TCP composite scaffold incorporating bioactive phytomolecule icaritin for enhancement of bone defect repair in rabbits

Acta Biomaterialia 9 (2013) 6711–6722

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PLGA/TCP composite scaffold incorporating bioactive phytomolecule icaritin for enhancement of bone defect repair in rabbits S.-H. Chen a,f, M. Lei b, X.-H. Xie a,g, L.-Z. Zheng a, D. Yao a, X.-L. Wang a,h, W. Li b, Z. Zhao c, A. Kong a, D.-M. Xiao b, D.-P. Wang c, X.-H. Pan d, Y.-X. Wang e, L. Qin a,h,⇑ a

Department of Orthopaedics & Traumatology, The Chinese University of Hong Kong, Hong Kong Special Administrative Region, China Department of Orthopaedics, Shenzhen Hospital of Beijing University, Shenzhen, People’s Republic of China Department of Orthopaedics, The Second Peoples’ Hospital, Shenzhen, People’s Republic of China d Department of Orthopaedics, The First Peoples’ Hospital, Shenzhen, People’s Republic of China e Department of Imaging and Interventional Radiology, The Chinese University of Hong Kong, Hong Kong Special Administrative Region, China f Department of Biochemistry and Molecular Biology, Harbin Medical University, Harbin, People’s Republic of China g Department of Orthopaedics, The First Affiliated Hospital of Soochow University, Suzhou, China h Translational Medicine R&D Center, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, People’s Republic of China b c

a r t i c l e

i n f o

Article history: Received 15 October 2012 Received in revised form 22 January 2013 Accepted 23 January 2013 Available online 1 February 2013 Keywords: Bone scaffold Icaritin Bone defect Osteogenesis Angiogenesis

a b s t r a c t Bone defect repair is challenging in orthopaedic clinics. For treatment of large bone defects, bone grafting remains the method of choice for the majority of surgeons, as it fills spaces and provides support to enhance biological bone repair. As therapeutic agents are desirable for enhancing bone healing, this study was designed to develop such a bioactive composite scaffold (PLGA/TCP/ICT) made of polylactide-co-glycolide (PLGA) and tricalcium phosphate (TCP) as a basic carrier, incorporating a phytomolecule icaritin (ICT), i.e., a novel osteogenic exogenous growth factor. PLGA/TCP/ICT scaffolds were fabricated as PLGA/TCP (control group) and PLGA/TCP in tandem with low/mid/high-dose ICT (LICT/MICT/HICT groups, respectively). To evaluate the in vivo osteogenic and angiogenic potentials of these bioactive scaffolds with slow release of osteogenic ICT, the authors established a 12 mm ulnar bone defect model in rabbits. X-ray and high-resolution peripheral quantitative computed tomography results at weeks 2, 4 and 8 post-surgery showed more newly formed bone within bone defects implanted with PLGA/TCP/ICT scaffolds, especially PLGA/TCP/MICT scaffold. Histological results at weeks 4 and 8 also demonstrated more newly mineralized bone in PLGA/TCP/ICT groups, especially in the PLGA/TCP/MICT group, with correspondingly more new vessel ingrowth. These findings may form a good foundation for potential clinical validation of this innovative bioactive scaffold incorporated with the proper amount of osteopromotive phytomolecule ICT as a ready product for clinical applications. Ó 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction Innovation of therapies for enhancing osteogenesis and angiogenesis remains a critical challenge in reconstruction of large bone defects. Currently, the most successful graft materials used for this purpose are autograft and allograft. There have been 2.2 million bone grafts used annually worldwide [1]. However, problems still exist, such as resorption of the allograft, fatigue failure, fracture, secondary infection and limited supply of autograft [2,3]. Prostheses for reconstruction of bone defects are widely used in orthopaedic tissue engineering. The overall concept focuses on the use of conductive scaffold materials possessing the desired mechanical ⇑ Corresponding author at: Department of Orthopaedics & Traumatology, The Chinese University of Hong Kong, Hong Kong Special Administrative Region, China. Tel.: +852 2632 3071. E-mail address: [email protected] (L. Qin).

function and tissue regeneration potential, in combination with potential delivery of endogenous osteoinductive or osteopromotive growth factors [4–9]. Icaritin (ICT), an exogenous semisynthesized small phytomolecule [9–11], is an intestinal metabolite of epimedium-derived flavonoids (EF) discovered in serum [12,13]. EF is a known ‘‘bone strengthening Chinese herb’’ exerting an anabolic effect on osteoporotic bone [14] and promoting angiogenesis attributed to its estrogenic effect [15]. Recent work demonstrated that its metabolite ICT was the potent one to enhance differentiation and proliferation of osteoblasts and facilitate matrix calcification; and meanwhile, ICT was also found to be able to inhibit adipogenic differentiation of mesenchymal stem cells (MSC) [9,11,12,16]. Scaffold biomaterials have been developed for various orthopaedic applications [17]. Polymeric materials such as polylactideco-glycolide (PLGA) can be dissolved with organic solvent to form paste and therefore used as a basic biomaterial for forming

1742-7061/$ - see front matter Ó 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.actbio.2013.01.024

