Life Sciences 121 (2015) 174–183

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Critical-size bone defect repair using amniotic fluid stem cell/collagen constructs: Effect of oral ferutinin treatment in rats Manuela Zavatti a,⁎, Laura Bertoni a, Tullia Maraldi a, Elisa Resca a, Francesca Beretti a, Marianna Guida b, Giovanni B. La Sala c, Anto De Pol a a b c

Department of Surgical, Medical, Dental and Morphological Sciences with Interest in Transplants, Oncology and Regenerative Medicine, University of Modena and Reggio Emilia, Modena, Italy EURAC Research, Center for Biomedicine, Bolzano, Italy Unit of Obstetrics & Gynecology, IRCCS-Arcispedale Santa Maria Nuova, Reggio Emilia, Italy

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Article history: Received 23 June 2014 Accepted 18 October 2014 Available online 5 November 2014 Keywords: Ferutinin AFSCs Bone regeneration Collagen scaffold Rat

a b s t r a c t Aims: This study aims to evaluate the bone regeneration in a rat calvarias critical size bone defect treated with a construct consisting of collagen type I and human amniotic fluid stem cells (AFSCs) after oral administration of phytoestrogen ferutinin. Main methods: In 12 week old male rats (n = 10), we performed two symmetric full-thickness cranial defects on each parietal region, and a scaffold was implanted into each cranial defect. The rats were divided into four groups: 1) collagen scaffold, 2) collagen scaffold + ferutinin at a dose of 2 mg/kg/5 mL, 3) collagen scaffold + AFSCs, and 4) collagen scaffold + AFSCs + ferutinin. The rats were sacrificed after 4 weeks, and the calvariae were removed, fixed, embedded in paraffin and cut into 7 μm thick sections. Histomorphometric measures, immunohistochemical and immunofluorescence analyses were performed on the paraffin sections. Key findings: The histomorphometric analysis on H&E stained sections showed a significant increase in the regenerated area of the 4th group compared with the other groups. Immunohistochemistry performed with a human anti-mitochondrial antibody showed the presence of AFSCs 4 weeks after the transplant. Immunofluorescence analysis revealed the presence of osteocalcin and estrogen receptors (ERα and GPR30) in all groups, with a greater expression of all markers in samples where the scaffold was treated with AFSCs and the rats were orally administered ferutinin. Significance: Our results demonstrated that the oral administration of ferutinin is able to improve the bone regeneration of critical-size bone defects in vivo that is obtained with collagen–AFSCs constructs. © 2014 Elsevier Inc. All rights reserved.

Introduction A great challenge for regenerative medicine is the repair of bone loss due to a wide range of diseases, including osteoarthritis, osteoporosis, osteogenesis imperfecta as well as traumatic injury and orthopedic surgery. Critical-size bone defects are not capable of repairing themselves. Recently the gold standard treatments for critical-size bone defects were autologous bone grafts. However, critical-size bone defects present several limitations and complications, such as donor site pain, paresthesia, inflammation and infection [4,46,50]. Another option is the use of allografts (from humans) or xenografts (from non-

⁎ Corresponding author at: Department of Surgical, Medical, Dental and Morphological Sciences with Interest in Transplants, Oncology and Regenerative Medicine, University of Modena and Reggio Emilia, Via Del Pozzo 71, 41124 Modena, Italy. Tel.: + 39 0594224853; fax: +39 0594224859. E-mail address: [email protected] (M. Zavatti).

http://dx.doi.org/10.1016/j.lfs.2014.10.020 0024-3205/© 2014 Elsevier Inc. All rights reserved.

humans), although these methods are associated with potential infections and immune responses [5,49]. To address these issues, many scaffolds have been investigated as potential alternatives to bone grafts for bone defect repair [34]. Scaffolds are divided in two main categories: biological (collagen type I and demineralized bone matrix) and synthetic materials (porous metals, bioactive glasses, polylactic acid, polyglycolic acid, hydroxyapatite, tricalcium phosphates) [2,21,30,52]. Biological scaffolds have significant advantages, i.e., biocompatibility, biodegradability and regenerative characteristics [25]. Among these scaffolds, collagen type I, the major component of the extracellular matrix (ECM), is the most popular biologic material used to produce tissue-engineered grafts because of its high availability, easy purification from living organisms, nonantigenicity, non-toxicity and biological plasticity [22,24,38]. Bone tissue engineering merges scaffolds with cells and growth factors to create a tissue engineered construct to enhance bone regeneration [39]. Adipose-derived stem cells (ASCs), bone marrow mesenchymal stem cells (BM-MSCs), amniotic fluid stem cells (AFSCs) and dental

