Demineralized Bone Matrix Scaffolds Modified by CBD-SDF-1Î±

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Demineralized Bone Matrix Scaffolds Modified by CBD-SDF-1# Promote Bone Regeneration via Recruiting Endogenous Stem Cells Jia Jia Shi, Jie Sun, Wen Zhang, Hui Liang, Qin Shi, Xiaoran Li, Yanyan Chen, Yan Zhuang, and Jianwu Dai ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b08685 • Publication Date (Web): 30 Sep 2016 Downloaded from http://pubs.acs.org on September 30, 2016

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ACS Applied Materials & Interfaces

Demineralized Bone Matrix Scaffolds Modified by CBD-SDF-1α Promote Bone Regeneration via Recruiting Endogenous Stem Cells Jiajia Shi1,2 ‡, Jie Sun1,3, ‡, Wen Zhang4, Hui Liang1, Qin Shi5, Xiaoran Li1, Yanyan Chen1, Yan Zhuang1, Jianwu Dai*,1,2,6. 1

Key Laboratory for Nano-Bio Interface Research, Suzhou Key Laboratory for

Nanotheranostics, Division of Nanobiomedicine, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou 215123, China 2

School of Nano Technology and Nano Bionics, University of Science and Technology

of China, Hefei 230026, China 3

Institute of Combined Injury, State Key Laboratory of Trauma, Burns and Combined

Injury, Chongqing Engineering Research Center for Nanomedicine, College of Preventive Medicine, Third Military Medical University, Chongqing 400038, China 4

Affiliated Hospital of Soochow University, Orthopedic Institute, Soochow University,

Suzhou 215007, China；

5

Orthopedic Department, the First Affiliated Hospital of Soochow University, Suzhou

215006, China;

6

State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China

‡

Jiajia Shi and Jie Sun contribute equally to this work

Keywords: bone regeneration, CBD-SDF-1α, demineralized bone matrix scaffolds, sustained release, endogenous stem cell 0

ACS Paragon Plus Environment


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ABSTRACT The reconstruction of bone usually depends on substitute transplantation, which has drawbacks including the limited bone substitutes available, comorbidity, immune rejection and limited endogenous bone regeneration. Here, we constructed a functionalized bone substitute by combining application of the demineralized bone matrix (DBM) and a collagen-binding stromal cell-derived factor-1α (CBD-SDF-1α). DBM was a poriferous and biodegradable bone substitute, which derived from bovine bone and consisted mainly of collagen. The CBD-SDF-1α could bind to collagen and be controllably released from the DBM to mobilize stem cells. In a rat femur defect model, CBD-SDF-1α-modified DBM scaffolds could efficiently mobilize CD34+ and c-kit+ endogenous stem cells homing to the injured site at 3 days after implantation. According to the data from micro CT, CBD-SDF-1α-modified DBM scaffolds could help the bone defects rejoin with mineralization accumulated and bone volume expanded. Interestingly, osteoprotegerin (OPG) and osteopontin (OPN) were highly expressed in CBD-SDF-1α group at an early time after implantation, while the osteocalcin (OCN) was more expanded. H&E and Masson's trichrome staining showed that the CBD-SDF-1α-modified DBM scaffold group had more osteoblasts and that the bone defect rejoined earlier. The ultimate force of the regenerated bone was investigated by three-point bending, showing that the CBD-SDF-1α group had superior strength. In conclusion, CBD-SDF-1α-modified DBM scaffolds could promote bone regeneration by recruiting endogenous stem cells.

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INTRODUCTION The bones are among the few organs that possess the ability to regenerate after injury or during skeletal development, even during adult life1-2. However, when a bone defect is critical, it cannot recover by itself. Autologous bone grafts are preferred, but they entail drawbacks, such as the high prevalence of complications, the need for multiple surgical sites and the limited amount of bone available. To avoid these drawbacks, metal, paste, and chemical molecules have been utilized as bone substitute materials3-6. Unfortunately, none of these substitutes have been shown to promote endogenous bone regeneration, and they might even cause immune rejection7. Demineralized bone matrix (DBM) was first proposed for use in bone implantation by Marshall Urist in 19658. DBM is allograft bone with the bone mineral materials removed of which the major component is collagen. DBM was investigated for its hardness, function as a scaffold for drug assembly, and osteoconductive ability. Subsequent findings proved that DBM is a successful bone graft material owing to its low immunogenicity, sufficient hardness, and high bioactivity and availability9-11. As a bone-like component with low immunogenicity and fine biocompatibility, DBM is more acceptable in bone implantation, especially for large-area bone defects. It is well known that bone is a metabolic organ that exhibits cell proliferation and apoptosis. A mass of osteoclasts forms when bone tissue is injured, which accelerates the bone diffusion. Bone remodeling is a process in which osteoblasts differentiate and osteoclasts disappear12. Many researchers have demonstrated that mesenchymal stem cells (MSCs) and stem cells in vivo can differentiate into osteoblasts13-15, which 2



