Endothelial and Osteoblast Differentiation of Adipose-Derived

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Characterization, Synthesis, and Modifications

Endothelial and Osteoblast Differentiation of Adipose-derived Mesenchymal Stem Cells Using a Cobalt-doped CaP/Silk Fibroin Scaffold nesa fani, Mehdi Farokhi, mahmoud azami, amir kamali, Nasrin Lotfi Bakhshaiesh, somayeh ebrahimi-barough, jafar ai, and Mohamadreza Baghaban Eslaminejad ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b01372 • Publication Date (Web): 11 Apr 2019 Downloaded from http://pubs.acs.org on April 12, 2019

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ACS Biomaterials Science & Engineering

Endothelial and Osteoblast Differentiation of Adipose-derived Mesenchymal Stem Cells Using a Cobalt-doped CaP/Silk Fibroin Scaffold Nesa Fania,b, Mehdi Farokhic, Mahmoud Azamia, Amir Kamalid, Nasrin Lotfi Bakhshaiesha, Somayeh Ebrahimi-Barougha, Jafar Aia*, Mohamadreza B. Eslaminejadb* a. Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran b. Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, 1665659911, ACECR, Tehran, Iran c. National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran d. Department of Pathology, School of Veterinary Medicine, Shiraz University, Shiraz, Iran

Corresponding authors: Dr Ai, Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran. Tel: +9821- 43052110, E-mail address: [email protected] Dr Mohamadreza B. Eslaminejad, Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, 1665659911, ACECR, Tehran, Iran. Tel: +9821-23562524, E-mail address: [email protected]

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Abstract A major problem in the treatment of large bone defects is the inability to provide an adequate blood supply to the implantation site. Therefore, a bone regeneration strategy that provides an adequate supply of vessels would address this need. Cobalt (Co2+), due to its ability to induce hypoxia, has been used to accelerate new vessel formation. In this study, we used a freeze-drying technique to fabricate a scaffold that consisted of Co2+-doped calcium phosphate (CaP) [e.g., hydroxyapatite (HA)] and natural silk fiber through an optimized alternate mineralization process. The composition and structure of the scaffold were confirmed by X-ray diffraction (XRD), Fourier transform infrared (FTIR), inductively coupled plasma (ICP), and scanning electron microscope (SEM). The data showed that the scaffolds promoted differentiation of adipose-derived mesenchymal stem cells (ADSCs) toward endothelial and osteoblast linages. We observed improved angiogenesis and bone formation with the fabricated scaffolds compared to the control groups. Computed tomography (CT) scans and radiographic imaging, in addition to histology and immunohistochemical analyses showed the presence of angiogenesis and bone regeneration after implantation of the ADSC-seeded scaffolds in a critical size calavarial bone defect in a Wistar rat model. We obtained the best in vitro and in vivo results by doping 2% Co2+ in HA. Taken together, we propose that the Co2+-doped HA/silk fibroin (SF) scaffold would be a good candidate to induce angiogenesis and bone formation both in vitro and in vivo. Keywords: Cobalt, Silk fibroin, Angiogenesis, Osteogenesis, Bone Tissue Engineering 1. Introduction The reconstruction of critical size skeletal defects is a major orthopedic challenge. Autologous trabecular bone is widely considered as the standard for bone reconstruction. However, insufficient


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donor tissue, in conjunction with donor site morbidity, limit this process for large-scale applications 1. Allografts and xenografts are also problematic because of immune rejection and the risk of disease transmission 2-4. These limitations have encouraged researchers to develop suitable synthetic or natural grafting materials that could be used as bone substitutes for reconstruction of bone defects

4-6.

Bone scaffolds offer a promising strategy for healing bones because of their

osteoconductivity, limitless source, long shelf-life, and decreased risk of diseases or infections 7. Among various scaffold fabrication methods, biomimicry is a technique that synthesizes substrates which are similar to the natural extracellular matrix (ECM) of the defect tissues 8-9. Until recently, biomimicry has proven to be a good strategy for bone augmentation. Most are based on calcium phosphates (CaP), such as hydroxyapatite (HA), which is the main mineral component of bone 1011.

Bone matrix also consists of collagen type 1 (Col1), which acts as a template for bone

mineralization

12.

It may be possible to replace the organic phase of bone matrix (Col1) by a

polymer that, despite providing complete bone architecture, has an off-the-shelf character with the advantages of decreased cost. Silk fibroin (SF) is a favorable matrix for tissue engineering because of its suitable biocompatibility, good cell adhesion, and low immunogenicity

13-14.