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pulp stem cells (DPSCs) have been demonstrated as good candidates for in vitro and in vivo bone regeneration [7,26,27,37,40,42,54,57]. Recently, in our laboratories, we used a bioengineered construct of collagen scaffold-AFSCs to reconstruct a critical-size bone defect in an animal model [27]. We demonstrated that cell-seeded scaffolds have better and faster bone reconstruction capabilities than collagen alone and this improvement was due to the osteogenic potential of AFSCs [27]. Many compounds and growth factors are known to promote osteogenic differentiation; among them, 17β-estradiol plays an important role in bone metabolism. It has previously been demonstrated that 17β-estradiol enhances osteoblastic activity and bone formation [10, 41]. Moreover, it is known that the lack of 17β-estradiol after menopause in women results in enhanced bone resorption, which is accompanied by impaired bone formation [43]. In this condition, the estrogen administration exerted a positive effect on bone mineral density, preventing and reducing the progress of osteoporosis [15,28]. However, estrogen treatment is associated with an increased risk of breast cancer as well as cardiovascular diseases [6,44]. In this context phytoestrogens have attracted much attention among researchers because of their estrogenic activities and lack of adverse side effects associated with estrogens [16]. Many natural compounds promote the osteogenic differentiation of mesenchymal stem cells, such as the isoflavonoids genistein and daidzein, resveratrol, kaempferol, xanthoumol, with the involvement of estrogen receptors (ER) signaling [45]. Moreover, these compounds exerted a positive effect in vivo, thereby preventing ovariectomy-induced bone loss [19,31,53]. Ferutinin is one such phytoestrogens that is a daucane sesquiterpene found in the roots of plants in the Ferula genus, particularly Ferula hermonis Boiss [1]. Ferutinin has a binding affinity for human ERα and ERβ that is approximately 10% of estradiol for both receptors [3]. Different from the majority of phytoestrogens, which have a higher affinity for ERβ than ERα, ferutinin affinity is higher for ERα (IC50 = 33.1 nM) than ERβ (IC50 = 180.5 nM) [20]. Moreover, the estrogenic activity of ferutinin after oral administration has been widely demonstrated [47, 55,56]. We demonstrated that ferutinin was able to prevent, as well as treat, osteoporosis induced by estrogen deficiency in ovariectomized rats [13,36]. In particular, in the preventive protocol, ferutinin had the same anti-osteoporotic effects as estradiol benzoate [36] without exerting negative effects on the uterus or mammary glands [14]. Recently, we evaluated the role of ferutinin on the osteoblastic differentiation of AFSCs and DPSCs [57]. After 14 days of culture in an osteogenic medium in the presence of ferutinin, we observed a greater expression of osteoblast phenotype markers, an increased calcium deposition and osteocalcin secretion in the culture medium [57]. Accounting for the osteogenic potential of AFSCs and the enhancing properties of ferutinin in promoting the osteogenic differentiation of these stem cells, in this study we investigated the role of the oral administration of this phytoestrogen on bone regeneration in vivo. For this purpose, we employed a collagen scaffold seeded with AFSCs and implanted it in place of the parietal bone of rat calvaria. The aim of this study was to compare the capability of the collagen scaffold and the construct collagen scaffold + AFSCs, in the presence or absence of orally administration of ferutinin, to repair a critical-size bone defect using a well-established animal model and to evaluate the involvement of the estrogen receptors ERα and GPR30 in this regeneration process.

