Efficacy of prevascularization for segmental bone defect repair using β-tricalcium phosphate scaffold in rhesus monkey

Biomaterials xxx (2014) 1e9

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Efficacy of prevascularization for segmental bone defect repair using b-tricalcium phosphate scaffold in rhesus monkey Hongbin Fan a, 1, Xianli Zeng b, 1, Xueming Wang b, 1, Rui Zhu c, Guoxian Pei a, * a

Department of Orthopedic Surgery, Xijing Hospital, The Fourth Military Medical University, 17 West Changle Road, Xi'an, China Department of Orthopaedics & Traumatology, Nanfang Hospital, Nanfang Medical University, Guangzhou, China c Collage of Science, Engineering University of Air Force, Xi'an, China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 7 May 2014 Accepted 14 May 2014 Available online xxx

Although small animal model (rabbit) showed successful bone defect repair using prevascularized tissueengineered bone grafts (TEBG), large animal (rhesus monkey) studies are still needed to extrapolate the findings from animal data to humans. In current study, we investigated the efficacy of prevascularized TEBG for segmental bone defect repair in rhesus monkey. The segmental diaphyseal defects were created in both tibias. In group A, the defect was filled with prevascularized MSCs/scaffold prepared by inserting saphenous vascular bundle into the side groove and a fascia flap coverage; In group B, the defect was filled with MSCs/scaffold with a fascia flap coverage; In group C, the defect was filled with MSCs/scaffold; In group D, the defect was filled with only scaffold. The angiogenesis and new bone formation were compared among groups at 4, 8, and 12 weeks postoperatively. The results showed the prevascularized TEBG in group A could augment new bone formation and capillary vessel in-growth. It had significantly higher values of vascularization and radiographic grading score compared with other groups. In conclusion, the in vivo experiment data of prevascularized TEBG was further enriched from small to large animal model. It implies that prevascularized TEBG has great potentials in clinical applications. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Tissue engineering Bone defect Vascularization Tricalcium phosphate

1. Introduction The bone healing process usually fails due to large segmental bone defects under complicated conditions such as pathological fractures or severe trauma, [1,2]. For achieving the complete bone healing, grafts (autograft or allograft) are needed in surgery. It is estimated that more than 500,000 bone-grafting procedures are performed annually in the United States [3]. However, autograft or allograft transplantations are hampered by the limited supply, donor site morbidity, prolonged recovery, and pathogen transfer [4,5]. In recent years, tissue-engineered bone graft (TEBG) has offered a promising alternative for the treatment of large defect without many undesirable side effects associated with conventional therapies [6e8]. The TEBG was prepared by loading mesenchymal stem cells (MSCs) on scaffold. In this procedure, the survival and functions of MSCs on scaffold, especially in the central field of scaffold, are

* Corresponding author. Tel.: þ86 29 84773524. E-mail addresses: [email protected], [email protected] (G. Pei). 1 These authors contributed equally to this study.

crucial for the healing efficacy of TEBG. In native bone tissue, the distribution of cells is usually limited to a distance of 200 mm away from the nearest capillary, which is the effective diffusion distance of oxygen and nutrients [9]. Therefore, the vascularization is thought to play a significant role in the healing process of TEBG. Although the spontaneous vascularization can occur in the peripheral field of scaffold neighboring native tissue, the vascular ingrowth is limited to several tenths of micrometers per day. It's too slow to provide enough nutrients for cells in the central field of scaffold, leading to the compromised healing results [10]. So, how to regenerate the neo-vessels at early stage after implanting TEBG is of great importance for achieving the satisfactory healing. Several strategies to improve vascularization have been proposed in the past few decades. These strategies include modification of scaffold design, delivery of angiogenic factors, and surgical prevascularization [11,12]. The commonly used prevascularization methods included periosteal flap coverage, arteriovenous loop graft, and vascular bundle insertion, which provided a stable and instantaneous perfusion. Many studies demonstrated the efficacy for new bone formation and vascularization within the implants [13e16]. In our previous study, TEBG was prevascularized by inserting femoral vascular bundle for segmental defect repair in

http://dx.doi.org/10.1016/j.biomaterials.2014.05.035 0142-9612/© 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Fan H, et al., Efficacy of prevascularization for segmental bone defect repair using b-tricalcium phosphate scaffold in rhesus monkey, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.05.035

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Fig. 1. (A) Gross morphology of cylindrical b-TCP scaffold. Inset showed the diameters of side groove and central tube. (B) Porous structures of scaffold observed by SEM (30). Inset showed the macro pores and interconnected pores. (C) Positive alkaline phosphatase staining of osteo-differentiated MSCs (100). (D) Alizarin red S staining of osteo-differentiated MSCs (100).

