Increased bone remodelling around titanium implants coated with chondroitin sulfate in ovariectomized rats

Acta Biomaterialia 10 (2014) 2855–2865

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Increased bone remodelling around titanium implants coated with chondroitin sulfate in ovariectomized rats Jan Dudeck a,b,⇑, Sebastian Rehberg a, Ricardo Bernhardt c, Wolfgang Schneiders a, Oliver Zierau d, Manjubala Inderchand e,g, Jürgen Goebbels f, Günter Vollmer d, Peter Fratzl g, Dieter Scharnweber c, Stefan Rammelt a,h a

Department of Trauma and Reconstructive Surgery, University Hospital ‘‘Carl Gustav Carus’’, Technische Universität Dresden, Fetscherstrasse 74, Dresden 01307, Germany Institute of Immunology, Medical Faculty ‘‘Carl Gustav Carus’’, Technische Universität Dresden, Fetscherstrasse 74, Dresden 01307, Germany Max Bergmann Center of Biomaterials, Technische Universität Dresden, Budapester Str. 27, Dresden 01069, Germany d Institute for Zoology, Chair for Molecular Cell Physiology and Endocrinology, Technische Universität Dresden, Zellescher Weg 2b, Dresden 01062, Germany e Biomedical Engineering Division, School of Bio-Sciences and Technology, VIT University, Vellore, Tamilnadu 623014, India f BAM Federal Institute for Materials Research and Testing, Unter den Eichen 87, Berlin 12205, Germany g Max Planck Institute of Colloids and Interfaces, Department of Biomaterials, Potsdam-Golm Science Park, Am Mühlenberg 1, Potsdam 14476, Germany h Center for Regenerative Therapies Dresden (CRTD), Tatzberg 47, Dresden 01307, Germany b c

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Article history: Received 21 June 2013 Received in revised form 25 January 2014 Accepted 31 January 2014 Available online 15 February 2014 Keywords: Osseointegration Scanning nanoindentation Chondroitin sulfate Osteoporosis Estrogen replacement therapy

a b s t r a c t Coating titanium implants with artificial extracellular matrices based on collagen and chondroitin sulfate (CS) has been shown to enhance bone remodelling and de novo bone formation in vivo. The aim of this study was to evaluate the effect of estrogen deficiency and hormone replacement therapy (HRT) on the osseointegration of CS-modified Ti implants. 30 adult female, ovariectomized Wistar rats were fed either with an ethinyl-estradiol-rich diet (E) to simulate a clinical relevant HRT or with a genistein-rich diet (G) to test an alternative therapy based on nutritionally relevant phytoestrogens. Controls (C) received an estrogen-free diet. Uncoated titanium pins (Ti) or pins coated with type-I collagen and CS (Ti/CS) were inserted 8 weeks after ovarectomy into the tibia. Specimens were retrieved 28 days after implantation. Both the amount of newly formed bone and the affinity index (P < 0.05) were moderately higher around Ti/CS implants as compared to uncoated Ti. The highest values were measured in the G-Ti/CS and E-Ti/CS groups, the lowest values for the E-Ti and G-Ti controls. Quantitative synchrotron radiation micro-computed tomography (SRlCT) revealed the highest increase in total bone formation around G-Ti/CS as compared to C-Ti (P < 0.01). The effects with respect to direct bone apposition were less pronounced with SRlCT. Using scanning nanoindentation, both the indentation modulus and the hardness of the newly formed bone were highest in the E-Ti/CS, G-Ti/CS and G-Ti groups as compared to C-Ti (P < 0.05). Coatings with collagen and CS appear to improve both the quantity and quality of bone formed around Ti implants in ovarectomized rats. A simultaneous ethinyl estradiol- and genistein-rich diet seems to enhance these effects. Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction Postmenopausal osteoporosis is a highly relevant disease, caused by naturally or surgically induced restriction of the ovarian function, resulting in estrogen deficiency, pronounced bone loss, increased fracture risk, severe accessory symptoms and high social and financial burdens [1]. In patients with such a compromised ⇑ Corresponding author at: Institute of Immunology, Medical Faculty "Carl Gustav Carus", Technische Universität Dresden, Fetscherstrasse 74, Dresden 01307, Germany. Tel.: +49 3514586528; fax: +49 3514586316. E-mail address: [email protected] (J. Dudeck). http://dx.doi.org/10.1016/j.actbio.2014.01.034 1742-7061/Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

bone metabolism, aseptic loosening of metallic implants remains an issue of concern in trauma, orthopedic and maxillofacial surgery because osseointegration of Ti implants is impaired in osteoporotic bone [2–4]. HRT is the standard treatment of post-menopausal, estrogen-dependent symptoms of old age. HRT is also known to positively effect bone mass and fracture risk. But long-term HRT may bear substantial health risks, as indicated by the Woman’s Health Initiative (WHI) and other clinical studies [5,6]. In this context alternative therapies based on the nutritionally relevant phytoestrogens became the focus of attention. Isoflavone such as genistein are promising candidates to replace HRT due to their