Materials and methods Cell culture Human amniotic fluid stem cells (AFSCs) were obtained from supernumerary amniocentesis provided by the Laboratorio di Genetica, Ospedale Santa Maria Nuova (Reggio Emilia, Italy). All samples were collected with the informed consent of patients according to Italian

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law and the Ethical Committee guidelines of Modena and Reggio Emilia University. AFSCs were isolated as previously described [26]. Human amniocentesis cultures were harvested by trypsinization and submitted to cKit immunoselection using MACS® technology (Miltenyi Biotec, Cologne, Germany) [26]. cKit positive AFSCs were subcultured at a 1:6 dilution and were not allowed to reach 80% confluence. AFSCs were cultured in minimum essential medium (αMEM) supplemented with 5% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (all reagents, EuroClone, Milan, Italy) at 37 °C and 5% CO2. In vitro osteogenic differentiation Collagen disks that had 13 mm diameters and 1.5 mm heights (horse-derived collagen—Condress, Istituto Gentilini, Pisa, Italy) were used as the 3D scaffold and were placed in 12-well culture plates. The scaffolds were washed twice with culture medium (1 hour for each rinse). The cells were seeded on each scaffold at density of 1 × 106 cells per disk and cultured for 24 hours with 2 mL culture medium. Then, the culture medium was changed to osteogenic medium supplemented with 100 nM dexamethasone, 10 mM β-glycerophosphate and 100 μM ascorbic acid-2-phosphate (all reagents, Sigma-Aldrich, St Louis, MO, USA). The cell-scaffold constructs were maintained in the osteogenic medium for 1 week before in vivo implantation, according to a previous protocol [27]. Selected samples were stained with 6-carboxyfluorescein diacetate (CFDA, Sigma-Aldrich, St Louis, MO, USA) to detect viable seeded cells. The cells were observed for green fluorescent staining with a Nikon A1 confocal laser scanning microscope (Nikon Instruments S.p.A., Firenze, Italy). Surgery, implantation procedure and treatments CD® IG5 male rats that were 12 weeks old were purchased from Charles River Laboratories (Lecco, Italy). They were housed one per cage and maintained in standard conditions with a 12:12 light/dark cycle, at temperature of 22 ± 1 °C and 55%–60% relative humidity. Commercial rat pellets (Global Diet 2018, Mucedola Srl, Milan, Italy) and drinking water were available ad libitum. For the implantation procedure, the animals were anesthetized with an intraperitoneal injection (0.2 mL/100 g body weight) of ketamine hydrochloride (Ketavet 100®, Farmaceutici Gellini SpA, Aprilia, Italy). We performed two symmetric full-thickness cranial defects (5 mm × 8 mm) on each parietal region of 10 animals. A midline skin incision was performed from the nose-frontal area to the external occipital protuberance. The skin and underlying tissues, including the periosteum, were reflected laterally to expose the full extent of the calvaria. The cranial areas to be removed were marked at the parietal bones using stereotaxic coordinates using the cranial structures as references. The cranial defect was created with a micromotor drill under constant sterile saline solution irrigation to prevent bone overheating [27]. One scaffold (5 × 8 × 1.5 mm size) was implanted into each cranial defect and adapted to fill the entire defect area. Each animal received two constructs. To evaluate the bone regeneration in different conditions, the animals were divided into 4 groups: -

Group 1: collagen type I scaffold Group 2: collagen type I scaffold + ferutinin oral administration Group 3: collagen type I scaffold seeded with AFSCs Group 4: collagen type I scaffold seeded with AFSCs + ferutinin oral administration.

After scaffold implantation, the incisions were sutured with prolene 4-0 sutures (Ethicon, Roma, Italy). The animals were immunocompromised using cyclosporine A (Sandimmun, Novartis SpA, Origgio, Varese, Italy) at a dose of 15 mg/kg body weight administered 4 hours before