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has suggested to us that taking advantage of endogenous stem cells might be perfect for resolving bone defects. To utilize endogenous stem cells to promote intrinsic bone regeneration, scientists have investigated how to recruit host stem cells such as mesenchymal stem cells or hematopoietic stem cells to the site of the injury16-17. Stromal cell-derived factor 1α (SDF-1α), known as C-X-C motif chemokine 12 (CXCL12), is a chemokine of the CXC family that exhibits the capacity to recruit cells both in vivo and in vitro. Recently, SDF-1α was verified to be strongly chemotactic for mesenchymal stem cells and to have the capacity to mediate their suppressive effect on osteoclastogenesis18-19. As many studies on the use of SDF-1α in bone defects have shown that the native protein is of low concentration and easily diffuses, the delivery of the protein is a challenge. In a previous study, medicinal molecules were added onto the materials by physical absorption or crosslinking by a chemical method5, 20. The physical absorption was not effective and the chemical method might introduce poisonous or detrimental molecules into the body. To resolve this problem, we utilized a collagen-binding domain (CBD)21-23 that is biocompatible and biodegradable. Using a genetic engineering method, collagen-binding SDF-1α (CBD-SDF-1α) was constructed that has the capacity to bind to collagen and exhibits SDF-1α chemotactic function. The natural SDF-1α in this study was called NAT-SDF-1α. In this study, we utilized CBD-SDF-1α-encapsulated DBM scaffolds as implants in a rat femur defect model and tested whether this method could promote endogenous bone regeneration. 3


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EXPERIMENTAL METHODS Macroscopic and Microscopic Structures of DBM Scaffolds DBM scaffolds were manufactured according to a previously described method 24-25. Briefly, bovine bones were harvested, and the peripheral tissues were removed. The bones were cut into blocks and soaked in acetone for 2 days to remove the fatty components from the samples. Then, the bone samples were washed with H2O and completely decalcified in 0.6 N HCl for 2 days. After decalcification, the samples were washed in H2O and freeze-dried. The DBM scaffolds were manufactured in dimensions of 10 mm × 10 mm × 10 mm and cut into blocks of 3 mm × 3 mm × 3 mm when used. The microstructure of the DBM scaffolds was studied using scanning electron microscopy (FEI, USA, Quanta 250FEG). Protein Expression and Purification The expression and purification of CBD-SDF-1α and NAT-SDF-1α were carried out according to previous studies in our lab26. Briefly, the CBD gene was fused with SDF-1α by polymerase chain reaction (PCR), and the recombinant gene was inserted into the pET-28a+plasmid (Novagen, Madison, WI). The recombinant vector was transformed into the BL-21(DE3)-strain of Escherichia coli (Novagen, Madison, WI), and the protein expression was induced with 1 mM isopropyl β-D-thiogalactopyranoside (IPTG) for 5 h. Then, the protein was purified using a nickel affinity chromatography column (GE Healthcare, Chalfont St Giles, UK). Sustained Release Assay from DBM Scaffolds DBM scaffolds with dimensions of 3 mm × 3 mm × 3 mm were prepared to be incubated with 10 µM of CBD-SDF-1α or 4



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NAT-SDF-1α for 30 minutes and phosphate-buffered saline (PBS) as a control27-28. The DBM scaffolds loaded with cytokines were transferred into a 48-well plate that was prepared for the sustained release assay; 10 µM of CBD-SDF-1α /NAT-SDF-1α was dissolved into PBS. The cytokines were added to each DBM scaffold in the 48-well plate, and the 48-well plate was then placed in an incubator (37°C) for 30 min. After incubation, the DBM scaffolds loaded with CBD-SDF-1α or NAT-SDF-1α were ready for the sustained release assay. The prepared DBM scaffolds in the 48-well plate were covered with 500 µl PBS in each well. During the assay, the PBS was changed every 12 hours, the harvested PBS at each time point was preserved at -20°C, and the concentrations of the cytokines released from the DBM scaffolds of each group were tested using a human SDF-1 ELISA kit (Blue Gene) according to the manufacturer’s instructions. The remaining amount of the protein was calculated from the total amount and released amount, and a curve was generated by Origin Pro 8.0. Mesenchymal Stem Cell Isolation and Culture Umbilical cords were obtained from full-term infants delivered by Caesarean section, with ethical approval obtained from the Institutional Ethical Review Board. The human umbilical cord mesenchymal stem cells (hMSCs) were harvested from the umbilical cords by enzymatic digestion, following previously described procedures for isolation and culturing29. Briefly, the umbilical cords were rinsed with PBS to remove the blood and then disinfected with 70% alcohol. Under aseptic conditions, the umbilical cord was cut into small pieces and subsequently incubated in 0.05% (m/v) collagenase type II solution at 37°C for 1 h. The tissue suspension was shaken every 10 min and crushed for 2 min before being 5