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structural and functional similarities with the endocrine estradiol [7]. But the relevance of phytoestrogens in preventing osteoporotic fractures and implant loosening is still insufficiently studied. The increasing number of elderly patients suffering osteoporotic fractures leads to a higher demand for surgical techniques and the need for implants with improved osteogenic properties. One method of improving osseointegration is implant coating with inorganic [8] and organic [9] components of the physiological extracellular bone matrix (aECM). Several preclinical studies have investigated the in vivo effects of implant coating with type I collagen [10,11]. Other promising components of the ECM are glycosaminoglycans (GAGs) such as chondroitin sulfate (CS). Coating titanium implants with collagen and CS resulted in a significantly increased bone implant contact (BIC) around intramedullary Ti pins in the tibiae of healthy rats [12] and around dental implants in the mandible of healthy minipigs [13] when compared to uncoated implants. Similar results were shown in studies using CS as an additive in calcium-phosphate-based bone substitutes in small and large animal models [14]. However, in all these studies, healthy subjects with intact bone metabolism were used, hardly reflecting the situation in elderly, osteoporotic patients. Remarkably, the effect of collagen and CS on osseointegration of Ti implants under estrogen deficiency has not yet been investigated. GAGs are ubiquitous components of the ECM and cell membrane in all eukaryotic organisms bridging extracellular stimuli and intracellular signalling. GAGs are negatively charged, linear polysaccharide chains composed of repeating disaccharide units which carry different amounts of sulfate groups at varying positions. Most of the GAGs have a molecular weight ranging between 10 and 100 kDa and are commonly present as components of proteoglycans (PGs) in various tissues [15]. CS consists of N-acetylgalactosamine and uronic acid. It is the most abundant GAG in cartilage, tendons, ligaments, brain and cancellous and cortical bone. In bone and cartilage CS mostly exists as a component of the PGs decorin, biglycan and aggrecan [16]. Furthermore, CS is abundantly present in mineralized cartilage but mostly restricted to calcified nodules and to the surface of osteocytes, osteocyte lacunae and canaliculi in mineralized bone in rats [17]. Among other GAGs, CS is able to bind cytokines and growth factors involved in bone regeneration [18,19]. Osteoblast attachment, collagen deposition and matrix mineralization have been reported to be directly dependent on GAGs [20,21]. Furthermore, sulfated GAGs are capable of binding calcium and calcium phosphates, including hydroxyapatite [17,22]. Recently, it has been suggested that GAGs also play a functional role in osteoclastogenesis [23]. These functional heterogeneity differences might be related to the dosage, type, molecular weight and sulfatation of GAGs, influencing their capability to bind cytokines and growth factors as well as to interact with cells and ECM components [24,25]. However, the molecular mechanisms leading to these effects are not completely understood. Anchorage and stability of endosseous implants depend on the quantity and quality of peri-implant bone formation [26–28]. Methods such as synchrotron-radiation micro-computed tomography (SRlCT) and nanoindentation provide information about both the bone quantity and quality at the micro-scale, making them beneficial in assessing osseointegration [29,30]. Conventional lCT systems use polychromatic X-rays that cause image artifacts by beam hardening and interface scattering, especially at the implant–bone interface, resulting in a detection of higher absorbing species up to a distance of 200 lm around metal implants that are not present in reality [31]. This impairs the quantification of peri-implant bone structures and leads to a prominent overestimation of bone around metal implants. Therefore the use of standard lCT is limited for the evaluation of metal osseointegration [32]. A key benefit of SRlCT is the elimination of such image artifacts due

to the high primary X-ray photon flux of the synchrotron radiation that enables the use of intensive, monochromatic X-rays. SRlCT provides therefore more sensitive detection of bone close to metal implants than standard lCT and a reliable quantification of periimplant bone up to a distance of 18 lm to the implant surface. Only directly at the implant–bone interface does SRlCT show a difference of 10% in newly formed bone (BIA) as compared with histology, which may be attributed to the partial volume effect [31]. Nanoindentation enables spatially resolved measurements of mechanical properties such as Young’s modulus and hardness at the micro-scale [33]. Due to the strong correlation between the micromechanical properties and both the bone mineral density and constitution of the organic bone matrix [34,35] this technique allows for conclusions regarding the peri-implant bone quality and the potential stability of implant anchorage [26]. The quality of bone formed around aECM modified Ti implants has not yet been investigated. The present study was therefore performed to evaluate the effects of CS-coated Ti implants on the periimplant bone quantity and quality using SRlCT, nanoindentation, histomorphometry and histology. The impact of CS coatings on the osseointegration under estrogen deficiency was investigated using ovarectomized rats as an osteoporosis model. To test for the effects of an HRT on the osseointegration of CS-coated Ti implants under estrogen deficiency, an ethinyl-estradiol-rich diet was used to simulate a clinical relevant HRT. Furthermore, a genistein-rich diet was used to evaluate an alternative therapy based on the nutritionally relevant phytoestrogens. 2. Materials and methods 2.1. Implant preparation Titanium (Ti6Al4V) pins of 0.8 mm diameter were used for the immobilization of various ECM components as described previously [12]. Briefly, the pins were cleaned with 1% triton X-100, acetone and 96% ethanol, rinsed in distilled water and dried. Type I collagen was obtained from bovine skin (IBFB Pharma GmbH, Leipzig, Germany) and dissolved in 10 mM acetic acid. Fibrillogenesis was allowed to take place overnight under physiological conditions (37 °C, pH 7.4 in 60 mM sodium phosphate buffer) in the presence of 10 mass% chondroitin-4-sulfate from bovine trachea (Sigma-Aldrich, Steinheim, Germany). Fibrils were collected by centrifugation and suspended. The Ti pins were incubated in the suspension at 25 °C for 1 min and air dried. The collagen and CS concentration adsorbed at the titanium implant was quantified using the Sirius red and Pieper method as recently described [36,37]. The final collagen amount on the implant surface ranged between 15 and 30 mg cm 2. The properties of collagen type I and CS immobilized on titanium surfaces have been described in detail previously [36,37]. Uncoated Ti pins served as control implants. The coated implants were sterilized with gamma irradiation of 25 kGy, uncoated pins with ethylene dioxide. We found in preliminary studies that the degradation of lyophilized GAGs with molecular masses between 30 and 50 kDa (as used in this study) is minimal under gamma irradiation of 25 kGy in comparison to those of higher molecular weight (unpublished data). 2.2. Animal housing and diet All animal handling and experimental conditions were licensed by the local animal care committee and carried out according to the Institutional Animal Care and Use Committee guidelines as regulated by the federal law governing animal welfare. 30 adult female Wistar rats (Charles River Laboratories, Sulzfeld, Germany) with a mean BW of 250–300 g were housed under controlled