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transplantation and then daily for 2 weeks. During the last weeks, the daily dose was gradually reduced to 6 mg/kg. Ferutinin (Indena SpA, Milan, Italy) was solubilized in Tween 80 (0.5%) and water, which was administered daily for 4 weeks by oral gavage at a dose of 2 mg/kg/5 mL. Rats were sacrificed 4 weeks after the surgical implantation and the calvarias were rapidly explanted and fixed in 4% paraformaldehyde in PBS (Phosphate Buffered Saline) for 3 hours. All procedures were permormed according to the guidelines approved by the Committee of Use and Care of Laboratory Animals of the University of Modena and Reggio Emilia. Animal care, maintenance and surgery were conducted in accordance with Italian Law (D.L. No. 26/2014) and European legislation (2010/63/UE). Histology, histomorphometry and immunohistochemistry The fixed samples of the explanted calvarias were treated with 0.5 M ethylendiamine tetraacetic acid (EDTA), pH 8.3 until complete decalcification. Then, the parietal bones were rinsed in PBS, dehydrated with graded ethanol, diaphanized and embedded in paraffin. We performed transversal serial sections (10 μm thickness), cutting through the midline of the implant area. Routine hematoxylin/eosin (H & E) staining was performed to analyze morphological details. Histological images were obtained using a Nikon Labophot-2 optical microscope equipped with a DS-5 Mc CCD color camera (Nikon Instruments, Japan). Quantitative histomorphometry was performed to evaluate the level of bone regeneration in the different groups after 4 weeks of treatment. We measured at 10 different points (10 subsequent serial sections) the following parameters: - regenerated bone area (AR) between the two fracture lines - pre-existing bone area (AP) before the operation Then, we calculated the AR/AP ratio, which was expressed as a percentage. Immunohistochemistry was carried out using mouse anti-human mitochondrial protein (Millipore, Billerica, MA, USA), mouse anti-ERα (Millipore, Billerica, MA, USA) and rabbit anti-GPR30 (Genetex, Irvine, CA, USA) antibodies. Endogenous peroxidase activity was quenched by incubating sections in 10% methanol and 3% hydrogen peroxide (H2O2), followed by three washes in PBS 1× at pH 7.4 and incubation for 30 minutes in bovine serum albumin (BSA, Sigma-Aldrich, St Louis, MO, USA) in PBS 1× with 0.2% Triton X-100. The sections were incubated in the primary antibody (diluted 1:50 in 3% BSA) for 1 hour at room temperature (RT). After three washes (10 minutes each), the tissue sections were incubated for 1 hour at RT in peroxidase-labeled anti-mouse or anti-rabbit antibodies diluted 1:3000 (Pierce Antibodies, Thermo Scientific, Rockford, IL, USA). Following three washes in PBS 1×, staining was observed using diaminobenzidine (2 mg/mL) and H2O2 (0.3 μL/mL) (DAB Substrate System, Sigma-Aldrich, St Louis, MO, USA). Then, the sections were washed with water, dehydrated with graded ethanol, xylene and mounted with Eukitt® (Carlo Erba Reagenti Spa, Naples, Italy).

at RT with the secondary antibody diluted 1:200 in PBS containing 3% BSA: goat anti-mouse Alexa fluor® 488, donkey anti-rabbit Alexa fluor® 647 (all Life Technologies, Carlsbad, CA, USA) antibodies. After washing in PBS, the sections were stained with 1 μg/ml 4′,6-diamidino-2phenylindole (DAPI) in water for 5 minutes and then were mounted with anti-fading medium, 0.21 M DABCO (1,4-diazabicyclo[2.2.2]octane), and 90% glycerol in 0.02 M Tris, pH 8.0). Negative controls consisted of samples not incubated with the primary antibody. The multi-labeling immunofluorescence experiments were performed to avoid cross-reaction between the primary and secondary antibodies. Confocal imaging was performed using a Nikon A1 confocal laser scanning microscope. Spectral analysis was performed to exclude overlapping between two signals or the influence of autofluorescence on the fluorochrome signals. The confocal serial sections were processed with ImageJ software to obtain 3D projections and the image rendering was performed using Adobe Photoshop Software (Adobe System Software, Ireland). Statistical analysis We performed two cranial defects for each animal (n = 10). The data are expressed as the mean ± standard error (SEM). The statistical analysis was assessed with GraphPad Prism version 5.0 for Windows (GraphPad Software, San Diego, CA, USA). One way analysis of variance (ANOVA) with the Newman–Keuls post-test was used to compare histomorphometrical data. A value of P b 0.05 was considered the level of statistical significance. Results Cell viability in the collagen construct To evaluate the bone regenerative potential of the collagen scaffold seeded with AFSCs and the role of orally administered ferutinin, we created a critical-size bone defect in both the parietal bones of animals, as previously described. Before the implantation, the cell-scaffold constructs were maintained in osteogenic medium for 1 week and then we verified the presence of living cells in selected samples with the 6carboxyfluorescein diacetate probe. This test showed that the AFSCs