rabbit femur. The results indicated that prevascularized TEBG group had significantly higher volume of regenerated bone and new vessels [6]. Although the rabbit is one of the most commonly used animals for medical research (approximately 35% of musculoskeletal research studies), the drawbacks exist with the rabbit model for assessment of implanted materials [17]. In comparison with rhesus monkey, the rabbit has a very different microstructure of long bone (Haversian canal perimeter and bone density) from humans [18]. Furthermore, it has faster bone turnover compared with primates and some rodents [19]. Therefore, studies in rhesus monkey are still needed to extrapolate the findings from animal data to humans. In addition, the non-invasive and repetitive ways to quantitatively measure the blood flow in TEBG would be important to evaluate the vascularization [20]. To further investigate the angiogenesis of implanted TEBG in rhesus monkey, we propose to repair tibial defect using MSCs and b-tricalcium phosphate (b-TCP) scaffold. This can provide us insights into the histological changes of vascularization and new bone formation in TEBG. We hypothesize that (1) the prevascularized TEBG can successfully repair segmental bone defects in rhesus monkey; (2) the noninvasive assays including perfusion weighted MRI (PW-MRI) and single photon emission computed tomography (SPECT) can monitor the angiogenesis of TEBG. In order to prove this hypothesis, the study is designed (1) to prepare a prevascularized TEBG by inserting a vascular bundle into the side groove of scaffold seeded with MSCs, (2) to implant the prevascularized TEBG into a critical-sized segmental tibial defect with coverage of fascia flap in rhesus monkey, (3) to evaluate the vascular ingrowth and bone healing of the TEBG. 2. Materials and methods 2.1. Scaffold characterization The cylindrical b-TCP ceramic scaffold was prepared by Biocetis Company (Berck sur Mer, France) via the impregnation of a custom-made organic edifice

with b-TCP (Tokushima, Japan) suspension followed by sintering at 1110  C [21]. The cylinder scaffold (diameter: 12 mm; length: 20 mm) had a side groove (width: 2 mm) connecting the central tube (diameter: 3 mm), which passed through the scaffold along its long axis (Fig. 1A). The scaffold had a homogeneous porosity of 75 ± 10%. The diameter of macro pores and interconnected pores was 530 ± 150 mm and 150 ± 50 mm, respectively. The pores were well interconnected with each other and opened into the central tunnel and the outer surface of the scaffold (Fig. 1B). The scaffolds were sterilized by gamma irradiation at 25 kGy and conditioned with Dulbecco's Modified Eagle Medium (DMEM) for 1 h before cell loading. 2.2. Graft preparation Animal experiment was approved by Institutional Animal Care and Use Committee of Southern Medical University. Fourty-five rhesus monkeys (3~4 years old, 4.0e5.2 kg) were purchased from the South-China Primate Research & Development Center. MSCs were generated from bone marrow aspirates of rhesus monkeys and washed three times with Hanks' balanced salt solution (HBSS). Cells were re-suspended in 20 ml of DMEM supplemented with 10% fetal bovine serum (FBS) (HyClone Logan, Utah), L-glutamine (580 mg/L) and penicillin-streptomycin (100 U/mL). Cultures were incubated at 37  C and 5% CO2. After 72 h, nonadherent cells were removed by changing medium [22]. When reaching 70e80% confluence, adherent cells were freed from the flask with 0.05% trypsin and subcultured in osteogenic medium (DMEM supplemented with 10% FBS, 50 mg/ml L-ascorbic acid 2-phosphate, 10 nM dexamethasone, 10 mM b-glycerolphosphate, and 100 ng/ml BMP-2). After 3 weeks of culture, the osteogenic differentiation of MSCs (passage 3) was confirmed by positive results of alkaline phosphatase (Fig. 1C) and alizarin red S staining (Fig. 1D). Thereafter, autologous differentiated MSCs (5.0  106) were loaded onto each scaffold under a negative pressure of 0.25e0.5 atm, which facilitated cell infiltration. Finally, the MSCs/ scaffold constructs were cultured overnight in vitro for cell adhesion before implantation. 2.3. Cell proliferation and morphology on scaffold The MSCs/scaffolds were cultured and collected to assay cell proliferation and morphology on scaffolds. Cell proliferation were measured by 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT; Sigma, USA) staining. The cell-seeded scaffolds, at desired time points (day 2, 4, 6 and 8), were incubated in MTT solution (5 mg/mL MTT in cell culture medium) in a 5% CO2 incubator at 37  C for 2 h. The intense purple colored formazan derivative formed via cell metabolism was eluted and dissolved in 200 mL/well dimethylsulfoxide (DMSO; Merck, Germany). The absorbance was measured at 570 nm with a