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conditions (20 ± 1 °C, 50–80% humidity, 12:12 h light–dark cycles) in groups of five animals per cage with free access to water and a phytoestrogen-free diet (Harlan 2019 Rodent Breeding, Harlan Winkelmann, Borchen, Germany) for 14 days prior to the experiment. In all rats a standard bilateral ovariectomy was performed. The animals were randomly allocated to three experimental groups. The first group (n = 10) received an estrogen- and phytoestrogen-free diet (Sniff SMR/M-H, 10 mm, PE-free) purchased from Sniff GmbH (Soest, Germany) and served as an estrogen-deficient control (C). In the second group (n = 10) the control diet was substituted by an ethinyl-estradiol-rich diet (E), because estradiol is the commonly used standard HRT in treatment of postmenopausal, estrogen-dependent symptoms of old age. This dietary supplement resulted in an ethinyl estradiol content of the chow of 50 ppb (50 lg kg 1 feed). The third group received a soy extract substituted genistein-rich diet (G) with the genistein source Solgen 40 (Solbar Plant Extracts, Israel), because isoflavones such as genistein are promising candidates to replace the potentially harmful HRT. The genistein content of the diet was adjusted to 250 ppm (250 mg kg 1 feed), resulting in an exposure of 15 mg genistein kg 1 body weight (BW) per day. 2.3. Surgical procedure 28 days after ovariectomy all animals (n = 30) underwent intramedullary nailing with Ti pins. Five animals of each diet group (C, E, G) were randomized to receive either uncoated pins (Ti) or pins coated with type I collagen and CS (Ti/CS). After initial sedation with mixed O2/CO2 inhalation, the animals received intraperitoneal anesthesia with xylazine (10 mg kg 1 BW) and ketamine (90 mg kg 1 BW). Apart from the injection the animals experienced no pain throughout the experiments. After hindlimb shaving and disinfection, the pins were introduced via stab incisions through the tibial tuberosity into the medullary canal of the right tibia in the fashion of intramedullary nails (Fig. 1). The pins were cut at the bone surface and the skin was closed with absorbable sutures. Post-operatively the animals were allowed to move freely in their cages. All animals were sacrificed with lethal carbon dioxide

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inhalation after anesthesia with mixed O2/CO2 inhalation at days 28 post-implantation. The hindlimbs were disarticulated at the knee joint and the tibiae were freed completely from adherent soft tissues. The fibulae were removed and three to four transversal incisions were made into the tibia to facilitate the subsequent infiltration with methyl methacrylate (MMA). The tibiae with the implants were fixed immediately in 1.4% paraformaldehyde, washed, dehydrated in a graded series of alcohol then infiltrated with xylene and polymerized with MMA-based Technovit 9100 N (Heraeus-Kulzer, Friedrichsdorf, Germany). 2.4. Three-dimensional (3-D) SRlCT bone quantification SRlCT was used to quantify the bone formation and the affinity index (AI) around the implants three-dimensionally. The tibiae of three different animals per group were measured at the BAMline of the Federal Institute for Materials Research and Testing at the Berlin Electron Synchrotron (BESSY II, beamline 7TWLS) and at the Helmholtz-Zentrum Geesthacht at the Hamburg Synchrotron Laboratory (HASYLAB, beamline W2). At BESSY II the X-ray energy was set to 30 keV with a double multilayer monochromator. A GdOS scintillator and a CCD detector (VersArray:2048B, Princeton Instruments, USA) were used to measure the samples under 720 different projections angles with an image size of 2048  2048 pixels and an effective pixel size of 7  7 lm (zoom optics combined with CCD chip’s pixel size). At HASYLAB the photon energy was adjusted to 35 keV with a double-bent Laue monochromator (Si-111 reflex). A single crystal (CdWO4) fluorescence screen and a CCDcamera coupled with a zoom optic (KX2, Apogee Instruments, Inc., USA) were used to measure 720 projections per sample with an image size of 1274  1274 pixels (effective pixel size: 9  9 lm). A filtered back projection algorithm was used to obtain the 3-D data of X-ray absorption for the samples. The reconstructed SRlCT data were analysed with the SCANCO image evaluating software (SCANCO Medical, Brüttisellen, Switzerland). Per sample, an average of 450 slices perpendicular to the longitudinal implant axis with a slice distance of 20 lm were measured. The amount of newly formed mineralized bone at the implant surface was calculated along the whole implant surface by masking the implant outline and dilatating the mask by 18 lm to avoid the partial volume effect. At this position of the mask, the AI was defined as the length of the newly formed peri-implant-bone, normalized to the medullary portion of the implant circumference. The amount of BIA was measured in analogy to the histomorphometrical analysis with a second dilatation of the mask to a distance of 100 lm away from the implant surface and calculated as a percentage of the total tissue volume. Regions with direct implant contact to the tibial cortex were excluded from the BIA and AI analysis. 2.5. Scanning nanoindentation

Fig. 1. (A) Whole body radiograph of a rat with a tibial implant in situ and (B) lateral radiograph of a PMMA-embedded rat tibia including the Ti implant. The box shows the measured lCT volume where the sections for the subsequent analysis were prepared.

After SRlCT the specimens were cut into two sample blocks in the middle of the measured CT volume. The distal block was cut perpendicular to the bone length axis in 1 mm thick sections using an IsoMetÒlow speed saw (Buehler, Düsseldorf, Germany). Based on the implant position within the medullary channel, one section per sample was chosen to obtain comparable cutting levels. Subsequently the implants were removed carefully to receive a highly gloss-polished sample surface suitable for scanning nanoindentation of peri-implant bone structures. The implant cavity was then filled with Loctite 9434 (Henkel, Munich, Germany) and polymerized using ultraviolet light. These slices were ground and polished using a PM5 device (Logitech, Glasgow, UK) and abrasives of decreasing grain size down to 0.3 lm (Struers, Ballerup, Denmark). Finally all sections were sonicated with deionized water and

