Time-phase Sequential Utilization of ADSCs on Mesoporous Bioactive

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Biological and Medical Applications of Materials and Interfaces

Time-phase Sequential Utilization of ADSCs on Mesoporous Bioactive Glass for Restoration of Critical Size Bone Defects Jiahui Du, Peng Xie, Shuxian Lin, Yuqiong Wu, Deliang Zeng, Yulin Li, and Xinquan Jiang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b08563 • Publication Date (Web): 06 Aug 2018 Downloaded from http://pubs.acs.org on August 7, 2018

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ACS Applied Materials & Interfaces

Time-phase Sequential Utilization of ADSCs on Mesoporous Bioactive Glass for Restoration of Critical Size Bone Defects Jiahui Du, ¶,†,‡,₰ Peng Xie, ¶,§ Shuxian Lin, †,‡,₰ Yuqiong Wu, †,‡,₰ Deliang Zeng, †,‡,₰ Yulin Li, * ,§ and Xinquan Jiang, * ,†,‡, ₰

† Department of Prosthodontics, Shanghai Ninth People's Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine, 639 Zhizaoju Road, Shanghai 200011, China

‡ National Clinical Research Center for Oral Diseases, 639 Zhizaoju Road, Shanghai 200011, China

₰ Shanghai Key Laboratory of Stomatology & Shanghai Research Institute of

Stomatology, 639 Zhizaoju Road, Shanghai 200011, China

§ The State Key Laboratory of Bioreactor Engineering, Key Laboratory for Ultrafine Materials of Ministry of Education, Engineering Research Center for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237, People's Republic of China

Key words: critical size bone defect, adipose-derived stem cells, mesoporous bioactive glass, neovascularization, bone regeneration

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ABSTRACT The effective transportation of oxygen, nutrients and metabolic wastes through new blood vessel networks is key to the survival of engineered constructs in large bone defects. Adipose-derived mesenchymal stem cells (ADSCs), which are regarded as excellent candidates for both bone and blood vessel engineering, are the preferred option for the restoration of massive bone defects. Therefore, we propose to induce ADSCs into osteogenic and endothelial cells differently. A modified hierarchical mesoporous bioactive glass (MBG) scaffold with an enhanced compressive strength was constructed and prevascularized by seeding with endothelial-induced ADSCs (EI-ADSCs). The prevascularized scaffolds were combined with osteogenic-induced ADSCs (OI-ADSCs) to repair critical size bone defects. To validate the angiogenesis of the prevascularized MBG scaffolds in vivo, green fluorescent protein (GFP) was used to label EI-ADSCs. The labeled EI-ADSCs were demonstrated to survive and participate in vascularization at day 7 after subcutaneous implantation in nude mice by double immunofluorescence staining of CD31 and GFP. Regarding the restoration of critical size bone defects, early angiogenesis of rat femur plug defects was evaluated by perfusion of Microfil after 3 weeks. Compared to nonvascularized MBG carrying OI-ADSCs (MBG/OI-ADSCs) and non-cell-seeded MBG scaffolds, the prevascularized MBG carrying OI-ADSCs (Pv-MBG/OI-ADSCs) showed enhanced angiogenesis on the surface and interior. Through dynamic bone formation analysis with sequential fluorescent labeling and Van Gieson’s picro-fuchsin staining, we found that the Pv-MBG/OI-ADSCs exhibited the highest mineral deposition rate after


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surgery, which may be contributed by rapid vascular anastomosis facilitating increased survival of the seeded OI-ADSCs and by the recruitment function for bone mesenchymal stem cells. Therefore, the strategy of time-phase sequential utilization of ADSCs on MBG scaffolds is a practical design for the repair of massive bone defects.



1. INTRODUCTION Massive bone defects are a frequent, devastating and costly clinical problem. These defects are beyond the regenerative potential of human bone and can lead to severe deformation and dysfunction. Autogenous bone grafts are qualified with osteoconduction, osteoinduction and osteogenic cells; however, this is similar to “robbing Peter to pay Paul”, with potential complications of limited bone mass and injury at the donor site.1-2 Allogeneic bone grafts also present potential risks of disease transmission, contamination, and immunological rejection if donors are not adequately evaluated or serologically screened.3-4 Fortunately, engineered bones, which are lab-grown constructs of cells and supportive three-dimensional scaffolds, have become alternatives for patients with bone defects in recent years.5-6 However, the limited diffusion of oxygen and nutrients in the capillary can support the survival of cells only within 200 µm.7 Therefore, to guarantee the effective transportation of oxygen, nutrients and metabolic wastes, the early formation of new blood vessel networks inside engineered constructs is key to the success of the restoration of large bone defects.8-9 Many strategies exist for constructing vascularized bone scaffolds, including growth-factor-functionalized biomaterials, scaffolds with hyperporous or multichannel interiors and cell-laden approaches.9-11 As cellular approaches to engineering vascularized bone constructs, human umbilical vein endothelial cells (HUVECs), human blood-derived endothelial colony-forming cells (ECFCs) and pluripotent stem cells (hPSCs) have been used to construct microvascular networks.12-14 However, concerns still exist considering the limited expansion