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three-dimensional (3D) porous scaffold using spinning technology, together with addition of tricalcium phosphate (TCP), as TCP was able to buffer the lower pH value during degradation of PLGA in favour of reduction of inflammation in vivo, apart from enhancement of scaffold mechanical properties [18]. In this study, PLGA and TCP were used as basic carriers to fabricate porous composite scaffolds by incorporating or homogenizing bioactive phytoestrogenic ICT (PLGA/TCP/ICT composite scaffolds) at various dosages, using established low-temperature rapid-prototyping technology [6,19– 21]. ICT served as an exogenous growth factor incorporated into PLGA/TCP composite scaffold. In comparison with this novel approach, endogenous growth factor bone morphogenetic protein 2 (BMP-2) was incorporated, using the same method and in vitro degradation, the release and osteogenic potential were compared with ICT incorporating scaffold. The results indicated that BMP-2 incorporating scaffolds did not present desirable osteogenic ability for the cultured BMSC. This finding was explained by the fact that BMP-2 might have lost its original bioactivity, as organic solvent denatured the protein during scaffold fabrication and preparation [6]. So the incorporation of the exogenous growth factor ICT into PLGA/TCP to form porous composite scaffolds may become an attractive concept for further research and development of biomaterials incorporating relevant bioactive molecules. Recent in vitro studies [5,6,22] showed that PLGA/TCP/ICT porous composite scaffolds also functioning as a local delivery system of ICT were able to grant a slow release of the incorporated ICT from the composite scaffold to execute the bone anabolic effects. The present study was therefore designed to further investigate its potential in bone repair augmentation effects, using a standard rabbit ulnar segmental defect model, evaluated systemically using radiographs, high-resolution peripheral quantitative computed tomography (HR-pQCT) and histology. As it is also known that bioactive scaffold materials might promote angiogenesis and neovascularization if it involves underlying mechanisms related to its osteogenic potential [23,24], the current study also adopted micro-computed tomography (micro-CT) based angiography to study new vessel formation within the defects as well dynamic magnetic resonance imaging (MRI) for investigating its local perfusion functions [25–27]. 2. Materials and methods 2.1. Preparation of bioactive composite scaffolds PLGA/TCP incorporating phytomolecule ICT were fabricated at 28 °C with a low-temperature rapid-prototyping machine (CLRF-2000-II, Tsinghua University, China) using the established protocol [6]. Briefly, PLGA and TCP powders with a weight ratio of 4:1 were dissolved in organic solvent 1,4-dioxane to form a homogeneous solution. PLGA was added according to a published ratio of powder weight to solution volume of 13:100. Three doses of ICT were prepared, with 0.013:100 as the P/T/LICT group, 0.052:100 as the P/T/MICT group and 0.13:100 as the P/T/HICT group, respectively, as reported in a recent in vitro study [6]. The organic reagent 1,4-dioxane was volatile and therefore could be entirely removed by a 24 h drying process in a freeze dryer with an ice condenser temperature of 55 °C and a negative pressure of 500 Pa [19,21]. All scaffolds used in the animal model were trimmed down to 4  4  12 mm3 to fit into the bone defects. 2.2. Establishment of ulnar bone segmental defects in rabbits A segmental ulnar bone defect model was adopted into the current study in adult male New Zealand white rabbits [28,29]. A segment of ulnar midshaft with a length of 12 mm that was regarded as a sub-critical sized defect was surgically removed (Fig. 1A).

Briefly, bilateral mid-ulna osteotomy was performed on 5 month old New Zealand white male rabbits weighing 3.5–4 kg [30,31]. Four types of scaffolds were tested, including pure P/T scaffold as control and three experimental groups with different doses of ICT, i.e., P/T/LICT, P/T/MICT, P/T/HICT scaffold group (Fig. 1B). A total of 120 forelimbs in 60 rabbits were divided into five groups, with 24 ulna-defected forelimbs in 12 rabbits for each group: 16 forelimbs for decalcified histology (four samples harvested and analysed per time-point (weeks 1, 2, 4, 8); eight forelimbs for undecalcified histology (four samples harvested and analysed per time-point (weeks 4, 8). However, a total of six rabbits were used for monitoring defect healing by X-ray and HR-pQCT at weeks 2, 4, 8 from the animals with ending time-point of week 8, which were also used for decalcified and undecalcified histology. Under general anaesthesia with ketamine (2 mg kg 1 body weight) and xylazine (50 mg kg 1 body weight) (v:v = 1:1), bilateral forelimbs of rabbits were shaved, prepped with hibitane and 70% ethanol, and followed with a surgical incision measuring 20 mm in length above the ulna. Soft tissue was resected, and a 12 mm segment of ulna was removed using an oscillating saw (Synthes; Mathys AG, Bettlach, Switzerland) irrigated with saline. Scaffolds were press-fit into the ulnar defects, and the wound was closed with layers of sutures. Pressure bandages were used to protect the wound for 3 days post-surgery. Pain was managed by post-surgery injections of Temgesic three times in the first 72 h. Animals were fed with water ad libitum and allowed free cage activity. The Animal Experimental Ethics Committee of the corresponding author’s institution approved the study protocol (Ref. No.: 10/026/MIS-5).

2.3. Series radiographs and evaluation of new bone area fraction High-resolution radiographs (Medical X-ray Film, Fuji Photo Film Co., Japan) of the operated ulnae were taken immediately after implantation as baseline and then at post-operative weeks 2, 4 and 8, using a commercial X-ray machine (Faxitron X-ray Corporation, USA), with an exposure time of 3 s, a tube voltage 60 kVp, and at a X-ray source–object distance of 40 cm. Afterwards, X-ray films were scanned as TIF images, using a Photo Scanner (Epson Perfection 4990, Epson America, USA). The newly formed bone was identified for quantifying its size and calculating its area fraction within the proportional area of its original bone defect region using Adobe Photoshop CS5 software. New bone that formed within the bone defect region from week 8 radiographs was graded with score 1–4, i.e., area fraction of new bone: 0% to 25% (score 6 1); 25% to 50% (1 6 score 6 2); 50% to 75% (2 6 score 6 3); and 75% to 100% (3 6 score 6 4) based on a published work [31].

2.4. CT evaluation of new bone formation At the above given post-operative time points, newly formed bone was evaluated using HR-pQCT (XtremeCT, Scanco Medical, Brüttisellen, Switzerland) according to published protocol [25]. Briefly, the entire defect region was scanned at a spatial resolution of 40 lm, and the bony compartment was segmented from the marrow and soft tissue for subsequent analyses using a global threshold procedure. A threshold equal to or above 150 represented bony tissue; a threshold below 150 represented bone marrow, soft tissue and implanted composite scaffolds [32]. The new bone formed within the bone defect region was acquired for quantification of bone mineral density (BMD), tissue volume (TV) and bone volume (BV).