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cleaned with a cotton swab. Nanoindentation was performed on three independent samples per group using a nanoindenter (UB1, Hysitron Inc., Minneapolis, USA) equipped with a motorized x-y stage and a Berkovich diamond tip. The system was coupled with an atomic force microscope and an optical microscope. Loading was performed as described recently with a maximum load of 5000 lN at a loading rate of 1000 lN s 1 held for 60 s [38]. Hardness (H) and reduced E modulus (Er), which is referred to as indentation modulus, were calculated according to Oliver and Pharr [33]. Regions of interest were preselected from environmental scanning electron microscopy (ESEM) images obtained in backscattered electron (BSE) mode (Quanta 600, FEI, Hillsboro, USA), identified using the optical system of the nanoindenter and measured with an indentation pattern consisting of several indent lines with a spacing of 10 lm perpendicular and 50 lm in parallel to the peri-implant bone. The number and size of the patterns were adapted to the geometry of the tested bone structures (Fig. 2). This approach enabled us to measure the whole newly formed peri-implant bone precisely with numerous indents (60-120 indents per sample). After nanoindentation the indent patterns were controlled with ESEM. Indents situated on cracks, osteocyte lacunae, abrasive particles, both interfaces (implant–bone, bone-embedding material), incorporated trabeculae and within the embedding material were excluded from the data evaluation. Fused silica and the embedding material of each sample (poly(methyl methacrylate), PMMA) served as external and internal control.

2.6. Histology and histomorphometry In order to provide information about bone quality and quantity from exactly the same locations within the samples, ESEM in BSE mode was used to quantify the peri-implant bone formation and the AI in the sections prior used for nanoindention. The ESEM was operating in low vacuum (0.3 Torr) at 10 kV with a working distance between 13.6 and 6.4 mm. Depending on the mineralization degree, calcified tissue appeared in different grey levels, indicating either high (bright) or low (dark) mineral content [39]. ESEM-BSE enables therefore also conclusions regarding the

A

calcium content of bone. Histomorphometric analysis was performed with a light microscope and the Quantimed software (Leica, Bensheim, Germany). The de novo bone formation around the implants (BIA) was defined as the area occupied by newly formed peri-implant bone within a circle of 100 lm around the implant normalized to the total area in this zone (Fig. 3). The latter was set to 100%. Orthotopic cortical and attached trabecular bone were excluded. In sections where the implant was in direct contact with the cortex, the measurement and normalization were restricted to the medullary part of the 100 lm zone. The AI was defined as the length of the newly formed peri-implant bone, normalized to the medullary portion of the implant circumference. The latter portion was set to 100%. To confirm BIA and AI outside the sections previously used for nanoindentation, histomorphometric analysis was also performed in histological sections (data not shown). For this purpose three sections per sample (10–15 lm) were stained with hematoxylin and eosin and Goldner–Masson trichrome. Giemsa staining was used to further characterize the peri-implant bone with respect to calcified tissue and PG content. 2.7. Statistical analysis All data are presented as box plots, using the 25 and 75 percentile as box width. The mean value and the median are indicated as square and line within the box, respectively, and the standard deviation as whiskers. The differences between the groups were analysed for significance with SigmaPlot (Systat Software, Inc., San Jose, CA, USA) using a one-way analysis of variance with a Tukey post-hoc test for multiple comparisons of the mean values. Statistical significance was assumed at P < 0.05. 3. Results 3.1. Uterus weight Ovariectomy induced a decrease of the relative uterine wet weight measured at the time of explantation. This effect was antagonized partly by the ethinyl-estradiol-rich diet (E) while the

B

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Fig. 2. SEM pictures (BSE mode) of an exemplary sample used for nanoindentation with the implant removed. (A) Overview (48) and (B) close up (200) showing the newly formed bone surrounding a Ti implant. The dots depict the indent patterns used in scanning nanoindentation. (C) Close up (400) showing a bone trabecula attached tightly to the peri-implant bone sleeve and not included in the measurements. Note the typical septum of highly mineralized cartilage designating clearly the trabeculae (arrowhead).

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Fig. 3. (A) Exemplary cross-section of a rat tibia showing the typical osseointegration of a medullary Ti implant in a sleeve-like manner 4 weeks after implantation (Giemsa, implant in-situ). The scheme depicts the histomorphometric measurements (BIA). The amount of BIA was calculated as a percentage of the whole are at a range of 100 lm around the implant. (B, C) Enlarged sections of the bone sleeve. Note the thick osteoid layer deliminating the medullary surface of the bone sleeve and the thin connective tissue layer at the interface between sleeve and implant. (D) Cortical remodelling zone adjacent to the implant. (E, F) Details of the remodelling zone (o: osteocytes, arrowheads: osteoid, arrows: interface layer between sleeve and implant, asterisk: direct implant–bone contact).

soy extract substituted genistein-rich diet (G) had no significant effect on the uterine wet weight. The relative uterus wet weight averaged 0.35 mg kg 1 BW in the E group and 0.166 mg kg 1 BW in the untreated controls (P < 0.05). The relative uterus wet weight in the G group averaged 0.134 mg kg 1 BW.

3.2. Histological analysis All implants healed uneventfully without wound infection, hardware loosening or chronic adverse reaction. Histologically, no accumulations of inflammatory cells or foreign body giant cells

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A

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Fig. 4. (A) Overview (48) and (B,D) close up view (400) of a rat tibia with peri-implant bone formation showing the heterogeneity of the tissue composing the newly formed bone (SEM-BSE, implant removed). Close to the cortex, lowly mineralized, immature lamellar bone is frequently interspersed with highly mineralized woven bone trabeculae (arrowheads in B and D). In contrast, the medullary part of the bone sleeve is composed mainly of lamellar bone with occasionally occurring areas of woven bone. (C, E) Distribution of the hardness within the peri-implant bone sleeve distant (C) and close (E) to the adjacent cortical bone. The hardness values were obtained from scanning nanoindentation and represented by color coded contour plots ranging from 400-1000 MPa that were mapped directly onto the corresponding SEM-BSE-images (400). Note the high coincidence of the woven bone (arrowheads) with areas of high hardness both in the peri-implant bone sleeve and in the adjacent cortical bone.