Please cite this article in press as: Fan H, et al., Efficacy of prevascularization for segmental bone defect repair using b-tricalcium phosphate scaffold in rhesus monkey, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.05.035

H. Fan et al. / Biomaterials xxx (2014) 1e9 reference wavelength of 690 nm. Cell number was correlated to optical density (OD) (Fig. 2A). The cell morphology on scaffold was characterized using SEM (JEOL JSM-5600LV, Japan) on day 1 and 7 (Fig. 2B, C). 2.4. Surgical procedures Fourty-five rhesus monkeys mentioned above were used for bone defects repair and the operation was performed as reported (Fig. 2DeF) [23]. The bilateral tibias were exposed through longitudinal incisions. A 3.5 mm titanium seven-hole plate was then applied to the anterolateral cortex and fixated with screws. Three screws were inserted on either side of the proposed osteotomy site. The resection of segmental diaphysis (20 mm in length) was performed in each tibia. Ninety defects were randomly assigned into five groups. In group A (n ¼ 21), the defect was filled with autologous MSCs/scaffold. For pre-vascularization, the saphenous vascular bundle was freed from the muscular layer and inserted into the side groove of scaffold. Then the construct was covered with a fascia flap. In group B (n ¼ 21), the defect was filled with MSCs/scaffold followed by a coverage of fascia flap. In group C (n ¼ 21), the defect was filled with MSCs/scaffold. In group D (n ¼ 21), the defect was filled with only scaffold. In group E (n ¼ 6), the defect was untreated as blank control. The incision was closed in layers. All monkeys were allowed to move freely after surgery without plaster immobilization.

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2.5. X-ray analysis The monkeys were lightly anesthetized at 4, 8, and 12 weeks postoperatively. Then the anteroposterior and lateral radiographs of tibia were taken with exposure factors of 50 kV and 50 mA. The films were graded according to the scoring system reported by Yang's group [24]. The scale was composed of four categories: (1) the first category evaluated periosteal reaction graded from 0 to 4 points. (2) The second category evaluated osteotomy line graded from 0 to 4 points. (3) The third category evaluated remodeling graded from 0 to 2. (4) The fourth category evaluated graft appearance graded from 0 to 4 points (Table 1). The proximal, central, and distal part of graft was scored individually. A graft that had fully consolidated and had completely reorganized would receive a maximum total score of 42 points. The films were scored and compared among groups at different time points.

2.6. Single photon emission computed tomography (SPECT) examination SPECT examination was performed at 4, 8, and 12 weeks postoperatively. The monkeys were lightly anesthetized and 99mTc-methylene-diphosphonate (MDP) was injected into the cephalic vein at a dose of 370 MBq. Delayed SPECT images were obtained (512  512 acquisition matrix) 4 h after injection. The manually drawn rectangular region of interest (ROI, 1.2  2 cm2) was established on the implant site.

Fig. 2. (A) The cell proliferation of MSCs seeded on scaffold measured by MTT assay. (B) The cell morphology on scaffold was observed using SEM on day 1. (C) The cell morphology on scaffold was observed using SEM on day 7. (D) The resection of segmental diaphysis (20 mm in length) was performed in the tibia of rhesus monkey. (E) The defect was filled with prevascularized TEBG prepared by inserting the saphenous vascular bundle into the side groove of scaffold/MSCs. (F) The prevascularized TEBG was covered by a fascia flap.

Please cite this article in press as: Fan H, et al., Efficacy of prevascularization for segmental bone defect repair using b-tricalcium phosphate scaffold in rhesus monkey, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.05.035

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H. Fan et al. / Biomaterials xxx (2014) 1e9 deviation (SD) and difference was detected using one-way analysis of variance (ANOVA) test.

Table 1 Radiographic grading system. Score Periosteal reactiona None Minimal [localized to the gap] Medium [extends over the gap; <1/4] Moderate [1/2 but 3/4] Full Osteotomy sitea Osteotomy line completely radiolucent Osteotomy line partially radiolucent Osteotomy line invisible Remodelinga None apparent Intramedullary space Intracortical Graft appearancea Unchanged/intact Mild resorption Moderate replacement Mostly replaced Fully reorganized Maximum total score a

0 1 2 3 4 0 2 4 0 1 2 0 1 2 3 4 42

Proximal, distal, and central part of graft scored individually.

