking, and grafting copolymerization to enhance solubility. Moreover, the biodegradability of chitosan fibers increases with degree of acetylation (36). All these parameters should be taken into consideration when selecting a suture for implant surgical procedures. Although a suture with faster biodegradation rate might be preferable when suturing peri-implant tissues, it may result in rapid resorption at the wound causing loss of wound stability that can potentially affect the osseointe-gration process. Moreover, the degradation process leads to different degrees of inflammatory response depending on the suture material (8). A trend toward a higher amount of total RNA and protein content was found in the R group compared with the NR group. This may reflect a proliferative stage (37) at the implant interface and the presence of bone proteins at an early stage of bone healing (26). These results may be explained by

the higher tissue reactivity and proteolytic enzymatic degradation shown by the R material used in the present experiment. Thus, biodegradation of chitosan fibers may lead to a specific cell response that can influence healing in the peri-implant tissues. Chou et al. (16) found that chitosan-treated macrophages decreased expression of pro-inflammatory cytokines (TNF-α and IL-6). In the present study, a trend toward reduction of TNF-α and IL-6 was also found in the chitosan group. Moreover, a trend toward reduced expression of IL-10 was found in our study, while increased production of IL-10 by macrophages was reported by Chou et al. (16). Chitosan-coated culture plates have been reported to upregulate genes associated with Coll-1, osteopontin, osteonectin, and OC in osteoblasts (38). Similarly, it has been shown that chitosan-coated fibers increased cell attachment, ALP activity, and the expression of osteopontin on pre-osteoblasts, resulting in significant mineralization in a 3D construct (39) and new bone formation in a rabbit calvarial model (40). Our in vivo experiment differs from the in vitro results from Matthews et al. (38) and Yang et al. (39) as the expression of Coll-1 was not modified, the specific ALP activity was reduced, and a trend toward reduction in the expression of OC was found. OC is a marker for mineralization (41) and has been associated with osseointegration (37). Thus, this observed trend might be related to an ongoing remodeling in the peri-implant bone tissue that takes place during the resorption process of the suture material.

This study examined the effects of suture materials on early wound healing and showed that sutures may influence early peri-implant healing if filaments are left in close proximity to the bone-implant interface. Further investigations are required to determine the influence of a broader number of sutures materials on peri-implant bone healing.

AcknowledgmentsThis work was supported by the University of Oslo, the Norwe-gian Research Council and the Ministerio de Ciencia e Innovación del Gobierno de España (Ramón y Cajal contract to MM) and NILS Science and Sustainability Programme (ES07 EEA Grants; 013-Abel-CM-2013). The authors have no conflicts of interest to declare.

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