were seen around any of the implants. 28 days after surgery, all implants were surrounded by newly formed lamellar bone in a sleeve-like manner (Fig. 3A) containing numerous osteocytes that were oriented parallel to the implant surface (Fig. 3B and C). The outer surface of the bone sleeve, facing the medullary cavity, was to a great extent covered by a thick osteoid layer with cubic or slender-like cells (Fig. 3B and C). On the inner surface of the bone sleeve, facing the implant, a small interface consisting of collagen and fibrocyte-like cells was usually apparent, independent of the type of implant surface (Fig. 3B–F). Within the bone sleeve, lower mineralized, immature lamellar bone was frequently intervened with highly mineralized woven bone, as indicated by irregular areas with a high BSE yield (Fig. 4). Formation of both lamellar and woven bone was more pronounced at the implant surface (being closer to the cortical bone) than at the implant side oriented towards the medullary cavity (Fig. 4). In sections with a pronounced implant–cortex contact, the cortical bone adjacent to the implant was characterized by an intensive bone remodelling (Fig. 3E and F).

3.3. SRlCT analysis 3-D reconstruction of the SRlCT data revealed a mesh-like coverage of the titanium pins with mineralized bone (Fig. 5). The de novo bone formation for all coated implants (Ti-CS) averaged 36.0% as compared to 35.4% around the uncoated controls (Fig. 6). BIA proved to be highest around G-Ti/CS implants (40.1%; SD 10.0). The lowest BIA was found around the C-Ti

(30.8%, SD 16.4) and E-Ti/CS (30.1%, SD 13.1) implants. The AI for all coated implants (Ti-CS) averaged 53.7% as compared to 55.2% around the uncoated controls (P < 0.001). AI proved to be highest around G-Ti implants (67.4%, SD 12.3) compared to the lowest values around C-Ti (46.9%, SD 15.8). and C-Ti/CS implants (46.4%, SD 15.3). When comparing the three different diets, AI increased in both implant groups from estrogen-free diet (C) to the estradiol (E) and genistein (G) groups (Fig. 7).

3.4. Histomorphometrical findings The de novo bone formation, 100 lm around the implants, was moderately higher on average around the coated implants (Ti/CS: 37.3%) than around the uncoated controls (Ti: 33.3%) over all diet groups without reaching significance. BIA proved to be highest in the G-Ti/CS group with 41.7% (SD 1.4). The lowest amount with 30.4% (SD 9.0) was found around the uncoated implants of the ETi group (Fig. 8). The difference between these two groups was statistically significant (P < 0.05). The AI was significantly higher around the coated (Ti/CS) implants (91.7%, SD 6.0%) than around the controls (86.9%, SD 3.4%) over all diet groups (P < 0.05). AI was highest around G-Ti/CS implants with 94.1% (SD 2.7) and lowest around the uncoated G-Ti implants (85.7%, SD 1.5) (Fig. 9). The difference between these two groups was statistically significant (P < 0.05). When comparing the three different diets, both BIA and AI remained unchanged for uncoated implants but increased for CS coated implants from estrogen-free diet (C) to the estradiol (E) and genistein (G) groups.

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Fig. 7. Quantitative SRlCT: affinity index (AI) at 4 weeks after implantation in percentage of the total implant circumference: arithmetic mean (small square), median (horizontal line), upper and lower quartile (box), SD (whiskers).

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Fig. 5. SRlCT: visualization of reconstructed absorption values filtered for Ti (white) and mineralized bone (grey) showing the tibial osseointegration of the Tipins 4 weeks after implantation.

40 30 20 10 0 C(Ti)

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groups Fig. 8. Histomorphometry: amount of new bone around the implants (BIA) at 4 weeks after implantation: arithmetic mean (small square), median (horizontal line), upper and lower quartile (box), SD (whiskers). Statistically significant differences as indicated: ⁄P < 0.05.

18.2 GPa, H: 0.74 GPa) when compared to the C-Ti control (Er: 15.5 GPa, H: 0.65 GPa). This difference was statistically significant for Er (P < 0.01). When comparing the treatment groups among the animals that received uncoated Ti pins, the indentation modulus increased significantly but hardness insignificantly with G substitution as compared to the C-Ti group (Fig. 10 and 11). Contrary, in treatment groups that received Ti/CS implants, both parameters increased more pronounced with E substitution than with G substitution as compared to the C-Ti/CS group (P < 0.05) (Fig. 10 and 11). Fig. 6. Quantitative SRlCT: amount of new bone around the implants (BIA) at 4 weeks after implantation: arithmetic mean (small square), median (horizontal line), upper and lower quartile (box), SD (whiskers).

3.5. Nanoindentation The indentation modulus (Er) and hardness (H) of the newly formed peri-implant bone were highest in the E-Ti/CS group (Er:

4. Discussion Bone regeneration and osseointegration of implants are highly coordinated processes regulated by a complex and balanced interplay of bone cells, growth factors, cytokines and components of the ECM. The crucial role that PGs and their GAG constituents play in these processes has received increasing scientific recognition over

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Fig. 9. Histomorphometry: affinity index (AI) at 4 weeks after implantation in percentage of the total implant circumference: arithmetic mean (small square), median (horizontal line), upper and lower quartile (box), SD (whiskers). Statistically significant differences as indicated: ⁄P < 0.05.

indentation modulus (GPa)

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groups Fig. 10. Nanoindentation: indentation modulus (Er) of the peri-implant bone: arithmetic mean (small square), median (horizontal line), upper and lower quartile (box), SD (whiskers). Statistically significant differences as indicated: ⁄P < 0.05.

the recent years [40]. Several studies using different animal models demonstrated an increased bone remodelling and enhanced bone formation around implants coated with artificial ECM based on collagen and CS [12,13]. However, data showing the effect of such aECM on the osseointegration under compromised conditions are still lacking. Because of the increasing number of osteoporotic fractures and the prospective socioeconomic impact of these injuries [1] we aimed to investigate the osseointegration of Ti implants coated with collagen I and CS under estrogen deficiency.