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filtered through a 63-µm mesh. The obtained suspension was then centrifuged at 931 × g for 10 min, and the supernatant was discarded. The cell pellet was resuspended in LG-DMEM containing 12.5% fetal bovine serum (FBS; Hyclone, Logan), 100 U/mL penicillin

(Gibco),

100

mg/mL

streptomycin

(Gibco),

1

mg/L

Insulin-Transferrin-Selenium (ITS; Gibco) and 10 ng/mL basic fibroblast growth factor (bFGF; Gibco). The re-suspended cells were seeded in a cell culture dish and incubated at 37°C in a humid atmosphere containing 5% CO2. After 96 h of culture, the non-adherent cells were removed by PBS, and fresh medium was added. The medium was changed every other day, and passaging was performed when the cells reached confluence (above 80%). The hMSCs of passage 4 were harvested and used for the following experiments. The hMSCs were cultured on DBM scaffolds in the same condition as normal. The DBM scaffolds with hMSCs inside were fixed in 4% paraformaldehyde for 2 h at room time and then dehydrated in graded alcohols. After being dried in a critical point drier (Jinghong, DZF-6050, Shanghai), the samples were coated with gold and evaluated under a scanning electron microscope (SEM, USA, Quanta 250FEG). Biomineralization Activity of DBM Scaffolds The biomineralization of the DBM scaffolds was studied using a 10x simulated body fluid (10×SBF) solution that could mimic the environment in vivo. The preparation of the 10×SBF solution followed A. Cuneyt Tas et al30-31. Before soaking the DBM scaffolds in the 10×SBF, we raised the pH to 6.5 using NaHCO3. Microanalysis reports and microphotos of each scaffold were obtained using SEM at 1, 2 and 3 hours. 6



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Biological Activity Assay of NAT-SDF-1α α and CBD-SDF-1α α A modified Boyden chamber (Corning Costar, 8 µm) assay was performed to test the chemotactic activity of NAT-SDF-1α and CBD-SDF-1α. In brief, 600 µL of cell medium containing 500 ng of NAT-SDF-1α or CBD-SDF-1α was added to the lower chamber, with medium alone used as the control, and 200 µL of hMSCs (1×106 cells/mL) was seeded into the upper chamber. After incubation at 37°C for 12 hours, the samples were fixed and stained with crystal violet. The mean numbers of migrated cells from five fields were observed and counted under a microscope (200×). Femur Defect Model in Rat In the present study, all the procedures followed the guidelines for the ethical use of animals of the National Institutes of Health “Guide for the Care and Use of Laboratory Animals” (NIH publication 23-80, revised 2011). All protocols were approved by the Animal Care and Use Committee of the Institutional Animal Committee of the Chinese Academy of Sciences. In this experiment, the rats were divided into 4 groups: Control group (Ctrl); DBM scaffold group (DBM); DBM scaffolds loaded with NAT-SDF-1α group (NAT-SDF-1α); and DBM scaffolds loaded with CBD-SDF-1α group (CBD-SDF-1α). Fifty male Sprague–Dawley rats (220 to 250 g) were anesthetized with 50 mg/kg pentobarbital sodium by intraperitoneal injection. The operation was conducted on the side face of the femur, right below the trochlea, and the size of the injury was 3 mm × 3 mm × 3 mm32-34. The femur defect model in the rat and the implantation of the CBD-SDF-1α-modified DBM scaffolds were conducted as shown. The incision was sutured with 3-0 stylolite and sterilized with 75% ethyl alcohol. To measure whether 7


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the defect is of the critical size, the rat was scanned by micro CT 7 days after the operation. (Data are presented in S1.) Immunofluorescence Analysis To test whether the CBD-SDF-1α could recruit the stem cells in vivo, CD34 and c-kit were stained, which are confirmed as common stem cell markers. Three days after operation, the bone defect samples were harvested. The implantations were embedded in O.C.T. compound (Sakura, Japan), and cryostat sections (6 µm thick) were generated using a cryostat microtome (Leica, Germany). The cryostat sections were fixed in acetone for 30 minutes and then incubated with mouse-anti-rat-anti-CD34 (1:200, Abcam) and anti-c-kit-biotin (1:200, Abcam) antibodies at 4°C overnight. The cryostat sections were washed several times with PBS and incubated with the second antibodies, rabbit anti-mouse IgG-PE (1:100, Abcam) and FITC-streptavidin (1:200, Leagene), at room temperature for 1 hour. The cell nucleus was co-stained with DAPI, and immunofluorescence photos were then taken by confocal microscopy (Leica, Germany). The percentages of CD34 and c-kit positive stained cells in each photograph were calculated by Image-Pro Plus software (Media Cybernetics, Rockville, MD, USA). Histological Analysis The rats were euthanized by injecting 4% pentobarbital sodium into the cavum abdominis to collect all the femur samples 4, 8 and 12 weeks after operation. The samples were washed gently with PBS to remove the contaminants and were then chemically fixed with cold 4% paraformaldehyde solution on a rocker overnight. After being washed with distilled water several times, the fixed calcified bones were subsequently decalcified in PBS containing 15% EDTA on a shaker. 8