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Fig. 1. Rabbit ulnar segmental bone defect and implantation with porous composite scaffold. A 12 mm ulnar segmental defect was created in the midshaft in 18 week old New Zealand white rabbits: (A) PLGA/TCP-based porous scaffold with size 4  4  12 mm3 (B) was implanted into the defect region.

2.5. Histological evaluation of new bone After HR-pQCT scanning, the samples were fixed in 10% neutral buffered formaldehyde (pH 7.2) for 3 days, decalcified in 9% formic acid at room temperature for 4 weeks, dehydrated through an ethanol series, and embedded in paraffin using an Embedding Center (Thermolyne Sybron, Dubuque, IA, USA). Sections of specimens at a thickness of 5 lm were prepared along the long axis and coronal plane of the ulna defect region using a microtome (LEICA RM2165, Germany). Serial sections were stained with various methods, as described below, for microscopic evaluations. 2.5.1. Hematoxylin and eosin and Goldner’s trichrome staining for routine histology Sections with hematoxylin and eosin (H&E) and Goldner’s trichrome staining were digitalized into a microscopic system (Leica MPS 60, Germany) for both descriptive histology of the appearance of new bone and quantitative histomorphometry [33,34]. In addition, polarized microscope was used to study the collagen alignment of the mineralized bone tissue. The area of new bone and the total implant area within the bone defects were quantified separately using an Image-pro Plus software system (Media Cybernetics, Silver Spring, MD, USA) [34]. The area fraction of new bone within the bone defects was also determined. Four serial sections from each specimen were used for taking its average for statistical analysis. 2.5.2. Immunohistochemistry Immunohistochemistry was performed using a published protocol [35]. Briefly, decalcified 5 lm sections were mounted on amino-propyl-triethoxy-silane coated slides. These sections were then dewaxed in descending concentrations of alcohol and rehydrated.

The slides were immersed in 3% hydrogen peroxide to block endogenous peroxidases and rinsed in phosphate buffered saline (PBS). They were then immersed in 0.1% TritonX100 in PBS for 20 min to allow penetration of the membrane. Antigen retrieval was carried out in a 10 mM 60 °C warm citrate buffer for 15 min at pH 6.0. Afterwards, the slides were rinsed in PBS. Specific sites were saturated with normal goat serum, for 40 min at 37 °C. Sections were incubated with the specific antisera (all diluted 1:100) for overnight at 4 °C. Immunofluorescence staining of osteocalcin was performed: first, primary antibody (Protocol distributed by Lab Vision Corp., Newmarket, Suffolk, UK) was used for overnight incubating at 4 °C followed by washing with PBS before applying fluorescent donkey polyclonal second antibody IgG (diluted 1:200, Invitrogen) for 30 min at room temperature. Thereafter, the sections were incubated for 30 min with DAPI for counterstain. The slides were then examined microscopically. 2.6. Evaluation of rate of new bone formation 2.6.1. Sequential fluorescence labelling and sample harvesting Sequential fluorescence labelling was used to study bone dynamic remodelling within segmental bone defect using the established protocol [36,37]. In brief, two fluorescent dyes, xylenol orange (90 mg kg 1 body weight) and calcein green (10 mg kg 1 body weight; both Sigma–Aldrich GmbH), were injected subcutaneously and sequentially into the rabbits of weeks 4 and 8 groups at day 10 and day 3 before euthanasia. 2.6.2. Bone formation evaluated using fluorescence microscopy The samples of the ulnar bone defects were fixed in 10% neutral buffered formaldehyde (pH 7.2) for 3 days. The fixed samples were

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then dehydrated in successive alcohol concentrations from 70% to 100%, infiltrated and cleared with xylene, and embedded in methyl methacrylate (MMA; Mecck-Schuchardt, Germany). After hardening, samples were cut at mid-coronal sections in a thickness of 200 lm using a saw microtome (Leica SP1600, Leica Instruments, Nussloch, Germany). The sections then were glued onto a transparent plastic plate and polished to 100 lm by a polisher (Phoenix 4000, Buehler Ltd. USA) before digitalization using a fluorescence microscopic system (Leica Q500MC, Leica Cambridge Ltd, a digital camera of Leica MPS60, Germany). Fluorochromes are calcium-seeking molecules that bond to the mineralization fronts in bone formation sites. In sections, the areas of bone formation labelled with fluorochromes were visualized by specific fluorescence microscopy and quantified. Calcein green was positive, with green colour at a 480 nm excitation wavelength; xylenol orange was positive with red colour at a 560 nm excitation wavelength. The mixture of calcein green/xylenol orange was positive, with green and red colours at a 620 nm excitation wavelength. The area fraction of temporal bone formation was then calculated and interpreted as the ratio of areas labelled with calcein green and xylenol orange [38]. For further observation of the osteogenic process from osteoblasts to osteoid tissues and final mineralized bone tissues, Goldner’s trichrome staining was also performed on undecalcified MMA sections in 10 lm thickness (Leica SM2500E, Leica Instruments, Nussloch, Germany). 2.7. Evaluation of neovascularization using micro-CT-based microangiography 2.7.1. Microfil vessels perfusion Microfil perfusion was conducted in the forelimbs of rabbits. Briefly, under deep general anaesthesia, the bilateral arteria axillaries and vena axillaries of the animals were separated and inserted with scurf-needles that were linked to a pump apparatus (PHD 22/ 2000, Harvard Apparatus, USA) with a flow speed set at 20 mm min 1 for perfusion, based on the established protocol [25]. The vasculature was flushed with adequate pre-warmed heparinized saline (50 U ml 1) [39] and was pumped with 10% neutral buffered formalin for fixation followed by heparinized saline flushing, then the vasculature was injected with a confected radiopaque silicone rubber compound based on the manufacturer’s protocol (Microfil MV-122, Flow Tech, Carver, MA, USA). After that, bilateral forelimbs were harvested, fixed with 10% buffered formalin for 3 days, and then decalcified with 9% formic acid for 4 weeks for the following evaluations. 2.7.2. Micro-CT-based micro-angiography and histomorphometry of neovascularization The entire rabbit forelimb was scanned for micro-CT-based micro-angiography and quantified for connectivity density of angiographic structure using the established protocol [25]. In brief, the limb was fixed in the tube with its long axis perpendicular to the bottom of the tube for micro-CT scanning using viva-40 (Scanco Medical, Brüttisellen, Switzerland). According to the distance from proximal ulna to the defect site, the scan was then perpendicular to the shaft and initiated from a reference line 20 mm away from the bottom, with an entire scan length of 14 mm. The scan was performed at a resolution of 36 lm voxel 1 with 1024  1024 pixel images matrix [9,13]. For segmentation of blood vessels from background, noise was removed using a low pass Gaussian filter (Sigma = 1.2, Support = 2), and blood vessels were then defined at a threshold of 100. The density of newly formed vessels within the implants was quantified by calculating the number of new vessels under the microscope. Four slices in each sample were used for quantification under a magnification of 100, and 10 regions in bone defects in