4.1. Animal model Based on the similarities between the alterations in bone metabolism induced by estrogen deficiency in ovarectomized rats and post-menpausal women, we and others used the ovarectomized rat as a reliable and accepted model to study bone regeneration under estrogen deficiency [41,42]. This model benefits from the quick reaction to hormonal changes induced by ovarectomy (OVX) as reflected by rapid changes in bone metabolism shortly

C-Ti

E-Ti

G-Ti

C-Ti/CS E-Ti/CS

G-Ti/CS

groups Fig. 11. Nanoindentation: hardness (H) of the peri-implant bone: arithmetic mean (small square), median (horizontal line), upper and lower quartile (box), SD (whiskers).

after OVX. This resulted in a significant decrease of bone volume fraction and the connectivity density of the cancellous bone already 4 weeks after OVX [43]. Dempster et al. reported a boost of the osteoclast number already beginning 5 days after OVX, closely followed by an increase in bone formation rate and a marked reduction of bone density (BD), integrity and connectivity of the cancellous bone 4 weeks after OVX [44]. Recently we published a decline of the BD and connectivity of 30% and 50%, respectively, already 4 weeks after OVX in the femur of ovariectomized rat and pronounced upregulation of the osteoblast marker genes osteocalcin and type I collagen as well as the osteoclast marker genes cathepsin K and tartrate-resistant acid phosphatase together with significantly increased serum levels of osteocalcin and collagen degradation fragments [43]. It has been also shown that osseointegration of Ti screws in the tibiae of Wistar rats is impaired under estrogen deficiency as early as 3 weeks after OVX [4]. In agreement with these previous reports we observed an significant reduction of the uterus wet weight 4 weeks after OVX that was compensated by the ethinyl-estradiol-enriched diet. These results confirm both the successful depletion of the ovarian hormones, enabling osseointegration under estrogen deficiency already 4 weeks after OVX and the efficacy of the estrogen replacement therapy. Estradiol is the commonly used standard HRT in treatment of post-menopausal, estrogen-dependent symptoms of old age. Estradiol is also known to positively influence bone mass and fracture risk [5]. But long-term HRT may bear substantial health risks as indicated by the WHI and other clinical studies [5,6]. The extent to which HRT is actually beneficial or harmful for patients has now developed into a subject of heated discussions. In this context we aimed to test an alternative therapy based on the nutritionally relevant isoflavone genistein (G) with estradiol as positive control (E) and estradiol-free diet as negative control (C). Genistein belongs to the plant-derived phytoestrogens found in soy, nuts, beans, sesame and other oilseeds. Due to their structural similarities with endocrine estradiol, phytoestrogens may imitate and/or antagonize the functions of estrogen [7] and became therefore the focus of attention as an alternative for HRT. Based on in vivo and in vitro studies it has been suggested that phytoestrogens may also have a positive impact on bone by maintaining peak bone mass under premenopausal conditions and attenuating bone loss during the menopausal transition [45]. But the relevance of phytoestrogens in preventing osteoporotic fractures is still insufficiently studied. Data assessing the impact of phytoestrogens on the osseointegration of implants are missing completely up to now. Therefore we

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aimed to provide for the first time scientific findings regarding the influence of phytoestrogens on the osseointegration of aECMmodified implants in vivo. In the current study, all coated and uncoated implants healed uneventfully without clinical or histological signs of infection, loosening or adverse reaction, which is in accordance with earlier studies on Ti implants in the healthy rat tibia [12]. At 28 days after implantation, all implants were surrounded by newly formed lamellar bone in a sleeve-like manner. De novo bone formation around the implants were more pronounced close to the inner surface of the cortical bone, which seems to have a particularly high potential for bone regeneration. 4.2. Analysing quality and quantity of peri-implant bone formation In the present study we were able to consistently evaluate the peri-implant bone quality and quantity at the same defined area within the specimens for the first time. Anchorage and stability of endosseous implants depend on the amount of peri-implant bone and BIC but also on the quality of the surrounding bone tissue [26,27]. Bone quality, i.e. the material composition and mineralization as well as the structure and architecture, determines bone strength on all structural levels [29,46]. To analyse the impact of CS and HRT on peri-implant bone formation in terms of bone quantity and quality we used a consecutive and complementary approach consisting of SRlCT, nanoindenation, histomorphometry and histology. SRlCT analysis benefits from the ability to measure the complete newly formed mineralized bone around and at the surface of metal implants non-destructively, without artifacts and with high spatial resolution [30,31]. Compared to histology, SRlCT does not suffer from the loss of information caused by the cutting and grinding process. Moreover, SRlCT allows for the analysis of multiple slices at any position within the measured sample volume and provides statistical highly representative data [47]. The mechanical properties of bone are strongly correlated to the bone mineral density and to the constitution of the organic bone matrix [34,35]. Consequently, measuring the indentation modulus and hardness allows for conclusions regarding the peri-implant bone quality at the micro-scale [29,30,48]. So far, only a few studies have assessed osseointegration, via nanoindentation [49–51]. But the sampling in these studies was either restricted to line scans running from the implant–bone interface to the surrounding cortical bone or limited to a few indents close and distant to the implant. But the pronounced tissue heterogeneity of peri-implant bone [52] raises the need for multiple measurements, especially in the earlier stages of osseointegration when remodelling and maturation of the bone are still ongoing. Apart from scanning acoustic microscopy [29] only nanoindenation provides a resolution and precision sufficient to measure and detect variances in the micromechanical properties within the tiny, bony structures around implants in a small rodent model. Therefore scanning nanoindentation was used in this study to allow for a reliable detection and mapping of local heterogeneities in the tissue micromechanics and quality within the peri-implant bone. 4.3. Effects of HRT When analysing our data with respect to the diet, HRT seemed to enhance osseointegration around uncoated implants. Newly formed bone and AI, as measured with SRlCT, increased with ethinyl-estradiol- (E) and genistein-rich (G) diets, although not significantly (Figs. 6 and 7). When measured at the locations were nanoindentation was carried out, neither BIA nor AI seemed to be affected by HRT (Figs. 8 and 9). This discrepancy may be attributed to the ability of SRlCT to measure the complete newly formed mineralized bone around the implants, therefore providing more reliable and representative data than histology. The measured