Immunofluorescence and confocal microscopy The paraffin embedded sections were washed with xylene, decreasing concentrations of ethanol and water (5 minutes for each passage). To expose the antigens, the sections were treated with boiling EDTA buffer (EDTA 1 mM +0.05% Tween 20, pH 8.0) and maintained at 60 °C for 10 minutes. After reaching RT, the samples were blocked with 3% BSA in PBS 1× for 30 minutes. The sections were incubated with the primary antibodies diluted 1:50 in PBS containing 3% BSA: anti-human mitochondrial protein (Millipore, Billerica, MA, USA), mouse anti-ERα (Millipore, Billerica, MA, USA), rabbit anti-GPR30 (Genetex, Irvine, CA, USA) and rabbit anti-osteocalcin (Millipore, Billerica, MA, USA) antibodies for 1 hour at RT. After washing with PBS 3% BSA, the samples were incubated for 1 hour

Fig. 1. Amniotic fluid stem cells cultured in collagen scaffolds. After 7 days of culture, amniotic fluid stem cells are present in the collagen scaffolds, as shown by 6-carboxyfluorescein diacetate vital staining in vivo (green). Scale bar = 20 μm.

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Fig. 2. Hematoxylin/eosin staining for comparative histological analysis of critical-size bone defects 4 weeks post-surgery. Transversal sections through the central area of the construct. (A) cranial defect filled with collagen, (B) cranial defect filled with collagen in rats treated for 4 weeks post-implantation with ferutinin 2 mg/kg per os, (C) cranial defect filled with collagen colonized with AFSCs, (D) cranial defect filled with collagen colonized with AFSCs in rats treated for 4 weeks post-implantation with ferutinin 2 mg/kg per os (scale bar = 100 μm). Panels (E), (F), (G), and (H) are magnifications of the respective adjacent image (scale bar = 10 μm). Arrowheads indicate vessels; arrow indicates osteoblast rim.

were viable in all samples analyzed and successfully colonized the collagen scaffolds. In Fig. 1 we present a representative confocal image of the viable cells (stained in green), seeded at full-thickness in the collagen support. The cells are homogeneously distributed within the collagen sponge between its micro-architecture. All animals implanted with the construct remained healthy throughout the course of experiment (4 weeks). There was no evidence of side effects or infection in any animals and no animals died.

observed a greater reconstruction from the fracture edges, as well as islets of bone in the center of the scaffold (Fig. 2C–D, G–H); this newly formed bone had a mineralized matrix, and it was well organized with an osteoblast rim (Fig. 2H arrow) and osteocyte lacunae (Fig. 2G–H). The presence of AFSCs together with ferutinin oral administration was able to increase the number and size of these islets, resulting in a better reconstruction of the critical size defect. Regarding the histomorphometric data, in a previous study, we calculated the percentage of bone reconstruction in negative controls

Qualitative histology and quantitative histomorphometry Four weeks after implantation, the rats were sacrificed and their calvarias were removed. To perform histological evaluations, the samples were processed as described in the Materials and methods section and were observed by bright field microscopy. Representative histological sections are shown in Fig. 2 (A–D), where the bone resected areas and the islets of newly formed bone are clearly detectable. In all groups, we observed the presence of vessels, as indicated by the arrowheads in the magnification panels (Fig. 2E–H). In samples where the lesion was filled only with the collagen sponge, we observed the presence of morphologically and structurally organized fibrous tissue with small areas of newly formed bone (Fig. 2A, E). This bone was immature and lacking in osteocyte lacunae, but we observed many vessels near these areas (Fig. 2E). In this condition, when the phytoestrogen ferutinin was administered orally for 4 weeks, the collagen sponge persisted in the central area of the damage, but the areas of newly formed bone, near to the pre-existing, were greater than those in the collagen group (Fig. 2B, F). In animals implanted with collagen colonized by pre-differentiated AFSCs, we

Fig. 3. Percentage of bone reconstructed area in collagen constructs 4 weeks post-surgery. The values are expressed as the mean ± SEM (n = 5 for each group). The statistical analysis was performed using one-way ANOVA followed by the Newman–Keuls multiple comparison test: ⁎P b 0.05, ⁎⁎⁎P b 0.0001 vs. collagen group, ###P b 0.0001 vs. collagen + F group, §§P b 0.01 vs. collagen + AFSCs group. F = ferutinin.