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Briefly, the samples were incubated with 15% EGTA (pH 7.4) in PBS at 4°C35, and the solutions were changed every 4 days. The decalcification was considered complete when the samples were easily punctured by a pin. The tissues were then washed several times in distilled water before dehydration. The dehydration was conducted by progressive replacement with 20%, 50%, 60%, 70%, and then 100% ethanol. Then, the samples were processed for embedding in paraffin and cut into 6-µm sections. To evaluate the morphology of the regenerated bone, hematoxylin and eosin and Masson’s trichrome (Leagene, Beijing) were used for staining according to standard procedures. The paraffin sections were incubated with antibodies including anti-osteoprotegerin (1:2000, Abcam), anti-osteopontin (1:200, Abcam) and anti-osteocalcin (1:100, Abcam) at 4°C overnight, and after being washed with PBS, the second antibody was incubated at room temperature for 1 hour. The images were observed under a light microscope (Carl Zeiss, Jena, Germany). Micro CT Analysis The bone samples at each time point were harvested and scanned by a cone beam micro-CT system (SkyScan1176 In Vivo Micro-CT, BRUKER, Kontich, Belgium) at the Orthopaedic Institute, Soochow University. The x-ray generator was operated at an accelerated potential of 80 kV with a beam current of 300 µA. The x-ray source 2-D detector operated with a shutter speed of 290 ms and produced images with a voxel size of 18 µm. The scans were reconstructed, and three-dimensional digitized images were generated for each specimen using the supporting analyzing software (CT Analyser Version 1.10; Skyscan). Using the scanner, we can obtain the parameters of the bone volume, bone mineral density and 9


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the 3D reconstruction of the femur. Biomechanical Test The biomechanical properties of samples were evaluated by a three-point bending test36-38 using a microcomputer-controlled electrical universal testing machine (HY–1080, Hengyi Instrument, Shanghai, China) at a crosshead speed of 1 mm/min. The fixture consisted of a loading pin and two supporting pins separated by 20 mm. At the beginning of the test, the loading pin compressed the middle of the femur shaft until fracture occurred. The computer recorded the load– displacement curve and calculated the ultimate load. Statistical Analysis All data were analyzed using SPSS computer software, in which significant differences between the two groups were analyzed with Student’s t-test. Data were presented as the means ± standard deviation (SD). *P< 0.05, **P< 0.01 and ***

P< 0.001 were considered to represent statistical significance.

RESULTS CBD-SDF-1α Sustained Release from DBM Scaffolds To promote the endogenous regeneration of bone, the DBM scaffolds were modified with CBD-SDF-1α as a bone substitute for comparison to NAT-SDF-1α (Fig. 1A). As shown in Fig. 1B and C, the DBM scaffolds were porous and sponge-like. By comparing the optical density at 450 nm in a modified ELISA assay, it was found that the amounts of released CBD-SDF-1α were significantly lower than those of NAT-SDF-1α 36 h after the sustained test (Fig. 1D). The data showed that more than 60% of the CBD-SDF-1α was retained 84 hours after 10



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implantation. In contrast, the NAT-SDF-1α group had a very low concentration (approximately 20%). The results of the retained concentration show that the CBD-SDF-1α was effectively bound on the DBM scaffolds and could undergo sustained release from the DBM. DBM Scaffolds Provide a Suitable Microenvironment for Cell Attachment It is well known that a tissue substitute must be biocompatible, so the DBM scaffolds must be tolerated by the cells. We harvested human umbilical mesenchymal stem cells (hMSC) and seeded them on DBM scaffolds. Under the same conditions used for 2D culture, we found that the hMSC could attach onto the scaffolds. As shown in Fig. 1E, the DBM scaffolds provide an appropriate microenvironment for the hMSCs to attach and live. The hMSCs cultured on DBM scaffolds exhibit a suitable size and retain the morphology of the stem cells. DBM Scaffolds Have Suitable Degradation and Biomineralization Activities As an ideal bone substitute, DBM scaffolds have a suitable degradation, as was demonstrated by our previous study20. In the degradation assay, DBM loaded with ADSCs became smaller than DBM in PBS alone after 14 days in vitro. The percentages of the remaining weight of the DBM and the DBM seeded with ADSCs were analyzed after being cultured in cell culture medium and determined to be 84.9%±6.76% and 57.8%±5.54%, respectively. In the in vivo test, DBM was found to be smaller 14 days after implantation, at which point it was ruptured and degraded in some regions. This shows that the DBM scaffolds have a suitable degradation activity both in vivo and in vitro. To study the biomineralization activity of the DBM scaffolds, 11