each slice were randomly selected, the new vessels filled with microfil dye were counted and averaged. 2.8. Blood perfusion function using dynamic MRI The rabbits were anaesthetized for blood perfusion within ulna defect sites at weeks 2 and 4 post-surgery by dynamic MRI, using a high-field 1.5 T superconducting system (ACS-NT Intera; Philips, The Netherlands), which had a maximum gradient strength of 30 mT m 1. The body volume radiofrequency coil was used for signal transmitting, and a commercially available surface radiofrequency coil with a diameter of 4.7 cm (Micro 4.7, Philips Medical Systems, Best, The Netherlands) was placed on the middle ulna area for receiving signals. Multi-slice T1-weighted fast spin echo sequence (TR/TE = 425/20 ms, echo train length = 3) was employed throughout the plane of bone defect site created in the ulna, based on a previous protocol [25]. Imaging parameters included section thickness 3 mm, intersection gap 1 mm, field of view 120 mm and imaging matrix 256  128. After conventional MRI scanning, the abnormal signal of defects in the middle ulna was checked and compared with the normal baseline by two radiologists. Based on anatomical images, the dynamic MRI scan plane was decided. A bolus of gadopenteta dimeglumine (Magnevist; Schering, Berlin, Germany) (0.8 mmol kg 1 body weight) was rapidly injected manually via an ear vein, then immediately followed by a 6 ml saline flush at the same injection rate [25,40]. The dynamic scan started as soon as the injection of the contrast medium commenced. The signal intensity (SI) was then measured in operatordefined ellipse-like regions of interest (ROI) over the target site beneath the joint space in the mid-coronal T1-weighted images, using a cursor and graphic display device. The SI values derived from the ROI were plotted against time as a time–intensity curve (TIC) for calculating ‘‘maximum enhancement’’, an index of local perfusion function defined as the maximum percentage increase (SImax SIbase) in SI from baseline (SIbase) [25]. 2.9. Statistical analysis All quantitative data are presented as mean ± standard deviations. Significant differences were analysed by one-way ANOVA tests using SPSS version 16.0 (SPSS, Chicago, IL, USA). Statistical significance was set at P < 0.05. 3. Results All rabbits survived during and after surgery. No fractures or infections were observed in any of the rabbits post-operatively. 3.1. Radiographic area fraction of new bone within ulnar defect The results in Fig. 2A show that the defects without scaffold implantation are filled with scattered bony structure. Bone defects implanted with P/T scaffold reveal a partial yet homogeneous filling of bony structure. Complete bridging between the bony ends is seen along the border of the radius and extending approximately midway up throughout the defect after implantation with P/T/ICT scaffolds, where the P/T/MICT group presents the best outcome results. Quantitatively, the mean percentages of newly formed bone filling the segmental defect region was 32.68% for the defect group without scaffold implantation; while it was 48.67%, 79.39%, 86.27% and 69.65% for P/T, P/T/LICT, P/T/MICT and P/T/HICT groups, respectively. The two-dimensional (2D) X-ray grading scores (Fig. 2B) indicate that there were significant enhancements of bone regeneration in P/T/ICT scaffolds (score: LICT-3.18, MICT-3.45, HICT-2.79, especially MICT group) compared with the P/T (score:

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Fig. 2. (A) Representative radiographs of ulnar segmental defects with or without implantation of porous scaffolds taken at week 0 (baseline), 2, 4 and 8 post-operatively. Three doses of P/T/ICT scaffolds, especially the P/T/MICT group, showed more bone filling in the defects compared with that of P/T group. (B) Mean scores of X-ray in the incorporated composite scaffolds taken at weeks 2, 4 and 8 post-operatively. In three doses of P/T/ICT groups, the P/T/MICT group showed more bone formation in defects compared with the P/T group (⁄P < 0.05 and ⁄⁄P < 0.01, comparison made between P/T/ICT groups and P/T group at each time-point, n = 6).

1.95) at week 8 after scaffold implantation (⁄P < 0.05, n = 6).