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capability of differentiated cells, such as in the case of endothelial progenitors or endothelial cells, and the ethical controversy related to embryonic sources. Adipose-derived mesenchymal stem cells (ADSCs) possess the potential for multilineage differentiation into adipogenic, osteogenic, vascular, myogenic, chondrogenic and even neuronal cells.15-17 Their advantages in terms of plentiful quantity and easy, minimally invasive access make ADSCs clinically popular adult stem cells.18-19 During bone regeneration, ADSCs can differentiate into osteoblasts to form an osteoid in vitro under certain conditions and to repair critical size bone defects in vivo.20-24 Additionally, ADSCs can be used for blood vessel regeneration due to their capability to differentiate into vascular cells and to secrete proangiogenic growth factors to promote angiogenesis.25-30 Numerous studies have shown that vascular networks engineered in vitro can promote angiogenesis after in vivo implantation.10,

31-38

In our previous study, we

confirmed that in vitro prevascularization with HUVECs can anastomose with host vessels and promote the survival of the seeded stem cells.39 Given of the problem of immune compatibility, the limited expansion of HUVECs and the excellent characteristics of ADSCs for bone regeneration and angiogenesis, we attempted to replace the HUVECs with endothelial cells differentiated from ADSCs, and repair critical size bone defects synergistically with osteogenic-induced ADSCs (OI-ADSCs) in our present study. A suitable scaffold for a large bone defect should have the qualities of excellent biocompatibility and enhanced compressive strength, and it must also meet the design



requirements for osteoinduction and vascularization. Mesoporous bioactive glasses (MBGs) were discovered in 2004.40 The diverse preparation methods for MBG scaffolds, their flexibility of use in composite and coated systems, and their ability to be incorporated with a number of therapeutic elements (cations), drugs and growth factors make them a special class of multifunctional biomaterials in bone tissue engineering.41 Such biodegradable scaffolds also have significant potential to stimulate osteogenesis and angiogenesis and to regulate immune responses both in vitro and in vivo.41-43 Moreover, no histopathological distortion caused by the degradation products of MBG scaffolds have been found in systemic toxicological studies in vivo and in vitro, thus providing a scientific basis for its potential clinical applications.44 In this study, trimodal porous MBG scaffolds with a uniform macroporous network and enhanced mechanical properties were obtained through a modified sol-gel casting and particle reinforced method.45 Highly interconnected macroporosity (200–500 µm) provides the basis for prevascular design for the ingrowth of new vessel/bone and transportation of nutrients/wastes. And the microporous topography was shown to activate FAK/MAPK, ILK/β-catenin signaling and to facilitate the adherence and osteoblast differentiation of rat bone marrow stroma mesenchymal stem cells (BMSCs).45-47 Moreover, Ca and Si ions released from MBG scaffolds can significantly promote osteogenesis and angiogenesis in vitro and in vivo.48-53 Therefore, we have combined the osteogenesis and angiogenesis advantages of ADSCs with modified MBG scaffolds to optimize the restoration of massive bone


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defects in this study, as illustrated in Figure 1. After the induction of ADSCs into osteogenic and endothelial cells using different methods (Figure 1A), we constructed prevascularized hierarchical MBG scaffolds with the EI-ADSCs (Figure 1B, C). After we evaluated the feasibility in vitro and in vivo, the OI-ADSCs were seeded onto the prevascularized hierarchical MBG scaffolds for critical size bone regeneration (Figure 1D). The rapid angiogenesis and bone regeneration outcomes observed in the critical size rat femur defect model demonstrate the effectiveness of the strategy of using time-phase sequential utilization of ADSCs in conjunction with trimodal macro/micro/nanoporous MBG scaffolds (Figure 1E).