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we used 10x simulated body fluid (10×SBF) solution to induce DBM scaffold mineralization in vitro. Photos taken by SEM displayed the mineralization of the DBM scaffolds treated with 10×SBF solution for 1, 2 and 3 hours (Fig. 2A, B). The DBM scaffolds were mineralized with time and the content of Ca2+ was shown by microanalysis reports to be clearly increased. The results demonstrate that DBM scaffolds can easily be mineralized and are fit for use as bone substitutes. Bioactivity of CBD-SDF-1α and NAT-SDF-1α To investigate the bioactivity of CBD-SDF-1α and NAT-SDF-1α, a transwell test was conducted according to the capacity of native SDF-1α to mobilize the migration of cells. As shown in Fig. 3A, the degrees of migration of the cells were similar for the CBD-SDF-1α and NAT-SDF-1α groups, and they were significantly larger than that of the control group. A quantitative analysis also demonstrated the similar bioactivities of CBD-SDF-1α and NAT-SDF-1α (Fig. 3B). All of the above prove that the collagen-binding domain did not affect the bioactivity of the SDF-1α. CBD-SDF-1α-modified DBM Scaffolds Can Recruit Stem Cells in vivo To determine whether the scaffolds of DBM and DBM loaded with CBD-SDF-1α or NAT-SDF-1α could recruit stem cells in vivo, the implantations were harvested 3 days after operation (Fig. 4A, B) and were immunofluorescently stained (Fig. 4C). As previous studies proved that CD34 and c-kit are typical markers of the hematopoietic stem cells and mesenchymal stem cells and can be used to identify their populations, we stained the cryostat sections for these proteins39-40. The confocal images showed that all of the groups had recruited CD34+ and c-kit+ cells in vivo (Fig. 4C). The cell 12



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nuclei visualized by DAPI staining showed that with the same relative amounts of cells, the DBM group had fewer CD34 and c-kit positive cells (Fig. 4C). The confocal images showed that the CD34+ cells and c-kit+ cells were more abundant in the CBD-SDF-1α group than in the NAT-SDF-1α group (Fig. 4C). To quantify the analysis, the CD34+ and c-kit+ cells were calculated as the ratio of the CD34+ cell number/ total cell number and the c-kit+ cell number/ total cell number. The data showed that the population of c-kit positive cells in the CBD-SDF-1α group was significantly larger than that in the NAT-SDF-1α group or DBM group. Additionally, the CBD-SDF-1α group was relatively richer in CD34+ cells than the DBM group (Fig. 4D). Histological Results Fig. 5A shows H&E staining 4 weeks after implantation in representative sections of each group. From the H&E stained images, it can be clearly seen that the bone defects of the control groups and DBM groups were partly recovered, with the bone gap still unjoined. Compared with the NAT-SDF-1α group, the CBD-SDF-1α group had dense new bone, and more bone-like tissues could be seen near the border of the bone defect. At high magnification (Fig. 5B), the CBD-SDF-1α group had more osteoblasts and almost no osteoclasts, while the NAT-SDF-1α group and DBM group had similar populations of osteoclasts to the control group (Fig. 5C, D). Masson’s trichrome stain was used to show the regeneration of collagen by staining the newly generated collagen in light blue (Fig. 5E). In the CBD-SDF-1α group, the bone defects were filled with new collagen more deeply than in the NAT-SDF-1α group. The H&E and Masson staining at 8 and 12 13


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weeks after implantation are shown in S2 and S3, which display the results over time for the DBM loaded with NAT-SDF-1α and CBD-SDF-1α, respectively. Immunohistochemistry (IHC) staining results are provided in Fig. 6A, B and C, where the brown areas indicate positive staining. It was found that OPN (osteopontin) was more highly expressed in the CBD-SDF-1α group than in the other three groups at 4 weeks after operation. The osteogenic marker OCN (osteocalcin) was increasingly expressed in each experimental group with time, but the speed of the increase in the CBD-SDF-1α group was much faster than those of the NAT-SDF-1α and DBM groups. OPG (osteoprotegerin) was less expressed in the experiment, which shows the existence of mature osteoblasts. The results of the OPG staining showed that the CBD-SDF-1α group had more osteoblasts and bone-like tissues regenerated. Analysis of Micro CT The micro CT analysis was performed at 4 and 8 weeks after implantation. To better display the level of the bone recovery, we measured each sample’s bone volume (BV) and bone mineral density (BMD), as shown in VOI-1 and VOI-2. VOI-1 was selected at a size of 3 mm×3 mm×1.5 mm at the femur defect, and VOI-2 was the same size as VOI-1. Importantly, VOI-2 shifted towards the bone axis for 1.5 mm (Fig. 4B). CT images (Figure 7A, B) show coronary images of all experimental groups 4 and 8 weeks after implantation. The pictures show that the control group at 4 weeks after operation with no implantation still had bone gaps, whereas the bone gaps of the CBD-SDF-1α groups were almost completely sealed up. As shown in Fig.7B, The experimental groups NAT-SDF-1α and DBM still had different levels of bone defects. 14