The

combination of inorganic material based on CaP within SF based constructs could positively affect the biological behaviors of osteoblasts 15-17. After implantation, the new bone tissue needs an active blood supply because highly vascularized tissues are essential for successful clinical application of engineered bone grafts 18-19. Methods that may improve the angiogenic potential of biomaterials include seeding scaffolds with endothelial cells, in vitro prevascularization of matrices, and incorporation of angiogenic factors in fabricated substitutes 20-21.



Although angiogenic growth factors are suitable for in vitro and in vivo promotion of angiogenesis, disadvantages include increased cost, short half-life, and risk of tumor formation, which restricts their clinical use

22-23.

Therefore, alternative strategies are needed. A number of studies have

introduced ions that have the ability to promote angiogenesis 24-25. Cobalt ions (Co2+) have been used extensively to mimic a hypoxic environment in vitro that can stabilize hypoxia-inducible factor-1 (HIF-1α) and consequently activate vascular endothelial growth factor (VEGF) 26-27. Co2+ can be used as an inorganic angiogenic factor because of its increased greater stability, potentially superior safety in comparison with recombinant proteins or genetic engineering approaches, and decreased cost. Bone tissue scaffolds that have controllable Co2+ release are reported to enhance VEGF secretion, HIF-1α expression and bone-related gene expressions in mesenchymal stem cells (MSCs) 28-30. The incorporation of Co2+ into a biomimetic construct of CaP/SF may be an efficient way to expand angiogenesis for improving bone repair. The regeneration capability of tissueengineered constructs can be improved by either optimizing the biomaterial composition and properties, or by combining appropriate cells

31.

Adipose-derived mesenchymal stem cells

(ADSCs) are multipotent cells that have the capability to differentiate into osteocytes, adipocytes, vascular endothelial cells, and neural cells. Compared with other stem cells, procuring ADSCs requires minimally invasive techniques. Therefore, ADSCs are a useful resource for regenerative medicine applications 32-34. In this study, we mineralized silk fiber using Co2+-doped CaP and subsequently immersed the fibers in SF solution to fabricate a composite (SF/F/CaP-Co). We used various analytical techniques to evaluate the structure, composition, and biological properties of this scaffold both in vitro and in vivo.


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2. Materials and Methods 2.1. Materials and chemicals Sodium carbonate, lithium bromide, dialysis tube with a 3500 Da cutoff, Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), ascorbic 2-phosphate, penicillin, streptomycin, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma–Aldrich, USA. The phosphate-buffered saline (PBS) tablets were purchased from VWR (Radnor, USA). All antibodies and the Alkaline Phosphatase Kit were purchased from Abcam (Canada). The Calcium Colorimetric Assay Kit was purchased from Biovision (USA). All other reagents used in the cell culture experiments were purchased from Life Technologies (USA). Freshly prepared ultrapure demineralized water (Milli-Q System, Millipore, Billerica, MA, USA) was used for the preparation of the solutions. 2.2. Silk Fibroin/Fiber/CaP scaffold preparation According to our biomimicry concept, we designed a scaffold composed of mineralized fibers distributed within a polymeric matric. For this aim, we first extracted silk fibers, then mineralized them and finally added to a fibroin matric. In the next sections, each of these steps will be described in details. 2.2.1. Extraction and treatment of silk fibers The natural silk fibers were cut into lengths of 3-4 mm, degummed in 0.05% (w/v) boiled sodium carbonate for 60-70 min to remove the sericin, and then vacuum-dried at 40◦C for one day 35. In order to produce the SF solution, which is supposed to be used as the matric in the final scaffold, some parts of the degummed silk fibers were further processed. For this aim, these fibers were



soaked in 9.3 M LiBr at 70◦C for 4 h. Then, the solution was dialyzed against deionized water in a dialysis tube for 72 h to obtain a 5% (w/v) SF solution, which was further purified by centrifugation at 4000 rpm for 10 min. At this step, a fibroin solution was obtained and maintained at fridge for being used later as the scaffold matric. 2.2.2. Mineralization of silk fibers At this step, obtained silk fibers at the previous step were mineralized. For this aim, we used an optimized alternate soaking in 1M CaCl2.2H2O and 600 mM Na2HPO4 solutions, reported as the optimum concentration previously by Luickx et al.36. In this work, time of soaking (5-30 min), number of cycles (3-12) and sonicate washing time (0-60 sec) (Transsonic T700/H, Elma Ultrasonic, Ruiselede, Belgium) as shown in S1, were considered as variables to reach the best condition for mineralization. We assumed the best mineralization for the fibers covered homogeneously with a thin layer of minerals without having discrete minerals trapped between the fibers. We weighed the fibers before and after mineralization. To characterize the obtained fibers and quality of mineralization and choosing the best condition for fiber mineralization, we used scanning electron microscope (SEM) to assess the fiber morphology. All experiments were conducted in triplicate at room temperature to evaluate the reproducibility of the procedure. CaP coated silk fibers were assessed by recording X-ray diffraction (XRD) patterns with an X’pertproMPD (PANalytical) with X’Celerator counters. Fourier transform infrared (FTIR; Germany) spectrophotometer was also performed in the range of 650–4000 cm-1 with a spectral resolution of 4 cm-1 according to the KBr disk technique. 2.2.3. Final composite scaffold preparation