⁄⁄

P < 0.01,

3.2. New bone volume fraction and BMD measured by HR-pQCT The 3D images of HR-pQCT reconstruction in Fig. 3A demonstrate newly formed bone within the bone defect regions at weeks 2, 4, and 8 after scaffold implantation. These images present a similar tendency of new bone formation to that measured from the 2D radiographs in Fig. 3B, which described the quantitative data of new bone volume (BV/TV) and BMD within the bone defects. At week 2, there was no significant increase in BV/TV and BMD in P/T/ICT groups compared with the P/T group. After 4 weeks postimplantation, new bone was remarkably formed in the defects, though mainly next to the residual bone, and an increase in bone formation was seen in the P/T/ICT and P/T groups compared with the bone defect group without scaffold implantation. However, at week 8, the BV/TV and BMD in all groups increased significantly compared with those at week 4 (⁄P < 0.05); the BV/TV and BMD in P/T/ICT groups were significantly higher than those in P/T group (⁄P < 0.05, ⁄⁄P < 0.01, n = 6). 3.3. Histological new bone fraction 3.3.1. New bone area percentage There was no notable healing taking place at the bone defect region up to week 2 after surgery. The scaffold pores were filled with

loose fibrous connective tissue, but, in the first week, healing defects were mostly marked by soft tissue callus formation. In vivo osteogenesis of implants in bone defect sites at weeks 4 and 8 post-implantation was described in Fig. 4A1. Newly formed bone tissue in the defects was observed at week 4. Some bony bridging was seen in the defect region along the border of the radius 8 weeks after scaffold implantation. The serial radiographs demonstrated that the new bone originated from the resident bone edges and gradually formed along the external margins of the implant towards the centre of the scaffolds. The quantitative results (Fig. 4B) show that, at weeks 4 and 8, P/T/ICT groups presented more new bone in the defects compared with the P/T group (⁄P < 0.05), especially the P/T/MICT group (⁄⁄P < 0.01, n = 4). At week 8, P/T/LICT, P/T/HICT scaffolds had more and larger isolated bone islands and new bone along the radius margins and within the scaffolds pores. P/T/MICT scaffold had large bone islands and partial bone formation within the scaffolds pores. The defects without any implant treatment had only concave-shaped fibrous tissue. For the scaffolds, newly formed tissues consisting of viable osteocytes within lacunae embedded in bone matrix were deposited directly on the pores surface pores of scaffolds. Osteoblast-like cells lined the surface of newly formed bone, producing a bone matrix in scaffolds. For biodegradation evaluated at week 8, the decalcified sections stained with H&E presented evidence of new bone ingrowth into the spores of the implanted scaffolds in all scaffold groups, suggesting good osteoconductivity and biocompatibility of the scaf-

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Fig. 3. (A) Representative HR-pQCT 3D images of ulnar segmental defects reconstructed at week 2, 4 and 8 post-implantation. Three doses of P/T/ICT, especially the P/T/MICT group, presented more new bone in the defects compared with the P/T group. (B) Bone volume within the bony defects and BMD in the implanted scaffolds evaluated by HRpQCT at week 2, 4 and 8. Three P/T/ICT groups showed more new bone formation, new bone density and relative new bone volume in defects compared with P/T group (⁄P < 0.05 and ⁄⁄P < 0.01, compared between P/T/ICT groups and P/T group, n = 6).

fold materials. In addition, the results of Goldner’s trichrome staining at week 8 also demonstrated significantly more new bone formation in the defects treated with P/T/ICT scaffolds (⁄P < 0.05), especially in P/T/MICT scaffold (⁄⁄P < 0.01), compared with P/T scaffold (Fig. 5A). Polarized microscopy presented remarkable parallel arrangement collagen in the newly formed bone, which significantly differentiated bone matrix with collagen alignment and implanted materials without collagen (Fig. 5B). 3.3.2. Osteogenic markers expression in situ In order to demonstrate osteogenic differentiation in the implants within bone defect sites, later stage osteogenic marker osteocalcin was identified by immunofluorescence staining in decalcified paraffin sections. The expression of osteocalcin was found in newly mineralized bone matrix, as shown by the larger fluorescence stained area. By week 8, more evident fluorescence signals indicated the higher expression of osteocalcin in P/T/ICT groups than that in P/T group. P/T/MICT presented more obvious osteocalcin expression on the newly formed bone with a larger and stronger fluorescence signal than that of the P/T/LICT and P/T/HICT groups (n = 4; Fig. 4A2). 3.4. New bone formation Fluorescence microscopic evaluation revealed increased new bone formation, as evidenced by a higher ratio of the calcein green/xylenol orange labelled area in P/T/ICT treatment specimens compared with that of the P/T group at both week 4 and week 8 (Fig. 6A1). A comparison of the amount of fluorescence labelling in all groups at week 4 showed that P/T/ICT groups had more calcein green than xylenol orange labelling (⁄P < 0.05). The significant difference increased in the week 8 samples (⁄⁄P < 0.01, n = 4; Fig. 6A2). Goldner trichrome staining could differentiate the miner-

alized bone from osteoid tissues and investigate the osteogenic process. The results implied that the differentiated cuboidal osteoblasts lined red-stained osteoid tissues that surrounded the subsequently formed green-stained mineralized bone tissues, and mature osteocytes existed in internal mineralized bone tissues (Fig. 6B). 3.5. Evaluation of neovascularization using micro-CT-based microangiography The 3D images of neovascularization within the bone defect regions were demonstrated in Fig. 7A (newly formed vessel-like structure (0–300 lm). There were more newly formed vessels observed in the defects implanted with P/T/ICT scaffolds with three ICT doses, especially with P/T/MICT, compared with P/T scaffold at weeks 2 and 4 post-implantation. Fig. 7B1 shows that new vessel ingrowth increased in all groups from week 2, reaching a peak at week 4 before decreasing at week 8. Compared with the P/T group, the P/T/ICT groups of three different ICT concentrations presented many more new vessels in total volume growing into the defects, especially the P/T/MICT group (⁄P < 0.05, ⁄⁄P < 0.01, n = 4; Fig. 7B1). Vessel number or density located within the scaffolds at weeks 2 and 4 post-implantation is described in Fig. 7B2. Newly formed vessels with perfused microfil were located within the pores of the scaffolds that became visible at week 2 and increased at week 4, with a significant difference between the P/T/MICT group and the P/T group (⁄P < 0.05, n = 4), but without a significant difference in vessel number in P/T/LICT and P/T/HICT groups compared with the P/T group at weeks 2 and 4 (P > 0.05). 3.6. Dynamic blood perfusion function The low signal area on T1-weighed MR images and intensity curves are presented in the defect sites at weeks 2 and 4, and no