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(empty cranial defects with or without ferutinin oral administration) and observed that these values were significantly lower compared with the collagen and collagen + F groups; however, these groups were but not significantly different from each other (data not shown). For this reason, we referred to the collagen scaffold as the control group. Fig. 3 shows the percentage of the bone reconstructed area as the ratio between the regenerated bone area (AR) and the preexisting bone area (AP) before surgery × 100. The cell-free groups (collagen and collagen + F) showed a reconstruction percentage of approximately 40% (36.3 ± 3.4 and 42.2 ± 2.8, respectively). When the collagen sponge was colonized by AFSCs, the AR/AP value was

significantly greater compared with the collagen group (P b 0.05, 51.9 ± 2.9 vs. 36.3 ± 3.4). Ferutinin treatment in the animals implanted with the collagen + AFSCs construct led to a reconstruction of approximately 70%; this value was statistically significant compared with both the collagen cell-free construct groups (P b 0.0001) and collagen + AFSCs group (P b 0.01). Immunohistochemistry and immunofluorescence Four weeks after the surgery, cells of human origin were detectable in all samples derived from animals implanted with collagen + AFSCs

Fig. 4. Immunohistochemical analysis of anti-human mitochondria (anti-h-mit) in constructs colonized by AFSCs. Diaminobenzidine (DAB) staining of human cells using an anti-human mitochondria antibody in the collagen + AFSCs group in (A) connective tissue (collagen sponge implanted in the cranial defect) and (B) newly formed bone tissue and in collagen + AFSCs in rats treated for 4 weeks post-implantation with ferutinin 2 mg/kg per os in (C) connective tissue and (D) newly formed bone tissue. Negative control for anti-h-mit in a cell-free scaffold in (E) connective tissue and (F) newly formed bone tissue. Scale bar = 50 μm.

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constructs, as showed by immunohistochemistry for the anti-human mitochondria protein (h-mit, Fig. 4A–D). We examined the presence of h-mit in the connective tissue (collagen sponge) between the two edges of the bone and in the newly formed bone near fracture edges. In the collagen + AFSCs and collagen + AFSCs + F groups, human cells were localized both in connective tissue (Fig. 4A, C) and newly formed bone (Fig. 4B, D). As expected, in the cell-free scaffolds, the presence of h-mit protein was negative (Fig. 4E, F). In all groups included in this study, we evaluated the expression of the most important marker for bone deposition, osteocalcin (OCN). A double immunofluorescence analysis for OCN (red) and h-mit (green) was performed in samples colonized with AFSCs. We detected the presence of the osteoid protein OCN in the extracellular matrix of all samples (Fig. 5A, B, D, G). In particular, OCN was highly expressed in the collagen + AFSCs group (Fig. 5D) and even more highly expressed when ferutinin was administered (Fig. 5G). The immunofluorescence analysis for h-mit confirmed the presence of human cells in both the connective tissue and the newly formed bone in the collagen + AFSCs and collagen + AFSCs + F groups (Fig. 5C, F). In those groups, the

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double staining for h-mit and OCN indicated that there was greater OCN expression near h-mit positive cells. Moreover, a higher magnification showed that many cells in the connective tissue near the newly formed bone were positive for h-mit (Fig. 5E, H). Specifically, in this area, high OCN expression was observed in the scaffolds colonized by AFSCs when ferutinin was orally administered (Fig. 5H). Immunohistochemistry and immunofluorescence experiments were performed to detect the expression of estrogen receptors (ER) α and GPR30 in the connective tissue and newly formed bone near the fracture edges (Figs. 6 and 7). In the immunohistochemistry analysis, positive cells for ERα were detectable in all samples (Fig. 6A, D, G, J). In AFSCs-free conditions, few cells showed marked staining for ERα (Fig. 6A), whereas the presence of ferutinin increased ERα positivity (Fig. 6D). In both groups where the scaffolds were colonized by AFSCs, ERα staining was highly diffuse (Fig. 6G–J). In the immunofluorescence analysis, the confocal images in Fig. 6 confirmed these results. In the collagen and collagen + F groups, we observed nuclear positivity for ERα (green) in some osteocytes (Fig. 6B, C, E, F). The number of osteocytes expressing ERα was higher in samples with constructs colonized by