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During the test, the bone defects in each group healed at different speeds. The images displayed that the CBD-SDF-1α groups had fast healing, while the DBM groups still had bone gaps at 8 weeks after implantation. With the help of micro CT, the DBM implanted in vivo was investigated by displaying directly the DBM scaffolds inserted in the fracture after a 4-week healing process (S4). The bone mineral density (BMD) of bone samples at 4 weeks after implantation is shown in Fig. 7C, and that of 8 weeks is displayed in Fig. 7D. At 4 weeks after operation, we can observe that the BMD of the CBD-SDF-1α group was remarkably higher than that of the control group, and it was also significantly greater when compared to that of the NAT-SDF-1α group. We calculated the BMDs of all the groups at 8 weeks after operation and found that the BMDs of all groups were greater than that at 4 weeks. The difference between the experimental groups was insignificant. The bone volume was another parameter used to describe each sample. The bone volume of the CBD-SDF-1α group at 4 weeks after operation was significantly larger than that of the NAT-SDF-1α group for both VOI-1 and VOI-2 (Fig. 7E, F). The bone volume of VOI-1 that is shown in Fig. 7E was significantly different between the CBD-SDF-1α group and the other experimental groups. CBD-SDF-1α-modified DBM Can Enhance the Biomechanical Properties of Regenerated Bone. As shown in Fig. 8A, each bone sample was tested by three-point bending. The rectangle directs the bone loss in the femur. The ultimate force was analyzed for bone samples at 4 weeks, 8 weeks and 12 weeks after implantation, as 15


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shown in Fig. 8B, C and D, respectively. At 4 weeks after implantation, the ultimate force that the bone sample could resist was significantly different. The CBD-SDF-1α group resisted a significantly higher ultimate force than the NAT-SDF-1α group. The difference between the CBD-SDF-1α group and the NAT-SDF-1α group at 8 weeks and 12 weeks after implantation was significant but not extremely so. The data showed that there was almost no difference between the DBM group and the control group during the bone defect recovery.

DISCUSSION Bone is a tissue that possesses the capacity to regenerate in a complex and well-orchestrated physiological manner throughout life. Autologous regeneration may be useless if a bone defect is critical. The traditional treatment of a bone defect is to implant autologous bone or a bone substitute. An autologous bone graft suffers from high morbidity and blood loss at the donor site and requires multiple surgical sites, and there is limited bone available. The substitute graft seems more acceptable but it also has drawbacks including immune rejection and degradation problems. In this study, we constructed a tissue-engineered bone system consisting of DBM scaffolds and CBD-SDF-1α that could recruit the host stem cells to promote endogenous bone regeneration. Compared with previous implantation substitutes, the tissue-engineered bone system provides biological function without the drawbacks. Demineralized bone matrix has been proven to be a successful bone implantation material in which exposed cells and 16



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inorganic mineral are removed. Composed primarily of collagen (93%) 41, DBM is an osteoconductive and osteoinductive biomaterial

10, 42

. As a material for implantation

for bone defects, DBM has been made in many forms. Some researchers only implant DBM powder to induce intrinsic bone regeneration

43

. Therefore, DBM can be

obtained from both allogeneic and xenogenic bone tissue with no construction

32

. In

this study, DBM was made into a poriferous scaffold that provides cells with a suitable microenvironment to adhere and connect

44-45

. DBM scaffolds used as bone

substitutes were investigated further. To study the characteristics of DBM scaffolds, we analyzed their degradation and biomineralization. The degradation of DBM scaffolds in vitro and in vivo was analyzed in our previous studies

20

. The results

showed that cells could promote the degradation of the DBM in vitro the same as in vivo. The DBM scaffolds were also tested with respect to their mechanics to ensure that they had a sustainable hardness. This property highlights that DBM scaffolds could act more bone-like and support the body in a sturdier manner in daily life. The problem that remains in bone implants is how to control the release of the cytokines during implantation in vivo. Good implants in bone defects must have the capacity to hold the medicinal molecules at a sustained concentration before operation, and when implanted, they should release the functional molecules at a slow and controllable rate. In this cell-free tissue bone engineering system, cytokine SDF-1α was modified with a unique peptide from a collagen-binding domain. CBD was investigated in our previous study, and it was used to modify cytokines to help them bind to collagen. Compared with physical adsorption, CBD improves the efficiency of 17