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Fig. 4. Representative sagittal sections of decalcified histology of ulnar segmental bone defects. (A1) H&E stained sections at week 4 and 8 showed more newly formed bone in P/T/ICT scaffolds groups, especially in P/T/MICT group, than that in P/T group (yellow ⁄, scaffold; black #, new bone; black or white bar = 200 lm). (A2) Representative immunohistochemistry of osteocalcin expression in osteoblasts and mineralized sites at week 8. Immunohistochemistry staining demonstrated osteocalcin expression (white arrows) on the newly formed bone with more and stronger fluorescence signals within P/T/ICT scaffolds, especially P/T/MICT, than that in P/T scaffold in the segmental defects, indicating more osteoblasts (white bar is 200 lm). (B) New bone area quantified at weeks 4 and week 8. In P/T/ICT groups, there was more newly formed bone in segmental defects compared with that of P/T group. P/T/MICT showed the largest area of newly formed bone among three P/T/ICT groups (⁄P < 0.05 and ⁄⁄P < 0.01, compared between P/T/ICT groups and P/T group, n = 4).

Fig. 5. Representative sagittal sections of decalcified histology of ulnar segmental bone defects. (A) Sections with Goldner’s trichrome staining at week 8 presented more new bone ingrowth (yellow arrows) in P/T/ICT scaffolds groups, especially in P/T/MICT group, compared with the P/T group. (B) Polarized microscopic images at week 8. Collagen fibers (yellow arrows) presented parallel alignment in newly formed bone. More collagen alignment showed in P/T/ICT groups, especially in P/T/MICT group (white or black bar = 200 lm).

suspicious signal area was found throughout the experimental period (Fig. 8A). ‘‘Maximum enhancement’’ at the examined bone de-

fect sites from baseline increased in the P/T/MICT group at weeks 2 and 4 (Fig. 8B).

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Fig. 6. Representative sagittal sections of undecalcified histology in ulnar segmental bone defects. (A1) Fluorescent micrographs showing new bone formation in bone defects at weeks 4 and 8 after implantation. More evident fluorescent deposition indicated more new bone formation and remodelling in P/T/ICT, especially P/T/MICT, than that in P/T group (yellow , scaffold; white #, new bone; white bar = 400 lm). (A2) Quantitative analysis of new bone formation within bone defects region at weeks 4 and 8. There was more bone formation in the P/T/ICT groups, especially the P/T/MICT group, compared with the P/T group (⁄P < 0.05 and ⁄⁄P < 0.01, compared between P/T/ICT groups and P/T group, n = 4). (B) Goldner’s trichrome stained section at week 8 demonstrated the osteogenesis process from osteoblasts (yellow arrows) to osteoid (white arrows), then finally to newly mineralized bone (NB) within the implanted scaffolds (S).

Fig. 7. (A) Representative microCT-based micro-angiography of vessels formed within the ulnar segmental defect region at weeks 2 and 4 after implantation. The obvious increase in vessel volume within the defects was presented at weeks 2 and 4 after implantation, where the P/T/MICT group presented more vessels than the P/T group. (B1) Quantitative analysis of volume of new vessels formed in defect regions at week 1, 2, 4 and 8 after implantation. The volume of new vessels reached a peak at week 4 after implantation, and there was significantly higher vessels volume in the P/T/ICT groups than in the P/T group (⁄P < 0.05 and ⁄⁄P < 0.01, compared between P/T/ICT groups and P/T group at each time-point, n = 4). (B2) Vessel numbers in the implanted scaffolds in the defects: significantly more new vessel ingrowth was found in the P/T/MICT scaffold compared with that in the P/T scaffold (⁄P < 0.05, compared between P/T/ICT groups and P/T group, n = 4).

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4. Discussion The present study investigated a unique biodegradable PLGA/ TCP-based porous scaffold incorporating bioactive phytomolecule ICT for enhancing bone regeneration within ulnar segmental defects in rabbits. In a recently accepted work, the present authors demonstrated the safety of both P/T with or without ICT incorporation using in vitro BMSC proliferation testing [41]. In fact, this is not surprising, as both PLGA and TCP are medical grade used for clinical applications, and ICT is identified as an intestinal metabolite of EF, discovered in serum after oral administration of herbal compounds with EF as the main components [13,41]. Therefore, ICT can be regarded biologically safe and non-toxic for applications if its concentration is within a physical range suggested by the recently published in vitro cell culture results using BMSC [6]. In this study, three different dosages of ICT were incorporated into scaffolds for the evaluation of in vivo biocompatibility and biosafety, which would be a rational foundation for potential clinical application. In this in vivo study, the ulnar bone healing was monitored and assessed by in vivo radiographs, HR-pQCT and MRI, and ex vivo histology and histomorphometry, with focuses on the osteogenic and angiogenic potentials of P/T scaffolds incorporating ICT. The results of radiography and HR-pQCT demonstrated that, compared with the defects without scaffold implantation, more bone formation and advanced bone remodelling progress was shown in the bone defects with scaffold implantation at both week 4 and week 8 after segmental bone defect surgery, indicating that