The uptake ratios of 99mTc-MDP (T/NT) among groups at different time points were compared. 2.7. Perfusion weighted MRI (PW-MRI) analysis MR imaging was performed by using a 1.5 T clinical MR imaging system (SIEMENS). The monkeys were anesthetized and an 18-gauge heparinized catheter was inserted into the cephalic vein. The tibia was placed in the coil. Following a coronal scout acquisition, T1-weighted image of the regenerated bone was obtained. A dynamic single-section short T1-weighted gradient-echo MR series (repetition time msec/echo time msec, 4/1.4; flip angle, 40 ; matrix, 256  128) was obtained. A gadolinium-based contrast agent was injected into the vein (0.2 mmol/kg; 3 ml/s). It took approximately 80 s to acquire 40 dynamic MR images. The images were processed and analyzed in a workstation. Region of interest was drawn over the field of regenerated bone. Signal intensity enhancement over time was recorded and plotted as a signal intensityetime (SIeT) curve. From this SIeT curve, two MR perfusion indexes were analyzed: maximum steepest slop (SS) and maximum signal-intensity increase (MSI). SS was calculated as follows: SS ¼ [(SIendSIprior)/ (SIbaseline  T)]  100 (%S). MSI was calculated as follows: MSI ¼ SIpeakSIbaseline. 2.8. Histological examination The monkeys were sacrificed at 4, 8, and 12 weeks postoperatively. Before sacrifice, the femoral artery was cannulated using a 24-gauge catheter and flushed with 100 mL heparin solution in Ringer (100 IE/mL). The femoral vein was severed to allow blood and heparin solution to drain. Then, the India ink solution (50% v/v India ink, Rohrer, Leipzig, Germany) was injected into the femoral artery until it was drained from vein. Then the tibias were macroscopically inspected, collected, and immediately kept at 80  C. The samples were fixed in 10% neutral buffered formaldehyde for 2 weeks. Five samples from groups A, B, C and D were dehydrated through gradient alcohols and embedded in polymethylmethacrylate (PMMA) without decalcification. The samples were cut into slices perpendicular to the long axis with the thickness of 200 mm using a saw microtome (Leica SP1600, Leica Microsystems GmbH, Germany). Thereafter, the sections were glued onto a plastic support and polished to 80 mm thickness with an Exakt Grinder (Norderstedt, Germany). The central tunnel (diameter: 3 mm) and its neighboring area (diameter: 2 mm) of scaffold was defined as central graft, the rest of scaffold was indicated as peripheral graft. With the help of Leica Qwin 500 image analysis system, the images from nine microscopy fields of each slide were collected. During the gridding process, the area of each field was divided into 1024 units. The ink-staining area was automatically recognized and the percentage of staining area was calculated. The other two samples from groups A, B, C and D were decalcified in 50 mM ethylene diaminetetraacetic acid (EDTA), embedded in paraffin, and sectioned at 5 mm thickness. The slides were stained with hematoxylin and eosin (HE) to observe the new bone formation. The quantitative analysis was not performed in decalcified samples due to small sample size. 2.9. Statistical analysis All data were analyzed using SPSS 13.0 software and statistically significant values were defined as p < 0.05. The data were expressed as mean ± standard