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the binding of cytokines and materials. There are thousands of cells moving throughout the peripheral blood every minute in our body, which makes the peripheral blood the most promising cell reservoir for injured tissue regeneration. Bone regeneration fails when stem cells are not rapidly recruited to an injury, while inflammation causes inflammatory cells to become active46. SDF-1α is a cytokine that can generally mobilize various stem cells in vivo. It has been reported that SDF-1α is chemotactic for mesenchymal stem cells and mediates osteoclastogenesis by inhibiting the suppression of MSCs47. In this study, it was found that DBM scaffolds loaded with CBD-SDF-1α could recruit stem cells to an injury in a short time. Fluorescence images showed that the CBD-SDF-1α groups recruited the hematopoietic stem cells 3 days after operation. On DBM scaffolds, the CBD-SDF-1α groups mobilized many more stem cells to the defect site when compared with the NAT-SDF-1α groups using CD34 and c-kit positive cell staining (Fig. 4C). The CBD-SDF-1α group promoted the stem cells’ movement without delivering cells to the injury and prevented immune rejection. Thus, at the current time, it is a challenge to conduct a controlled release method to mediate the concentration of the medicinal molecule. Based on this work, we could increase the concentration of SDF-1α to engage the recruitment of stem cells to the bone injury so that we could utilize the intrinsic bone regeneration. Notably, from the SDF-1α retention curve, we found that the DBM scaffolds can efficiently maintain the CBD-SDF-1α at a relatively high concentration. The modified SDF-1α does not significantly bind to the DBM scaffolds without chemical crosslinking20. 18



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Bone defects occur with the development of osteoclasts and the accumulation of inflammatory cells,

48

while bone regeneration occurs through a series of steps in

which osteoblasts secrete cytokines and rebuild the bone structure13, 49-50. The images of H&E staining show that the bone defects of the CBD-SDF-1α group have many more osteoblasts and fewer osteoclasts in the early stage after implantation. The images of IHC verified that the positive expression of OPG and OPN in the CBD-SDF-1α groups was significantly higher than that of the NAT-SDF-1α groups and DBM scaffold groups in the primary recovery stage. These data prove that the CBD-SDF-1α loaded DBM scaffolds have the ability to mobilize cells, especially osteoblasts, to the bone defect site. To demonstrate the level of recovery, we stained the OCN and investigated the bone mineral density and bone volume of each bone sample47. The OCN of each experimental group showed a developing expression process, although the CBD-SDF-1α group had the deepest staining. With the accumulation of OCN, the bone defect accumulates more mineral, which will aid in the recovery of bone function. In summary, our study demonstrated that the CBD-SDF-1α-modified DBM could recruit stem cells to the bone defect and promote bone injury regeneration itself. This eliminated the need for cell transplantation by recruiting endogenous cells and inducing the stem cells to differentiate and secrete cytokines. With the controlled release of CBD-SDF-1α, we enhanced the local concentration of cytokines and had less diffusion. Unlike chemical medicine, the use of CBD-SDF-1α-modified DBM opens a new window for bone regeneration with no side effects. Importantly, the 19


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underlying molecular mechanism of CBD-SDF-1α-modified DBM scaffolds in the promotion of bone regeneration is still unknown, and we will continue to study it further. CONCLUSIONS Because endogenous regeneration is generally accepted, the treatment of bone defects was considered to efficiently promote endogenous regeneration51-52. In this study, CBD-SDF-1α-modified DBM scaffolds, which is a novel cell-free bone system, was used for bone regeneration. Sustained release was achieved in CBD-SDF-1α encapsulated DBM scaffolds. When the scaffolds were implanted in femur bone loss defects in rats, it was found that the functionalized scaffolds could recruit endogenous stem cells at a fast rate and promote the intrinsic bone regeneration. The ultimate force data showed that the CBD-SDF-1α group regained hardness in a relatively short time. Additionally, the micro CT results revealed that the CBD-SDF-1α-modified DBM scaffolds help to regenerate more bone-like tissue.

SUPPORTING INFORMATION A further display of bone defect healing after CBD-SDF-1α modified DBM scaffolds inserted in vivo along time as follows, micro CT images of bone defects at 7 days after operation, H&E staining of bone samples at 8 and 12 weeks after operation, Masson’s trichrome stain of bone samples at 8 and 12 weeks after operation, and micro CT images of a live rat at 4 weeks after treatment.

20



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AUTHOR INFORMATION Corresponding Author: Jianwu Dai, PhD., Professor, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, 3 Nanyitiao, Zhongguancun, Beijing 100190, China. 86-010-82614426 (phone/fax) *E-mail: [email protected] Notes The authors declare there is no competing ﬁnancial interest.