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SRlCT volume included parts of the proximal tibial metaphysis, which is characterized by more orthotopic trabecular bone than the diaphysis. Within the metaphysis the implants were in closer contact with bone trabeculae than in the proximal diaphysis, potentially resulting in a higher peri-implant bone volume. Indentation modulus and hardness of the BIA were increased around uncoated Ti implants under both forms of HRT. The mechanical properties of bone are strongly correlated to the bone mineral density and to the constitution of the organic bone matrix [34,35]. The improved mechanical properties may therefore reflect the capability of HRT to improve the peri-implant bone quality at the microscale and suggest an enhanced implant stability at the macro-scale. Estrogen plays a crucial role in regulating bone turnover in humans and rodents through its capability to influence the interplay between receptor activator of nuclear factor-kappa B ligand (RANKL) and osteoprotegerin (OPG), as well as cytokines known to affect bone resorption such as TNF-a, IL-1 and IL-6 [53,54]. Stimulatory effects of estrogen on osteoblast differentiation and function were indicated by increased osteoblast markers such as collagen-I, alkaline phosphatase, osteocalcin, osteopontin and TGF-b as well as augmented bone matrix segregation and mineralization in vitro and in vivo [55,56]. Several authors have demonstrated indirect effects of estrogen deficiency on the osteoclastogenesis through increased RANKL expression, decreased OPG release and elevated TNF-a, IL-1 and IL-6 segregation by osteoblasts and bone marrow stromal cells, as well as T and B lymphocytes, thus promoting osteoclast recruitment, differentiation, activation and survival [57–59]. Moreover, estrogen deficiency restricts the inhibition of bone resorption through compromised release of TGF-b by osteocytes [60] and suppresses osteoclast apoptosis through diminished TGF-b segregation by osteoblasts precursors [61]. Osseointegration of Ti implants is decreased in cancellous bone of ovarectomized rats, accompanied by a reduced BIC, BIA and BD around the bone substitutes [4]. Some clinical reports from dental applications indicated both a significantly impaired osseointegration in postmenopausal woman and a positive effect of HRT on implant stability [3], while other studies argued that type I osteoporosis has no significant influence on osseointegration [62]. Treatment with 17b-estradiol caused a reduction of the osteoclast number and an increase of the osteoblast number and osteoid volume in trabecular bone of ovariectomized rats [63]. Direct effects of HRT on osteoclasts such as diminished osteoclast activity, decreased bone resorption and incomplete degradation of the organic ECM within the resorption pits have been demonstrated in vitro [64,65]. The observed improvements of the peri-implant bone quantity and quality may therefore be attributed to the capability of HRT to inhibit osteoclastogenesis and to stimulate osteoblastogenesis. In the present study, HRT with genistein (G) resulted in a more pronounced bone formation than ethinyl estradiol (E) around uncoated implants. The positive effects of G are in accordance with recent studies that suggested a positive correlation between phytoestrogen supplement and bone regeneration [66]. At the same time G is discussed as a potential agent for the prevention and treatment of post-menopausal osteoporosis [67]. It was surprising, however, that E resulted in less enhanced AI, indentation modulus and hardness than G, which may be due to the different dosages of the substances. Alternatively, it might be explained by effects of G that were not mediated via the estrogen receptor. It is known that G is able to induce alternative pathways besides the estrogen receptor, which mediate a shift towards osteogenesis [66]. 4.4. Effects of implant coatingand HRT Implant coating with collagen I and CS alone had no effect on the AI and tissue hardness but resulted in a moderately increased peri-implant bone formation and indentation modulus in the tibiae

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of ovariectomized rats. Remarkably, this trend became more pronounced with both types of HRT, especially with ethinyl estradiol supplement. Under these circumstances CS-coated implants were characterized by an increased AI and significantly stiffer and harder peri-implant bone, indicating a higher mineralization degree and a more mature bone as compared to the uncoated Ti pins. Thus, the combination of CS-containing aECM coatings, with their potential for accelerating bone regeneration, and HRT appears to exert synergistic effects on the quality of peri-implant bone under estrogen deficiency. Surprisingly the amount of peri-implant bone was not increased substantially. The positive effects of CS on osteogenesis and bone remodelling have been shown in few in vivo studies using healthy rodents [12] and large animal models [68]. In a rat tibia model both osteoclast and osteoblast numbers were increased significantly around CS-coated Ti pins 1 and 2 weeks after implantation, respectively, but decreased thereafter as compared to uncoated pins [12]. It has been shown in vitro that PGs attract calcium (Ca) via their negatively charged GAG chains [69]. Moreover, PG degradation and subsequent structural alteration of their GAG components is considered to be crucial for initiation of the bone mineralization process through delivering of precursory stimuli [17,70]. Transmission electron microscopy (TEM) has detected large, CS-containing PG aggregates both in the osteoid and around matrix vesicles. In contrast, smaller obviously degraded but still CS-containing PGs were concentrated in initial calcified nodules and in the periphery of expanded nodules, colocalized with alkaline phosphatase [17]. Elemental mapping of Ca and phosphorus (P) using energy-filtered TEM revealed colocalization of Ca and P in areas of ongoing mineralization but different localization patterns in uncalcified areas, where Ca frequently mapped to PG sides and P were often found close to the collagen fibrils [17]. The spatial separation of Ca and P seems to prevent mineralization within the osteoid. Therefore, organic ECM components seem to be crucial in initiating matrix calcification. CS and collagen immobilized at the implant surface could therefore contribute to Ca and P accumulation at the implant surface facilitating matrix calcification. This may lead to an increased mineralization degree and accelerated bone maturation, as reflected by the enhanced indentation modulus and hardness of the peri-implant bone in the CS groups. Besides being a scaffold and substrate for bone regeneration, GAGs within the ECM are known to directly interact with cytokines and growth factors essential for bone regeneration like TGF-b, FGF2 and BMPs [71]. Stadlinger et al. found even more bone formation around implants coated with collagen and CS than around those coated with collagen and TGF-b1 or BMP-4 [13]. These findings support the observations from prior in vitro experiments, suggesting the capability of GAGs to bind growth factors and cytokines as well as to improve and accelerate the differentiation of osteoblasts [36]. Osteoblast attachment, collagen deposition and matrix mineralization have been reported to be directly dependent on GAGs [20,21]. Thus, CS as part of aECM implant coatings may trap and bind growth factors and cytokines released from the injured bone, thereby enhancing their bioavailability and local activity by preventing them from being degraded. Moreover, GAGs amplify the signal transduction induced by growth factors and cytokines in vitro [72]. In this way CS could also contribute to signal transduction in cells accumulating around the implant.