3. Results 3.1. Cell proliferation and morphology on scaffold MTT assay indicated that proliferation of MSCs on scaffold increased rapidly with culture time at early stage. The OD value was 0.18 ± 0.01 on day 2 and 0.63 ± 0.08 on day 6 after cell seeding. The proliferation curve showed a steep slope initially and reached the climax on day 6. Then it fluctuated and reached a plateau on day 8 with the value of 0.61 ± 0.10 (Fig. 2A). The SEM images showed the cells adhered to scaffold and spread after 1 day of culture. Then cells extended to bridge the neighboring pores within scaffold. The morphology of adherent MSCs showed spherical or spindle shape (Fig. 2B). After 7 days of culture, the scaffolds were fully covered with multi-layered cell sheet and ECM-like substance (Fig. 2C). 3.2. Roentgenographic analysis of bone defects repair After 4 weeks of implantation, X-ray films showed the scaffolds were securely fixed at defect sites in all groups. The osteotomy lines were completely radiolucent in all groups. No callus formed around scaffolds to bridge defects. There was no significant difference discerned among groups. After 8 weeks, the high-density radiopaque areas of implanted scaffolds were clearly identified in groups C and D. The osteotomy lines were still visible. In contrast, the abundant newly formed bone connected both ends of defects in groups A and B. The osteotomy lines were partially radiolucent. After 12 weeks, the newly formed bone appeared to have been remodeled into cortical bone with a bone marrow cavity in groups A and B. The osteotomy lines were invisible. Although the new bone was abundant in groups C and D, the osteotomy lines were still discerned. In group E, the defect was still filled with fibrous scar tissue after 12 weeks (Fig. 3). The radiographic grading score showed a time-dependent increase in all groups except group E. At 4 weeks postoperatively, there was no significant difference among groups A, B, C, and D. Then, the scores of group A increased steeply with the value of 24.0 ± 1.9 and 37.8 ± 1.3 at 8 and 12 weeks, respectively, which were all significantly higher than those of other groups. However, the score of group E (empty control) remained low throughout the implantation time (Table 2). 3.3. SPECT and PW-MRI analysis The uptake ratios of 99mTc-MDP in ROI showed similar trend in groups A, B, C, and D. The all values increased continuously with implantation time at initial stage. They reached the summit at 8 weeks with the values of 16.2 ± 1.6, 12.2 ± 1.7, 5.3 ± 0.7, and 3.3 ± 0.6 in groups A, B, C, and D, respectively. In all groups, the significant increase was recorded in groups A and C. Then, the values steadily declined in the following 4 weeks and reached the lower level at 12 weeks. For group E (empty control), the values remained low throughout the period of implantation (Table 3). In PW-MRI analysis, the base line of signal intensity increased with implantation time in all groups. The base lines of groups A and B were significantly higher than those of other three groups (p < 0.05) (Fig. 4A). The SS of groups A and B increased steeply at initial stage. The values of 8 weeks were both significantly higher those of 4 weeks (p < 0.05). Then, the SS showed a slight increase till 12 weeks. The SS of group A was significantly higher compared with that of group B. The SS of groups C and D showed similar increasing trend with lower values. The values showed no significant difference between these two groups (Fig. 4B). The MSI of

Please cite this article in press as: Fan H, et al., Efficacy of prevascularization for segmental bone defect repair using b-tricalcium phosphate scaffold in rhesus monkey, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.05.035

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Fig. 3. Roentgenograph of bone defects repair with prevascularized TEBG (group A) and other grafts (groups B, C, D, E) after 4, 8, and 12 weeks postoperatively.

groups A and B showed significantly higher values compared with those of groups C, D, and E. However, there was no significant difference between groups A and B (Fig. 4C). 3.4. Gross observation of regenerated bone At 4 weeks postoperatively, gross observation revealed that the bone defects were filled with newly formed fibrous or partially calcified tissue and scaffolds in all groups. The osteotomy lines were clearly discerned. No significant difference was detected among groups. At 8 weeks, the osteotomy lines were partially visible in groups A and B. The abundant newly formed bone bridged the broken parts. The regenerated bone of group A remolded and formed a smooth contour in defect. The scaffold couldn't be discerned. In group B, the scaffold was covered by a thick layer of new bone. In groups C and D, the osteotomy lines and scaffold were still observed although the new bone formed. At 12 weeks, the regenerated bone in groups A, B, and C remolded further. The smooth contour was achieved in defects areas, which was similar to that of native bone. However, the scaffolds were partially degraded and still observed in group D. In group E (empty control), the defects were filled with fibrous scar throughout the implantation period (Fig. 5). 3.5. Quantification of vascular regeneration Histological evaluation of implants demonstrated an increase of angiogenesis over time in all groups, especially in group A. At 4 weeks, the central vascular bundle and surrounding fascia flap gave rise to some capillaries and larger blood vessels in group A. The Table 2 Summary of radiographic grading score among groups (Data in mean ± SD, n ¼ 21 in groups A, B, C and D; n ¼ 6 in group E). Post-operation time

Group A

Group B

Group C

Group D

Group E

4 weeks 8 weeks 12 weeks

11.1 ± 2.4 24.0 ± 1.9 37.8 ± 1.3

10.5 ± 1.5 22.1 ± 1.4* 35.5 ± 1.2*

9.7 ± 1.7 20.6 ± 1.2* 28.1 ± 1.2*

10.1 ± 1.1 19.4 ± 1.3* 24.7 ± 1.5*

6.0 ± 0.9* 6.0 ± 0.8* 6.3 ± 0.8*

*p < 0.05 compared with group A.