Fig. 5. Confocal images of implants for the detection of osteocalcin (OCN) and anti-h-mit. Double immunofluorescence (IF) confocal images showing the signals from anti-OCN (red) and DAPI (blue) in sections of newly formed bone tissue in the (A) collagen, (B) collagen + F, (D) collagen + AFSCs and (G) collagen + AFSCs + F groups. Double IF for anti-hmit (green) and DAPI (blue) in the (C) collagen + AFSCs and (F) collagen + AFSCs + F groups. Magnification of triple IF for h-mit (green), OCN (red) and DAPI (blue) in the (E) collagen + AFSCs and (H) collagen + AFSCs + F groups. F = ferutinin. Scale bar = 50 μm.

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Fig. 6. Immunohistochemical and immunofluorescence analysis for ERα detection. Diaminobenzidine staining for ERα positive cells and immunofluorescence confocal images showing the signals from anti-ERα (green) and DAPI (blue) in constructs of the (A–C) collagen, (D–F) collagen + F, (G–I) collagen + AFSCs, (J–L) collagen + AFSCs + F groups. F = ferutinin. Scale bar = 50 μm.

AFSCs (Fig. 6H, I), and this receptor was strongly expressed in the nuclei of the osteoblast rims that constituted the deposition front in animals treated orally with the phytoestrogen ferutinin (Fig. 6K, L). A similar condition was observed for GPR30 (Fig. 7). Immunohistochemistry revealed that GPR30 was expressed in the connective tissue and newly formed bone of all groups (Fig. 7A, C, E, G), with a clear prevalence in the collagen + AFSC + F group (Fig. 7G). Interestingly, GPR30 (green) was expressed in newly formed bone, as shown by its localization in osteocytes in the cell-free constructs (Fig. 7B, D) and in osteoblast rims in the scaffolds colonized by AFSCs (Fig. 7F, H).

Discussion To treat critical-size bone defects, the gold standard remains the autograft despite several limitations [29]. Recently, new strategies have been designed to produce an efficient bone graft as an alternative method. For example, tissue engineering researchers suggest the use of scaffolds that have healing promotion factors and stem cells. An ideal tissue-engineered product should be a construct with properties similar to those of autografts but without their limitations.

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Fig. 7. Immunohistochemical and immunofluorescence analysis for GPR30 detection. Diaminobenzidine staining for GPR30 positive cells and immunofluorescence confocal images showing signals from anti-GPR30 (green) and DAPI (blue) in constructs of the (A, B) collagen, (C, D) collagen + F, (E, F) collagen + AFSCs, and (G, H) collagen + AFSCs + F groups. F = ferutinin. Scale bar = 50 μm.