5. Conclusions Coatings of titanium implants with type I collagen and CS enhanced osseointegration in the tibiae of ovarectomized rats and improved the mechanical properties of the newly formed peri-implant bone. Artificial ECMs of collagen and CS appear to create a

Ca- and P-trapping matrix on the implant surface, capable of facilitating matrix mineralization and retaining and modulating growth factor and cytokine activity during the initial stages of osseointegration. HRT both with ethinyl estradiol and with genistein could offset some of the negative effects caused by OVX such as reduced BIA, AI, tissue hardness and indentation modulus. The combination of HRT and CS-containing aECM coatings exerted synergistic effects and appears therefore to be beneficial for the quality and quantity of the newly formed peri-implant bone under estrogen deficiency. These findings are of potential clinical relevance because surface modification of implants with aECM may provide a useful additional strategy when treating osteoporotic patients. Further studies should aim at confirming these results in preclinical models and using GAG derivatives with different sulfation patterns. Acknowledgments The authors gratefully acknowledge the Deutsche Forschungsgemeinschaft (DFG) for financial support (TRR 67, SCHA 570/9-2), BESSY, HASYLAB and S. Manthey, A. Wenke, A. Dudeck, P. Leibner, A. Martens, F. Beckmann and G. Weidemann for technical assistance. We thank Solbar Plant Extracts, Israel, for the delivery of Solgen 40. Appendix A. Figures with essential colour discrimination Certain figures in this article, particularly Figs. 2–4 and 7, are difficult to interpret in black and white. The full color images can be found in the on-line version, at 10.1016/j.actbio.2014.01.034 References [1] Rachner TD, Khosla S, Hofbauer LC. Osteoporisis: now and the future. Lancet 2011;377:1267–87. [2] Yamazaki M, Shirota T, Tokugawa Y, Ohno K, Michi K, Yamaguchi A. Bone reactions to titanium screw implants in ovariectomized animals. Oral Surg Oral Med Oral Pathol 1999;87(4):411–8. [3] August M, Chung K, Chang Y, Glowacki J. Influence of estrogen status on endoosseous implant osseointegration. J Oral Maxillofac Surg 2001;59:1285–9. [4] Duarte PM, Neto JBC, Gonçalves PF, Sallum EA, Nociti FH. Estrogen deficiency affects bone healing around Titanium implants: a histometric study in rats. Implant Denstistry 2003;12(4):340–5. [5] Health, National Institute of. Findings from the WHI postmenopausal hormone therapy trial. Woman’s Health Initiative. [6] Beral V, Bull D, Green J, Reeves G. Ovarian cancer and hormone replacement therapy in the million woman study. Lancet 2007;369(9574):1703–10. [7] Yildiz F, editor. Phytoestrogens in functional foods. Boca Raton, FL: CRC Press; 2005. p. 210–1. [8] Moroni A, Toksvig-Larsen S, Maltarello MC, Orienti L, Stea S, Giannini S. A comparison of hydroxyapatite-coated, titanium-coated, and uncoated tapered external-fixation pins. An in vivo study in sheep. J Bone Joint Surg Am 1998;80:547–54. [9] Förster Y, Rentsch C, Schneiders W, Bernhardt R, Simon JC, Rammelt S. Surface modifications of implants in long bone. Biomatter 2012;2(3):149–57. [10] Rammelt S, Schulze E, Bernhardt R, Hanisch U, Scharnweber D, Worch H, et al. Coating of titanium implants with type-I collagen. J Orthop Res 2004;22:1025–34. [11] Morra M, Cassinelli C, Meda L, Fini M, Giavaresi G, Giardino R. Surface analysis and effects on interfacial bone microhardness of collagen-coated titanium implants: a rabbit model. Int J Oral Maxillofac Implants 2005;20:23–30. [12] Rammelt S, Illert T, Bierbaum S, Scharnweber D, Zwipp H, Schneiders W. Coating of titanium implants with collagen, RGD peptide and chondroitin sulfate. Biomaterials 2006;27(32):5561–71. [13] Stadlinger B, Pilling E, Huhle M, Mai R, Bierbaum S, Scharnweber D, et al. Evaluation of osseointegration of dental implants coated with collagen, chondroitin sulphate and BMP-4: an animal study. Int J Oral Maxillofac Surg 2008;37(1):54–9. [14] Schneiders W, Reinstorf A, Biewener A, Serra A, Grass R, Kinscher M, et al. In vivo effects of modification of hydroxyapatite/collagen composites with and without chondroitin sulphate on bone remodeling in the sheep tibia. J Orthop Res 2009;27(1):15–21. [15] Gandhi NS, Mancera RL. The structure of glycosaminoglycans an their interaction with proteins. Chem Biol Drug Des 2008;72:455–82. [16] Waddington RJ, Roberts HC, Sugars RV, Schonherr E. Differential roles for small leucine-rich proteoclycans in bone formation. Eur Cell Mater 2003;6:12–21.

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