scattered ink-filled blood vessels invaded the pores surrounding central tunnel and peripheral area of scaffold (Fig. 6A). In group B, the ink-filled vessels could be detected in the peripheral pores of scaffold due to fascia flap envelope. The ink-staining areas within the pores decreased as it progressed from the vessels of fascia towards the central area (Fig. 6D). No vessels could be observed in scaffolds of groups C and D (Fig. 6G, J). From 4 to 8 weeks, the vascularized tissue continued to grow toward the void pores of scaffold. The degree of angiogenesis increased considerably during this period. The maturation of the newly formed bone was obvious from 4 to 8 weeks in groups A and B. The ink-filled blood vessels displayed a vascular network within the pores. Group A showed a higher vascularization density compared with group B (Fig. 6B, E). In contrast, the density of blood vessels was considerably lower in groups C and D. Only scattered dots of ink-filled blood vessels could be observed in the area of interface between scaffold and native bone (Fig. 6H, K). After 12 weeks, the vascular network became much denser in groups A and B (Fig. 6C, F). Most of scaffolds were degraded and replaced with regenerated bone in group A. The vascularization increased and the network of ink-filled vessels began to form in groups C and D (Fig. 6I, L). Image analysis showed the vascular area of peripheral part of scaffolds increased continuously with 125.4 ± 24.7, 328.7 ± 70.8, 521.5 ± 77.8 units at 4, 8, and 12 weeks, respectively in group A. The values were significantly higher than those of groups B, C, and D (Fig. 7A). The vascular area of central part of scaffolds showed the similar increasing trend with the peak value of 308.6 ± 64.9 units at 12 weeks, which was also significantly higher (Fig. 7B).

Table 3 The uptake ratios of 99mTc-MDP in the region of interest (ROI) at 4, 8, and 12 weeks postoperatively (data in mean ± SD, n ¼ 21 in groups A, B, C and D; n ¼ 6 in group E). Group

4w

A B C D E

12.3 10.6 4.2 2.8 0.8

8w ± ± ± ± ±

2.2 1.5 0.8 0.6 0.1

16.2 12.2 5.3 3.3 0.8

12 w ± ± ± ± ±

1.6* 1.7 0.7* 0.6 0.1

11.5 9.5 3.8 2.3 0.9

± ± ± ± ±

2.2 1.1 0.8 0.8 0.1

*p < 0.05.

Please cite this article in press as: Fan H, et al., Efficacy of prevascularization for segmental bone defect repair using b-tricalcium phosphate scaffold in rhesus monkey, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.05.035

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Fig. 4. (A) The base line of signal intensity in different groups at 4, 8, and 12 weeks postoperatively. (B) The steepest slop in different groups at 4, 8, and 12 weeks postoperatively. (C) The maximum signal intensity increase in different groups at 4, 8, and 12 weeks postoperatively.

3.6. Histological assessment of bone regeneration There was no obvious inflammation found in the region of TEBG in all groups. At 12 weeks postoperatively, most of scaffold degraded and was not detectable in group A. With the absorption of scaffold the regenerated woven bone formed extensively. The new bone remolded and much thicker trabecular bone was observed (Fig. 8A). Group B showed the similar histological changes while with less newly formed bone. The scaffolds degraded mostly and the residual could be clearly identified. The trabecular bone was much thinner and sparser compared with that of group A (Fig. 8B). The scaffolds degraded partially and the thin trabecular bone connected each other in group C (Fig. 8C). In group D, granulation tissue in micro pores were mineralized and new bone formed along the pore walls. The new trabecular bone was separated by the residual scaffold (Fig. 8D). The group E (empty control) showed the defect was filled with fibrous tissue. 4. Discussion Although b-TCP ceramic scaffold has good osteoconductivity, it is not applicable for large bone defect repair due to avascularity. The autograft with superior osteogenic potential and abundant blood supply was conventionally used to treat large defect. However, this

technique has considerable disadvantages including donor site morbidity, limited availability, and prolonged recovery. Recently, prevascularized tissue-engineered scaffold has proved to be one of the most promising alternative therapies. Compared with other prevascularization methods such as flap coverage and endothelial cells co-culture [25,26], vascular bundle insertion has proved to be a safe, simple, and effective way [6,27,28]. In our previous report, the efficacy of prevascularized TEBG with vascular bundle insertion was investigated in bone defect of rabbit. The results showed the implanted prevascularized TEBG had faster capillary vessel infiltration in the defect region and more bone was regenerated with concurrently up-regulation of VEGF expression [6]. The selection of appropriate animal model is essential to assess the healing capability of prevascularized TEBG. Tissue-engineered devices are generally classified as class III medical devices requiring FDA approval for use. Class III is the most stringent regulatory category for devices. A Class III device is one for which insufficient information exists to assure safety and effectiveness solely through the general or special controls sufficient for Class I or Class II devices [29]. There are many hurdles in developing and testing proposed TEBG for the treatment of bone defect in humans. The general progression goes from success in cell culture or a small animal to a larger animal in an anatomically correct location and then to human trials. The rabbit has similar osteon diameter

Fig. 5. The gross observation of regenerated bone in different groups at 8 and 12 weeks postoperatively.