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In this study, we used a collagen-type I scaffold that was colonized with human amniotic fluid stem cells (AFSCs). This construct was implanted into the parietal bone defects of immunodeficient rats. A collagen sponge is currently used in several therapeutic practices because of its high availability, easy purification, biocompatibility and non-toxicity [11,34]. We have previously demonstrated that the collagen sponge is an excellent scaffold that facilitates cell adhesion and bone-forming cells in the defect site, even in a cell-free condition. However, better and faster bone reconstruction was obtained when the construct was colonized by AFSCs [27]. Our results in this study confirmed these previous observations. In fact, we reported that 4 weeks after implantation, the collagen–AFSCs construct was able to reconstruct approximately 52% of the critical-size defect with a bone healing process that moved from the edges of the fractures, as well as involved newly formed bone islets inside the collagen sponge. This percentage of bone reconstruction was significantly greater in compared with those obtained with the collagen scaffold in a cell-free condition. Before the implant procedure, the scaffolds colonized by AFSCs were maintained in osteogenic medium for 7 days. This treatment improved the reconstruction capability because the cells were committed to osteogenic differentiation. Moreover, we observed the presence of vessels in the scaffold area of all samples analyzed. The vascularization of the scaffold is very important for providing an environment suitable for bone healing and regeneration [29]. Our study was designed to use the phytoestrogen ferutinin as a healing promotion factor. Ferutinin is a daucane sesquiterpene that has estrogenic activity and showed in vitro enhancing capabilities on the proliferation and osteogenic differentiation of AFSCs [57]. Starting from the first day post-surgery, the rats were orally administered ferutinin at dose of 2 mg/kg for 4 weeks. The treatment was performed in rats implanted with only the collagen sponge and with the collagen colonized by AFSCs. The beneficial effects of ferutinin were evident in the presence of stem cells because ferutinin was able to act on the AFSCs, inducing osteogenic differentiation after 14 days of treatment [57]. The collagen + AFSCs + ferutinin construct led to an approximately 70% bone reconstruction area, which was significantly higher compared with other conditions (collagen, collagen + ferutinin, collagen + AFSCs). The presence of human cells was confirmed before and after implantation. Before surgery, some samples were treated with the 6carboxifluorescein diacetate probe; this treatment demonstrated that the scaffolds were colonized by viable cells. Post-surgery, we performed an immunohistochemical analysis on paraffin sections for human mitochondrial protein using an anti-human mitochondria antibody. Human cells were detectable in the connective tissue of the damaged area and in the newly formed bone islets, indicating that the bone regeneration was driven by the AFSCs. Osteocalcin (OCN) is a marker for the mature osteoblast phenotype, is involved in bone mineral deposition and is found in lamellar bone [17]. Our results demonstrated the presence of OCN inside the new bone in all samples; in particular, OCN expression was higher when the scaffolds were colonized by AFSCs. Ferutinin treatment led to a marked expression of OCN inside the new bone and in the extracellular matrix of the osteoblast rims and the connective cells near the deposition front. In this area, high OCN expression was accompanied by positivity for h-mit, confirming that human cells were responsible for bone deposition. Our data suggest that orally administered ferutinin was able in vivo to enhance the osteogenic differentiation of AFSCs, thereby increasing the bone reconstruction process. In this study, we also evaluated the expression of the estrogen receptors (ER) α and GPR30 because ferutinin is a phytoestrogenic compound that is able to interact with those receptors. It is well known that ferutinin acts as an agonist on ERα [20], but no data have been reported about its interaction with GPR30 and subsequent effects. Estrogens are important regulators of bone metabolism, particularly through ERα [48]. In vitro and in vivo studies have demonstrated that

ERα was expressed in osteoblasts, osteocytes and osteoclasts [8,12,23, 58]. Our data clearly showed the presence of ERα in all samples, in both the connective tissue and newly formed bone. Ferutinin treatment increased the ERα expression in the connective tissue and bone in the scaffolds colonized with or without AFSCs. These results suggest that ferutinin was able to up-regulate the amount of ERα expressed. GPR30 is an estrogen receptor that mediates non-genomic estrogen signaling with a predominantly intracellular localization that was identified by multiple groups in the late 1990s [9,32,35,51]. Recently, it was demonstrated that GPR30 is expressed in bone cells (osteoblasts, osteocytes and osteoclasts), although its role in these cells has not been completely elucidated [18]. Our data indicated that ferutinin did not alter GPR30 expression; however, greater GPR30 expression was obtained in samples with scaffolds colonized by AFSCs. Therefore, the presence of human cells, but not ferutinin, led to enhanced GPR30 expression, particularly near osteoblast rims. These results are in accordance with recent investigations performed in male mice that described the involvement of ERα in bone growth. However, GPR30, despite its capability to bind estrogens with high affinity, was not required for normal estrogenic responses on several well-known estrogen-regulated parameters, such as increased bone mass [33]. Conclusions In this study we demonstrated that in all treatment groups (collagen, collagen + ferutinin, collagen + AFSCs, collagen + AFSCs + ferutinin), critical-size defect repair was initiated. A daily administration of the phytoestrogen ferutinin, as a healing promotion factor, in association with pre-differentiated AFSCs seeded in the collagen sponge proved to be a good protocol that was able to enhance bone reconstruction from the edges of the fractures and directly from the collagen scaffold implant as islets of newly formed bone. In conclusion, ferutinin, acting through ERα binding, shows an osteoinductive potential and emerges as a good healing promotion factor in bone tissue engineering. Competing interest The authors declare that they have no competing interest.

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