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P/T scaffolds were suitable basic biomaterials for incorporating bioactive phyotomolecule ICT to form bioactive porous composite scaffolds. As expected, all three P/T/ICT groups with different ICT concentrations showed better treatment outcome in the defect sites than the P/T control group. Decalcified histological evaluations indicated that, in comparison with P/T control group, more osteoid tissue, mineralized bone tissue and mature lamellar bone were found in P/T/ICT scaffold groups, where the P/T/MICT group showed the best outcome results, including more new bone and vessel formation accompanied with up-regulated osteocalcin expression. Analysis of undecalcified MMA sections for area fractions of new bone revealed earlier and more bone formation in P/T/ICT scaffolds incorporating different ICT concentrations compared with the control P/T group at each evaluated post-implantation time point, implying a promising biotechnological and noncell bioengineering strategy for bone repair applications, attributed to their superior treatment efficacy, where ICT played a role in bone healing enhancement through its directly controlled release into local healing environment and the indirectly induced cell–matrix interaction in enhancement of osteogenesis, as verified in the recent in vitro study [6]. 4.1. PLGA/TCP composite scaffolds facilitated new bone formation and ingrowth Polymers/ceramic composites were the biomaterials used to assemble local delivery systems incorporating growth factors for

Fig. 8. (A) MRI images of T1W and TIC (arbitrary unit) by dynamic MRI acquired from the bone segmental defect of rabbit ulna. SI was measured in operator-defined ellipselike ROI over the target site beneath the joint space in the mid-coronal T1-weighted images, using a cursor and graphic display device. The SI values derived from the ROI were plotted against time as TIC (red arrows point to the bone defect sites). (B) Perfusion parameter ‘‘maximum enhancement (%)’’ defined as the maximum percentage increase (SImax SIbase) in SI from baseline (SIbase) was calculated according to TIC. (Only descriptive MRI information was available as averaged by two measurements owing to limited assessment of clinical dynamic MRI scanning for experimental animals).

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their controlled release [6,42], as they provided initially required mechanical strength, and their pore size and degradation rates also favoured new bone and vessel ingrowth [43–45]. In the present study, either P/T scaffolds without bioactive phytestrogenic molecule ICT or scaffolds with ICT incorporation at a macropore size of 400 lm and porosity of 70% were implanted into 12 mm long bone defects. HR-pQCT and histological results showed that new bone tissue and bone marrow could grow or migrate into the centre of scaffolds through the macropores, where P/T/ICT groups showed better osteogenic effects compared with either empty control or P/T scaffold alone. Mechanical properties and degradation rates are both important physical properties relevant for development of bone substitutes. During healing after scaffold implantation, the trabeculae of the scaffolds were degraded and separated by new bone, bone marrow and fibrous connective tissues, implying that the scaffolds underwent degradation after implantation over time, accompanied by new bone formation and its gradual replacement. Meanwhile, necessary mechanical support from the trabeculae of scaffolds can supply adequate space for newly formed tissue ingrowth surrounding the defects. In this study, compared with P/T scaffold group, there was more new bone formation in the P/T/ICT scaffolds groups, especially in the P/T/MICT scaffold. Although the in vivo degradation rate analysis of scaffolds indicated no significant difference in the remaining material’s area percentage of the total material area (Supplementary Fig. S1) from that for the whole framework structure of scaffolds, there was no more obvious collapse and shrinkage, but more sustained macropore structure in P/T/ICT scaffolds found up to week 8 post-implantation, when the faster new bone growth was shown, indicating that P/T/ICT scaffolds had adequate mechanical support that compromised the degradation accompanied by new bone formation and integration with the implanted porous scaffold materials [45]. The degradation rate of materials after week 8 might change, and P/T/ICT scaffolds would present increasing degradation and resorption rates, allowing a higher percentage of bone formation. Future specifically designed experiments will be able to delineate such observations.

4.2. PLGA/TCP composite scaffolds incorporating ICT for bone formation enhancement As an exogenous phytomolecule, ICT is known for its osteogenic potential and stable chemical structure [6,13,16]. Attributed to its bone anabolic effects [6,12,14,16,46], scaffolds incorporating ICT possessed a unique feature of its sustained release where the released ICT molecules still preserved its original bioactivity as confirmed in recent in vitro work [6]. The maintained bioactivity of ICT was also substantiated by in vivo HR-pQCT and histology analysis, where it demonstrated more significant osteopromotive efficacy in bone defect repair in P/T/ICT scaffolds compared with the control scaffold. First, as a small phytomolecule, the tertiary structure and molecular design of ICT for ligand-receptor-docking demonstrated its strong binding capability to estrogen-receptor in bone MSC (BMSC) [47], and ICT alone could enhance BMSC homing and differentiation in vitro, which might be directly involved in the BMP signalling pathway and associated with the Wnt signaling pathway [16]. After incorporation of ICT, the P/T/ICT scaffold became a type of ligand-functionalized material providing specific receptor-binding ligands that therefore significantly enhance local or circulating BMSC attaching efficiency to facilitate subsequent intracellular reactions in favour of osteogenic differentiation and bone formation. A recent study also suggested that P/T/ICT scaffolds could facilitate BMSC migration to the implanted scaffolds within bone defect sites [48].