2. MATERIALS AND METHODS Culture and Induction of Rat ADSCs. The animal experiments were licensed by the Institutional Animal Care and Use Committee of our hospital. The isolation and culture of the rat ADSCs were conducted as reported previously.54 The ADSCs obtained after three passages were used for further experiments. For osteogenic differentiation, the ADSC medium was changed to osteogenic differentiation medium.54 The cell morphology at 4 days after osteogenic induction was recorded by light microscopy (Nikon, Tokyo, Japan). After 7 and 21 days of osteogenic induction, alkaline phosphate staining kit and alizarin red staining was used to evaluate the alkaline phosphate activity and calcium deposition of the OI-ADSCs, respectively.54 To induce endothelial differentiation, ADSCs were exposed to 40 ng/ml of VEGF and 10 ng/ml of bFGF (Pepro Tech, Rocky Hill, NJ, USA) in complete culture medium.



Cell morphology changes were observed after 4 days of endothelial induction using a light microscope. The expression of CD31 and von Willebrand Factor (vWF) in the EI-ADSCs was visualized by immunofluorescence staining after 7 days of endothelial induction.39 Rhodamine phalloidin (Cytoskeleton, Denver, CO, USA) and DAPI (Sigma-Aldrich, St. Louis, MO, USA) were used to visualize the cytoskeleton and cell nuclei, respectively. Immunofluorescence staining images were recorded via confocal laser-scanning microscopy (CLSM, Nikon A1, Nikon, Japan). Moreover, the gene expression was further quantified and compared in the two types of induced ADSCs and noninduced cells using real-time PCR. The extraction and amplification procedures were performed as reported previously.54 The data were normalized to β-actin expression and analyzed using the comparative Ct (2-△△Ct) method. All primers are listed in Table 1. Fabrication of the Trimodal MBG Scaffolds. A modified sol-gel casting and double template method was adopted for the fabrication of the MBG scaffolds.45 In detail, 4.0 g of F127 (EO106PO70EO106, mesoporous template) was added to 50 ml of anhydrous ethanol and was dissolved with the help of HCl (1 M). After being stirred with Ca(NO3)2·4H2O, triethyl phosphate and tetraethyl orthosilicate at 40 °C for 1 day, part of the sol was dried in a fume hood and ground into particles; the other portion was rotary evaporated to obtain a viscous MBG sol. Then, the MBG particles and methyl cellulose (ethanol-insoluble microspheres as a microporous template) were added to the viscous MBG sol to acquire a uniform mixture slurry. The slurry was then impregnated into a polyurethane sponge (as a macroporous template with a


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designed shape). After being fully dried and calcined, tailored scaffolds were obtained. Scanning electron microscopy (SEM), transmission electron microscopy (TEM) and X-ray diffraction (XRD) were used to observe the interconnected macroporosity, microtopography and ordered mesoporous structures of the scaffolds.45 The mechanical properties of the samples (Φ5 mm and high 4 mm) were evaluated dynamically after incubation in a Tris-HCl buffer solution and PBS for 1, 2 and 4 weeks using a universal testing apparatus (AG-2000A, Shimadzu, Japan). To compare their ion dissolution capabilities, the trimodal MBG scaffolds (T-MBG) and the MBG scaffolds without microporous topography (nonmicro-MBG) were incubated in simulated body fluid (SBF) at a ratio of 200 mL/g.55 The ion concentration was quantified with inductive coupled plasma analysis (ICP, Optima 7000DV, PerkinElmer, USA) at 1, 3, 5 and 7 days. New dense apatite layer formation on the T-MBG and nonmicro-MBG scaffolds was detected using SEM after incubation in SBF for 1 and 3 days. The effect of the hierarchical MBG scaffolds on the osteogenesis and angiogenesis of the ADSCs was evaluated using real-time PCR. In detail, third-passage ADSCs were seeded onto the MBG scaffolds for cocultivation for 48 h, with ADSCs on a culture plate as a blank control. Then, the osteogenesis-related

gene

expression

of

COL1,

OPN

and

BMP-2,

the

angiogenesis-related genes expression of VEGF, VE-cadherin and ANG1, and the adipogenesis-related gene expression of adiponectin and adipocyte protein 2 (aP2) were quantified as mentioned above. The primer sets adopted are listed in Table 1.