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To fabricate the final scaffolds, selected optimized mineralized fibers were added to the fibroin solution obtained at the extraction step of silk fibers. Then, the mixture was transferred into an 8 mm diameter cylindrical tube and freeze-dried (Christ, Alpha 1-2 LD, Germany). Next, the samples were cross-linked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) in ethanol 37. After washing the crosslinked samples with distilled water, the substrates were cut into sections of 2 mm height and 8 mm diameter. The fabricated porous scaffolds were named SF/F/CaP scaffolds. The control scaffold (SF/F scaffold) that contained silk fibers without CaP was prepared by the same method. The pure SF scaffold (without fibers) was prepared as an extra control scaffold and referred to as “SF”. To find the best percentage of calcium phosphate within the scaffold, different amount of mineralized fiber were weighed and added to fibroin solution and adjusted to have a series of mineral content weight percent from 5 to 60. The selection was done according to the results obtained from MTT, ALP and Calcium assay. Supporting information file S3 shows the obtained results. Based on these results, we selected the scaffold containing 30 wt% calcium phosphate content of the whole scaffold mass. To induce angiogenesis potential within the scaffold, Co2+ ions was doped inside the crystal structure of depositing mineral on the silk fibers as a substitution for Ca2+ ions. For this aim, different concentrations of Co2+ ions were added to the calcium solution during precipitation as described above. Table 1 shows the theoretical Co/Ca ratio applied for mineralization as well as experimental obtained doped Co/Ca ratio at the final mineral. To find the most appropriate Co/Ca, MTT assay was done. Selected SF/F/CaP-Co scaffold with the best Co/Ca ratio was characterized further as will be described below.



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2.3. Characterization of the SF/F/CaP-Co scaffold We performed XRD patterns and FTIR to estimate the components of the scaffolds that contained different concentrations of Co2+. Inductively coupled plasma-atomic emission spectrometry (ICPAES: Liberty 200, Varian, Clayton South, Australia) was applied to determine the amount of Co2+, Ca2+, PO4 3- ions in the mineralized fibers (Table 1). The release pattern of ions from the SF/F/CaPCo scaffold were obtained by soaking 900 mg samples in 50 mL culture media (DMEM) at 0.5, 1, 3, and 7 days (S4). The solutions were also analyzed using ICP-AES to define the ion concentrations in the media. Table 1. Chemical composition of calcium phosphate (CaP) before and after substitution with various concentrations of cobalt ion (Co2+).

Sample Name Ceramic components

SF/F/CaP

SF/F/CaP-Co (2%)

SF/F/CaP-Co (4%)

SF/F/CaP-Co (6%)

Ca 10 (PO4)6OH2

Ca 9.6 Co 0.4 (PO4)6OH2

Ca 9.2 Co 0.8 (PO4)6OH2

Ca 8.8 Co 1.2 (PO4)6OH2

Theoretical values

Co/Ca

0

0.0412

0.0868

0.1351

experimental values (mM)

Ca Co Co/Ca

24.28±5.6 0 0

28.2575±6.4 0.7591±0.05 0.0268

29.8387±6.6 1.4317±0.1 0.0479

30.1073±7.3 1.873±0.6 0.06221

65

55

46

Substitution percent

Co/Ca( experimental ) Co/Ca(theoretical)

-

The surface and internal 3D architecture of the SF/F/CaP-Co scaffolds were examined by SEM (1540XB Crossbeam VR SEM, Zeiss). The mechanical properties of the scaffolds were also assessed. By using a Micromeritics AutoPore Mercury Intrusion Porosimeter 1100, the porosity measurements were obtained. Samples were sealed in penetrometers, weighed, and subjected to analysis.