Please cite this article in press as: Fan H, et al., Efficacy of prevascularization for segmental bone defect repair using b-tricalcium phosphate scaffold in rhesus monkey, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.05.035

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Fig. 6. Histological observation of ink-filled blood vessels in TEBG of different groups at 4, 8, and 12 weeks postoperatively (100).

(231 ± 45 mm) in comparison with rhesus monkey and humans (216 ± 52 mm and 235 ± 13 mm). However, the Haversian canal diameter of rabbit (27 ± 6 mm) was much smaller than those of rhesus monkey and humans (167 ± 46 mm; 195 ± 11 mm). Furthermore, the thickness of osteon wall in rabbits is greater than those of humans and rhesus monkeys, which can be associated with a different load determined by lifestyle [30,31]. The microarchitectures of long bones in primates are most similar to those of humans probably because of genetic similarity. Therefore, we tried to repair segmental tibial defect in rhesus monkey with prevascularized TEBG in this study after rabbit experiments yielded promising results [6]. The X-ray examination showed the prevascularized TEBG group (group A: vascular bundle insertion þ fascia flap coverage) exhibited more extensive osteogenesis. The radiological scores were better than those of groups B, C and D after 8 and 12 weeks. Consistent with radiographic results, histologic findings in group A revealed obvious osteogenesis. The new bone remolded and much thicker trabecular bone formed. This could be the result of vascularization mediated by vessel bundle insertion, which might deliver oxygen and nutrients for the constructs. Adequate oxygen tension and nutrient supply could allow formation of mineralized matrix in the central scaffold [21]. In contrast, the absence of vessel insertion in groups C and D resulted in retarded vascularization and poor osteogenesis. The scaffolds were not completely degraded during the period of implantation. This correspondingly led to a small amount of new bone formation. The fascia flap coverage technique in group B was also able to enhance vascularization and osteogenesis although it was less effective than use of vascular bundle insertion. These indicated that pre-vascularized TEBG could

effectively enhance a significant new bone formation. Compared with muscle flap coverage, the prevascularized technique (vascular bundle insertion and fascia flap) didn't sacrifice any motor system function. Furthermore, it could be used to treat bone defects almost anywhere in the trunk and extremities using local vessels. In combination with ink injection and image analysis, histological examination provided insight into the vascularization within scaffold. Vascularization was defined as the process of sprouting new blood vessels from established vessels. In this study, the higher degree of vascularization was demonstrated within the prevascularized scaffold in group A. The vascular area of central and peripheral scaffold increased continuously with implantation time and reached peak value (521.5 ± 77.8 and 308.6 ± 64.9 units) after 12 weeks, which were remarkably higher than those of groups B, C, and D. Correspondingly, the newly formed bone was abundant in group A probably due to enough perfusion. It was presented not only at surface layer but also in the central part of implant. Bone scintigraphy is considered as the gold standard for demonstrating successful grafting. The uptake of radiopharmaceutical depends on both adequate delivery system and living network of osteocytes. MDP, the most commonly used tracer, is a highly sensitive marker for blood flow and metabolic activity in bone tissue. In our experiment, uptake ratio was less than 1 in group E (empty control), which indicated there was no vascularization process happened. The ratios of groups A, B, C, and D increased continuously in the first 8 weeks and decreased from 8 to 12 weeks. At each time point, uptake ratios sequentially decreased from groups A to D (group A > group B > group C > group D), which indicated that osteogenesis and vascularization were best in group A. Taken together, these findings indicate that the vascularization of

Please cite this article in press as: Fan H, et al., Efficacy of prevascularization for segmental bone defect repair using b-tricalcium phosphate scaffold in rhesus monkey, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.05.035

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H. Fan et al. / Biomaterials xxx (2014) 1e9

Fig. 7. (A) The vascular area of peripheral part of graft in different groups at 4, 8, and 12 weeks postoperatively. (B) The vascular area of central part of graft in different groups at 4, 8, and 12 weeks postoperatively.