Preparation of Prevascularized MBG Scaffolds. The EI-ADSCs were collected and seeded on the hierarchical porous MBG scaffolds for an additional 7 days in endothelial inducing medium. In detail, to avoid intense fluctuations in the pH and to facilitate a uniform cell distribution, the scaffolds were preincubated in culture medium before being saturated in an EI-ADSC suspension (106 cells/ml). After a 2-h incubation to allow cell attachment, an adequate volume of endothelial inducing medium was added. SEM and CLSM were used to evaluate the cell adhesion and the spreading abilities 24 h after seeding. In detail, the fixed cells on the scaffolds were dehydrated and vacuum dried before observation under SEM and were stained with rhodamine phalloidin and DAPI before observation under CSLM. The cell survival and viability on the MBG scaffolds were evaluated using a Cell Counting Kit-8 assay (CCK-8, Dojindo, Kumamoto, Japan) at 1, 3, 5 and 7 days. After 7 days of endothelial induction, the canaliculization ability of the EI-ADSCs on the MBG scaffolds was assessed with immunofluorescence staining of CD31 and the cytoskeleton, as mentioned above. To verify the angiogenesis ability in vivo, green fluorescent protein (GFP) was used to label the EI-ADSCs with a lentivirus at an MOI of 25. After further prevascularization treatment on the scaffolds for 7 days, the scaffolds were subcutaneously transplanted into nude mice and compared to nonvascularized MBG scaffolds. On day 7, samples were removed and observed with a stereomicroscope (SZX16, Olympus, Tokyo, Japan). After decalcified, the samples were paraffin embedded and sectioned for double immunofluorescence staining of CD31 and GFP.


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Animal Surgery to Generate the Rat Femur Critical Size Defect Model. For the animal surgery, the prevascularized and nonvascularized MBG scaffolds were both seeded with OI-ADSCs at 2x107 cell/ml until saturation, and the non-cell-seeded MBG scaffolds were used in the control group. The rat femur plug defect experiments were performed using a total of twenty-four male Sprague-Dawley rats that were 12 weeks old. A linear skin incision was made, and the muscles were bluntly dissected to expose the femoral condyle in the distal femoral epiphysis after anesthesia and sterilization. Then, a plug defect of 3.5 mm in diameter and 5 mm in depth was constructed on the distal femoral cancellous bone using a slow speed bur under saline irrigation for cooling and rinsing. Eighteen rats were repaired with three types of scaffolds, including MBG (n=6), MBG carrying OI-ADSCs (MBG/OI-ADSCs, n=6) and prevascularized MBG carrying OI-ADSCs (Pv-MBG/OI-ADSCs, n=6), and an additional six rats were included as a blank group to test the critical bone defect model at different times. All animal experiment protocols followed our hospital’s Institutional Animal Care and Use Committee. Early Angiogenesis of the Rat Femur Critical Size Defect Model. Three weeks after implantation, Microfil (Flow Tech, Carver, MA, USA) was injected into the anesthetized rats (n=3 per group) using a cardiac approach.56 Then, the femurs were collected and fixed for 48 h. After dehydration, the undecalcified femurs were embedded in methyl methacrylate for sectioning with a microtome.56 Angiogenesis of the samples was shown by the blue Microfil on the sections and imaged using a microscope (BX51, Olympus, Tokyo, Japan).



Osteogenesis of the Rat Femur Critical Size Defect Model. For dynamic bone formation analysis at 2, 4 and 6 weeks after implantation, polychrome sequential fluorescent labeling with hydrochloride, ARS and calcein was performed, as described previously.57 Eight weeks after surgery, the animals (n=3 per group) were euthanized and the femurs were collected and fixed with paraformaldehyde. After dehydration, the undecalcified femurs were embedded in methyl methacrylate for sectioning with a microtome, as mentioned above. Fluorescence labeling for dynamic bone regeneration was observed using CLSM. Subsequently, the area of new bone formation in the whole bone defect was visualized using Van Gieson’s picro-fuchsin and quantified using an Image-Pro Plus system.56 Luciferase Assay and Coculture Test. To assess the survival of the seeded OI-ADSCs in vivo, lentivirus particles expressing luciferase were used to label the cells before subcutaneous implantation in nude mice. At days 7, 14 and 28 after surgery, the seeded OI-ADSCs were visualized with an in vivo fluorescence imaging system, as previously described.56 In the BMSC recruitment experiment, transwell migration assays (Corning Costar Corporation, Cambridge, MA) were performed. Briefly, 3×105 BMSCs were seeded on the upper insert with 8-µm pores, and the lower chamber either did or did not contain EI-ADSCs; the samples were incubated in medium with 2% FBS. After 24 h of coculture, the upper cells were erased, and the cells underneath the transwell were stained with DAPI and counted using the Image-Pro Plus system.


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Statistical Analysis. In this study, the results of repeated experiments were shown as the mean±standard deviation (SD). The significant differences between two or three data sets were analyzed using Student’s t-test or a one-way analysis of variance (ANOVA), respectively, and indicated by *p

Time-phase Sequential Utilization of ADSCs on Mesoporous Bioactive

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