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2.4. Biological characterization of the SF/F/CaP-Co scaffold 2.4.1. Viability assay We used the MTT assay to measure viability and proliferation rate of the ADSCs after 1, 3, and 7 days. The indirect MTT assay using extract solution was considered for cell viability and carried out according to the ISO 10993-5 States 21.We incubated SF/F, SF/F/CaP, and SF/F/CaP-Co in the appropriate culture media at a concentration of 10 mg/ml for each scaffold to achieve the extract solution. Culture media that did not contain any material were incubated at the same time points as the control group [tissue culture polystyrene (TPS)]. Approximately 3×104 ADSCs cells were incubated in 48 wells at 37°C. After a 24 h incubation period, we removed the cell culture media and replaced it with extract media for another 24 h. The cells were then stained with the MTT solution (5 mg/ml) for 2- 4 h at 37°C in a humidified atmosphere of 5% CO2. The viable cells metabolized the yellow tetrazolium salt and changed them to purple formazan crystals, which solvable in dimethyl sulfoxide solution (DMSO). Finally, optical density (OD) by using a spectrophotometer (Synergy HT, BioTek, USA) was measured at a 570 nm wavelength. 2.4.2. Cell attachment and morphology culture on scaffold ADSCs (1×106 cells/scaffold) were seeded on the surfaces of the scaffolds and incubated. We checked their morphology at day-7 post-seeding. The samples were fixed in 2.5% glutaraldehyde and dehydrated in increasing ethanol diluted concentrations of ethanol (20%, 40%, 60%, 80%, and 100%) followed by processing with osmium tetroxide. Finally, the samples were gold-coated under vacuum and prepared for SEM analysis.



2.5. In vitro osteogenic and angiogenic induction of adipose-derived mesenchymal stem cells (ADSCs) We evaluated the osteogenic and angiogenic potential of the SF/F/CaP-Co (2%) at 7 and 21 days. ADSCs were seeded onto the SF/F, SF/F/CaP, and SF/F/CaP-Co (2%) scaffolds. The cell-seeded scaffolds and the cells cultured in TPS were incubated in culture media (DMEM).

2.5.1. Alkaline phosphatase (ALP) activity We assessed the SF/F, SF/F/CaP and SF/F/CaP-Co (2%) scaffolds for their ability to support osteogenic cell differentiation during the in vitro studies. Briefly, ADSCs were seeded onto the scaffolds. The ALP assay (Colorimetric Alkaline Phosphatase Assay Kit, Abcam) was performed at 5, 10, and 15 days according to the manufacturer’s instructions. Total protein content was also determined by the BCA Protein Assay method (Merck Millipore). ALP levels were normalized to the total protein content at the end of the experiment.

2.5.2. Calcium (Ca2+) content assay We measured the amount of Ca2+ deposited on the SF/F, SF/F/CaP, and SF/F/CaP-Co (2%) scaffolds during osteogenic differentiation of the ADSCs. The ADSCs were seeded on the scaffolds in a 24-well tissue culture plate. After 5, 10, and 15 days of incubation, we added 0.5 N HCl to the scaffolds seeded with ADSCs. We used the supernatant to determine the amount of Ca2+ according to the Calcium Colorimetric Assay Kit (BioVision, Inc.). OD was determined with a microplate reader at a wavelength of 575 nm.

2.5.3. Quantitative real-time PCR (qRT-PCR)


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We performed quantitative real-time PCR (qRT-PCR) analyses of osteogenic [Col1, Runx2, and osteocalcin (OCN)] and angiogenic (VEGFα, CD31, and CD133) marker expression levels. Briefly, we extracted total RNA from the cells by using TRI Reagent® (Sigma-Aldrich, T9424). cDNA was produced by the Revert Aid First Strand cDNA Synthesis Kit (Fermentas, K1632) In accordance with the manufacturer's instructions.. To normalize the expression level of the target genes, the B2M selected as the reference gene. Analysis was performed by the comparative 2− (ΔΔCT)

method with B2M as reference. The primers of osteoblast and endothelial markers were

showed in S7.

2.5.4. Immunofluorescence analysis Initially, the ADSCs were seeded onto the SF/F/CaP and SF/F/CaP-Co (2%) scaffolds for 7 and 14 days. Next, the samples were fixed in 10% neutral buffered formalin (NBF, pH 7.26) for 48 h and embedded in paraffin. We prepared 6 µm thick sections of the samples and rinsed them in PBS for 5 min at room temperature. Primary VEGF, CD31, Col I and OCN antibodies were added to the samples at 1:100 dilutions. The samples were incubated with the AlexaFluor goat anti-rabbit conjugated secondary antibody at a 1:150 dilution in PBS for 1 h in the dark. The samples were washed 4 times and the stained with 4′, 6-diamidino-2-phenylindole (DAPI) for visualization of the nuclei. The samples were observed with an Olympus fluorescent microscope.