The present study showed dose-dependent therapeutic effects, as the P/T/MICT scaffold demonstrated better osteogenic potential compared with the P/T/LICT and P/T/HICT scaffolds. Similar dosedependent effects were also reported in a recent in vitro study on BMSC homing potentials [6]. ICT is bio-safe within a certain range of concentrations, unless ICT dosage were to increase to a non-physiological high dose, then the proliferation of BMSC could be inhibited, as shown in the in vitro results. A dosing study was therefore performed in vitro with three doses (low, 3.5  10 4 M; middle, 1.4  10 3 M; and high, 3.5  10 3 M) for incorporation of ICT into P/T matrix for fabricating porous scaffolds, as reported in recent systemic in vitro and in vivo studies. It was found that 1.4  10 3 M was the best in vitro concentration with regard to BMSC proliferation. However, the in vitro condition could be different from an in vivo environment, i.e., one should not simply translate the in vitro findings directly into in vivo outcome studies to achieve the best treatment efficacy. Systemic in vivo experimental studies were therefore designed and presented in the current animal experimental study for confirmation. The present in vivo results indicate that bone defects treated with P/T/HICT scaffold did not demonstrate the best bone formation results. In addition, published in vitro results indicated that P/T/MICT scaffold present more maintainable physical and mechanical properties compared with P/T/LICT and P/T/HICT scaffolds. So the P/T/MICT scaffold showed logically the best osteogenic potential compared with P/T/LICT and P/T/HICT scaffolds according to both in vitro and in vivo evaluations. 4.3. PLGA/TCP composite scaffolds incorporating ICT for vascularization enhancement It was reported that angiogenesis represents an important process during formation and repair of tissues and is essential for the nourishment and supply of reparative and immunological cells [49]. It has been demonstrated that angiogenesis is affected by alterations in the porosity of porous implantations and that a more interconnected pore structure is more conducive to tissue invasion and growth [50]. Relatively larger pores (e.g., 300–500 lm in diameter) lead to direct osteogenesis due to faster vascularization and higher local oxygenation [45]. In the present study, good blood perfusion was found in the bone defect regions in each group evaluated using dynamic perfusion MRI at weeks 2 and 4 postimplantation, as well as new vessel ingrowth into scaffold macropores from 3D micro-angiography analysis and 2D histological evaluations. These findings indicate that the porous scaffolds could facilitate vascularization, and thus could enhance the nutrients exchange and new bone formation. Numerous studies have shown that angiogenesis is essential for successful bone repair and regeneration. In tissue development and regeneration, vascularization precedes osteogenesis [51], suggesting that enhanced neovascularization accelerates bone formation even before local blood flow has been established [52]. The vasculature supplies nutrients and oxygen to develop and regenerate bone, as well as delivering critical biological signals to stroma that stimulate MSC differentiation and bone formation. However, bone also supplies growth factors, such as Fibroblast Growth Factor (FGF), Vascular Endothelial Growth Factor (VEGF) and BMP, as well as cells to enhance angiogenesis [53]. The results of the present study show that a significant number of new vessels ingrown into the bone defect region were already observed at week 2 after scaffold implantation and reached its peak at week 4, followed by better remodelling in terms of decrease in volume but increase in functionalization at week 8, with more connected vessels, a time point with faster bone regeneration. It was also demonstrated consistently that the scaffolds incorporating ICT induced significant blood vessel ingrowth into the pores of the implanted scaffold dur-

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ing the early stages of bone regeneration in this system compared with that of the control scaffold. This implies that a more newly formed vascular network might be one of the crucial indices for osteogenic enhancement, while enhanced osteogenesis results from up-regulated osteogenic differentiation of BMSC [54,55]. In addition, a recent in vitro study results indicated that the enhanced osteogenic differentiation of MSC by ICT might be related to Wnt/ beta-catenin in BMP signalling pathway [16], and unpublished data showed that ICT could recruit MSC to the bone defect repair site. However, the in vitro study did not show a direct stimulation effect of ICT on angiogenesis, while the in vivo findings demonstrated enhancement of angiogenesis after P/T/ICT treatment. So it was postulated that ICT regulated angiogenesis through in an indirect pathway, i.e., through promotion of angiogenesis and osteogenesis by enhanced MSC recruitment to the bone defect repair region, yet the involved cell signalling and exact molecular mechanisms are subject to future investigations. This study supports previous evidence that P/T/ICT biomaterial scaffolds could enhance neovascularization at week 4 after intramuscular implantation, compared with P/T control scaffold [13], and provide a favourable substrate for new vessel direct ingrowth in bone defect sites implanted with porous scaffolds during the bone healing process. Based on the dose-dependence of ICT incorporated into the scaffolds on osteogenic enhancement, the current study also showed the dose-dependence of ICT on promotion of vascularization that might similarly exist and be explained by associating with the difference in level of osteogenesis found in three types of P/T/ ICT scaffolds with different ICT concentrations. Some limitations still existed in the present study. From the published in vitro results, ICT could facilitate osteogenic potential through the modification of a lower pH value, which would benefit in vitro osteogenic differentiation of BMSC. In fact, the two factors we endeavored that in vivo local release of ICT and pH value microenvironment are coexistent that might jointly influence bone formation, although it was not possible to detect both factors in vivo in the present study. However, in order to definitively answer whether there are any systemic effects of ICT in this defect model, ICT content in serum should be detected, and this will be the subject of a future study. In addition, owing to larger variations in histomorphometrical data compared with HR-pQCT-based quantification of 3D new bone formation (volume), a larger sample size would be more suitable for better statistical analysis.

5. Conclusions The current study developed an innovative bioactive scaffold by combining the osteopromotive phytomolecule ICT with PLGA/TCP to form a composite scaffold for augmentation of bone regeneration within segmental bone defects, and systematically investigated its treatment efficacy in vivo. Due to their phytochemical stability and the unique features and bioactivity of ICT, PLGA/ TCP/ICT composite scaffolds presented the desired yet dose-dependent osteogenic and angiogenic potential, as evidenced in a rabbit ulnar bone segmental defect model. PLGA/TCP/ICT scaffolds may therefore be regarded as innovative bioactive scaffold materials for potential orthopaedic applications.

Acknowledgments This study was supported by ‘‘Hong Kong Innovation and Technology Support Program (GHP/001/08)’’, ‘‘Hong Kong RGC (473710)’’, ‘‘Guangdong Province Comprehensive Strategic Cooperation Project of the Chinese Academy of Sciences (2010B090300076)’’, and a collaborative research grant ‘‘Shenzhen

Bureau of Science SG200810200102A)’’.

Technology

&

Information

(No.

37,

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