ACKNOWLEDGMENTS This work was supported by grants from the Ministry of Science and Technologyof China (Grant Nos. 2011CB965001 and 2014CB965003), NationalScience Foundation of China (Grant Nos. 81200963, 81471276, and 51361130033), and Chinese Academy of Sciences (Grant No.KSCX2-EW-Q-24).

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Table of content Graphic

,

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Fig. 1 DBM scaffolds. (A) Schematic diagram for the construction of a bone substitute from which CBD-SDF1α could be released in a controlled manner (B) Photograph of DBM scaffolds on a macroscopic scale, with dimensions of 1 cm × 1 cm× 1 cm. (C) Scanning electron microscope (SEM) images of DBM scaffolds displaying the well-ordered pores inside. Scale bar, 1 mm. (D) Release curves of CBD-SDF-1α from DBM scaffolds. Data are shown as the means ± SD. *P< 0.05, **P < 0.01 (E) Scanning electron microscope (SEM) images of DBM scaffolds with MSCs inside, which show that MSCs could attach on DBM scaffolds. 151x193mm (300 x 300 DPI)



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Fig. 2 Biomineralization activity of DBM scaffolds. (A) SEM images of calcium phosphate coatings on DBM scaffold. The images were taken from different regions at 1, 2 and 3 hours after coating. The scale bars in the insets are 10 µm. (B) Results of microanalysis report calculations for each DBM scaffold, which showed that the DBM scaffold could induce biomineralization. 156x101mm (300 x 300 DPI)


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Fig. 3 Bioactivities of CBD-SDF-1α and NAT-SDF-1α recruit cells in vitro. (A) Comparison of the bioactivities of CBD-SDF-1α and NAT-SDF-1α with respect to the migratory ability of cells. (B) Quantitative analysis of recruiting stem cells, which showed that there was no significant difference between CBD-SDF-1α and NATSDF-1α groups. 144x105mm (300 x 300 DPI)



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Fig. 4 Femur defect model in a rat in which a CBD-SDF-1α-modified DBM scaffold recruited stem cells in vivo. (A) The location of the operation with a depth of bone defect of 3 mm, ready for the implantation of the CBD-SDF-1α-modified DBM scaffold. (B) Axial, coronal, and sagittal images of the femur defect using micro CT scanning. (C) Immunofluorescence images of the DBM scaffold group, NAT-SDF-1α loaded DBM scaffold group and CBD-SDF-1α-modified DBM scaffold group, which stained positive for CD34 and c-kit 3 days after operation. Scale bar, 50 µm. (D) Quantitative analysis of CD34 and c- kit positive cells. Data are expressed as the means ± SD, n = 5, *P< 0.05. 155x198mm (300 x 300 DPI)


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Fig. 5 H&E and Masson's trichrome staining of bone defect samples. (A) Images of H&E staining (50×). (B) 400× amplification of the bone defect section of the H&E stain. (C) Masson's trichrome stain of each group (50×). The arrow symbol in Fig. 4B shows an osteoclast and the blunt arrow symbol indicates an osteoblast. Scale bar, 100 µm. (D) Quantitative analysis of osteoblasts in each group 4 weeks after operation, with the bone defect group without treatment as a control. (E) Quantitative analysis of osteoclasts in each group 4 weeks after operation to demonstrate the changes in the types of cells. Data are shown as the means ±SD, N=5, *P< 0.05, **P < 0.01, ***P< 0.001. 157x93mm (300 x 300 DPI)



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Fig. 6 Immunohistochemical images of OPN, OPG and OCN staining. (A) Staining of OPN at 4 weeks after operation. (B) OPG-positive staining at 4 weeks after implantation. (C) Expression of OCN at 12 weeks after operation. The arrow symbol shows positive staining. Scale bar, 100 µm. 157x113mm (300 x 300 DPI)


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Fig. 7 Results of micro CT (A, B) Images scanned by micro CT at 4 and 8 weeks after operation displaying the coronal sites of the bone defect. Scale bar, 5 mm. (C, D) Bone mineral density of the areas of interest (VOI-1 and VOI-2) at 4 weeks and 8 weeks after operation were quantitatively analyzed. (E, F) Bone volume of the areas of interest (VOI-1 and VOI-2) at 4 weeks after implantation. Data are shown as the means ±SD, N=5, *P< 0.05, **P < 0.01, ***P< 0.001. 157x225mm (300 x 300 DPI)



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Fig. 8 Three-point bending test showing the functional recovery of the bone defect (A) Schematic of threepoint bending. The rectangle shows the site of the bone defect. (B, C, D) Ultimate analysis of each sample at 4 weeks, 8 weeks and 12 weeks after implantation. Data are shown as the means ±SD, N=5, *P< 0.05, **P< 0.01. 140x146mm (300 x 300 DPI)


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Demineralized Bone Matrix Scaffolds Modified by CBD-SDF-1Î±

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