the grafts was rapid during the first 8 weeks and entered a mature stage from 8 to 12 weeks after surgery. Vascularization was completed at approximately 12 weeks after surgery, which is consistent with the findings of other research [26]. With the introduction of the first gadolinium-based MR contrast agent to the clinical community in 1988, the necessity of contrastenhanced MRI has arisen in the study of oncology, infection, inflammation, and vascular abnormalities. The contrast agent remains extracellular and does not cross the intact bloodebrain barrier; it may be used to assess the blood flow and permeability in tissues. It provides a non-invasive and continuous way to assess the vascularization process within the implant. In this study, long-term monitoring of angiogenic activity within the TEBG in each individual monkey could be conducted at several time points using PW-MRI. Compared with histology staining (static evaluation), MRI could dynamically evaluate the vascularization within the

constructs. Because both vascular bundle insertion and fascia flap coverage could increase the blood flow within the constructs, the signal intensity base lines of groups A and B were significant higher than those of other groups. During the whole implantation period, the SS and MSI increased continuously and were recorded the peak value at 12 weeks in group A. The results were correlated with the findings of bone scintigraphy and histology staining. 5. Conclusions In this study, a rhesus monkey model which closely mimicked the human tibial defect was used to investigate the repair efficiency of prevascularized TEBG. The prevascularization of TEBG, which combined tissue-engineering strategies with plastic surgical techniques, could augment new bone formation and capillary vessel ingrowth. This concept may facilitate the generation of large volume

Fig. 8. Histological observation of regenerated bone in different groups by HE staining at 12 weeks postoperatively (100).

Please cite this article in press as: Fan H, et al., Efficacy of prevascularization for segmental bone defect repair using b-tricalcium phosphate scaffold in rhesus monkey, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.05.035

H. Fan et al. / Biomaterials xxx (2014) 1e9

of vascularized bone tissue suitable for large defect repair. The novelty of this study is that in vivo experiment data of prevascularized TEBG is further enriched from small to large animal model. It implies that prevascularized TEBG has great potential in clinical applications. Funding source This work was supported by National Natural Science Foundation of China (U0732003, 30600643, 30872638, 30900311, 31170936) and Major State Basic Research Development Program of China (2009CB930000). References [1] Myeroff C, Archdeacon M. Autogenous bone graft: donor sites and techniques. J Bone Jt Surg Am 2011;93:2227e36. [2] Muscolo DL, Ayerza MA, Aponte-Tinao LA. Massive allograft use in orthopedic oncology. Orthop Clin North Am 2006;37:65e74. [3] Cheung C. The future of bone healing. Clin Podiatr Med Surg 2005;22:631e41. [4] De Long Jr WG, Einhorn TA, Koval K, McKee M, Smith W, Sanders R, et al. Bone grafts and bone graft substitutes in orthopaedic trauma surgery. A critical analysis. J Bone Jt Surg Am 2007;89:649e58. [5] Calori GM, Mazza E, Colombo M, Ripamonti C. The use of bone-graft substitutes in large bone defects: any specific needs? Injury 2011;42(Suppl. 2): S56e63. [6] Wang L, Fan H, Zhang ZY, Lou AJ, Pei GX, Jiang S, et al. Osteogenesis and angiogenesis of tissue-engineered bone constructed by prevascularized btricalcium phosphate scaffold and mesenchymal stem cells. Biomaterials 2010;31:9452e61. [7] Bi L, Cheng W, Fan H, Pei G. Reconstruction of goat tibial defects using an injectable tricalcium phosphate/chitosan in combination with autologous platelet-rich plasma. Biomaterials 2010;31:3201e11. [8] Rezwan K, Chen QZ, Blaker JJ, Boccaccini AR. Biodegradable and bioactive porous polymer/inorganic composite scaffolds for bone tissue engineering. Biomaterials 2006;27:3413e31. [9] Lovett M, Lee K, Edwards A, Kaplan DL. Vascularization strategies for tissue engineering. Tissue Eng Part B Rev 2009;15:353e70. [10] Laschke MW, Harder Y, Amon M, Martin I, Farhadi J, Ring A, et al. Angiogenesis in tissue engineering: breathing life into constructed tissue substitutes. Tissue Eng 2006;12:2093e104. [11] Rouwkema J, Rivron NC, van Blitterswijk CA. Vascularization in tissue engineering. Trends Biotechnol 2008;26:434e41. [12] Phelps EA, García AJ. Engineering more than a cell: vascularization strategies in tissue engineering. Curr Opin Biotechnol 2010;21:704e9. [13] Hokugo A, Kubo Y, Takahashi Y, Fukuda A, Horiuchi K, Mushimoto K, et al. Prefabrication of vascularized bone graft using guided bone regeneration. Tissue Eng 2004;10:978e86.

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Please cite this article in press as: Fan H, et al., Efficacy of prevascularization for segmental bone defect repair using b-tricalcium phosphate scaffold in rhesus monkey, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.05.035