2.5.5. In vitro angiogenesis We used ECMatrix (Millipore, cat. no. ECM625) to evaluate in vitro angiogenesis. Briefly, the ADSCs were cultured in extract solution of the SF/F/CaP-Co (2%) scaffolds for 4, 8, and 12 h.



Same amount of cells cultured in Endothelial Cell Growth Basal Medium-2 (EBM-2) (Lonza, USA.) as positive group, and Same amount of cells cultured in DMEM as negative group. Tube formation by the samples was evaluated according to the manufacturer’s protocol. 2.6. In vivo study 2.6.1. Surgical and post-surgical procedures Adult male Wistar rats (n=6 per group) that weighed between 200 g and 250 g were anesthetized by intramuscular (IM) injections of 20 mg/kg ketamine hydrochloride and 2 mg/kg xylazine (both from Alfasan, Holland). The surgical area (calvaria) was shaved, washed, and disinfected with Betadine and 70% alcohol. One critical-size circular defect (8 mm diameter ×1 mm thick) for each of the scaffolds was created in the parietal bones of the skull. Scaffolds were implanted into the defect and divided into 5 experimental groups: (1) SF/F/CaP-Co (2%) scaffold with ADSC, (2) SF/F/CaP-Co (2%) scaffold without ADSC, (3) SF/F/CaP scaffold with ADSC, (4) SF/F/CaP scaffold without ADSC, and (5) negative control (blank), which was a defect without any construct. All animals received proper care in accordance with the Guide for Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1985). All procedures that involved the use of animals were done in compliance with the strategies of the Institutional Animal Care and Use Committee, Royan Institute, Tehran, Iran. We evaluated the lesions 12 weeks after implantation by computed tomography (CT) scan (Siemens Healthcare, Inc., PA, USA). The defect sites were scanned at multiple longitudinal and crosswise sections with 60 μm thicknesses. The 3D images of the new bone formation and gross profiles of the samples were prepared by Inveon Research Workplace software (Siemens


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Healthcare USA, Inc., PA, USA). The rate of gap (defect) closure (%) of the samples was calculated by the prepared images. 12 weeks post-surgery the animals were euthanized and the harvested tissues (implant sites) were decalcified in 19% EDTA and embedded in paraffin. We prepared 5 mm thick sections of these samples and stained them with hematoxylin and eosin (H&E) and Masson's trichrome (MT). The amount of the newly formed bone and the amount of implants that remained in the total area of the section were assessed. Inflammation was also assessed in the different scaffolds. Histomorphometric analysis of the numbers of cells (fibrocytes, fibroblasts, osteoblasts, osteons, osteoclasts, osteocytes) and other constituents such as blood vessels and new bone tissue formation were assessed by computer software Image-Pro Plus® V.6 (Media Cybernetics, Inc., Silver Spring, MD, USA). 2.6.2. Immunohistochemical analysis The sections from the defect site were prepared and analyzed for primary antibody expressions of anti-VEGF receptor 2 (KDR) and OCN. The slides were treated by citrate buffer (Dako, Glostrup, Denmark) solution at 60°C for heat-induced epitope retrieval (HIER). The slides were then blocked with 1% hydrogen peroxide/methanol for 30 min at room temperature, followed by an overnight incubation with primary antibodies at 4°C. By 3, 3′-diaminobenzidine (Dako liquid DAB color solution) the color reaction was improved and the slides were counterstained with hematoxylin. 2.7. Statistical analysis



All results were compared using one-way ANOVA. P-values less than 0.05 were considered statistically significant. Statistical analyses were performed using SPSS 18.0 software (Chicago, IL, USA). All experiments were performed in triplicate and all data were written as mean ± standard error. 3. Results 3.1. Characterization of the physical and chemical composition of the mineralized silk fibers Figure 1, (S1 and S2) provide summaries of the conditions used to obtain the optimized silk fiber/CaP. We determined that in order to fabricate an appropriate porous silk fiber/CaP based scaffold, a suitable fiber mineralization protocol that had the capability to provide the greatest weight uptake after mineralization, the shortest time period, and the ability to apply homogenous CaP coating without any excess CaP between the fibers would be needed. Figure 1A shows the best condition for preparation of the optimized CaP coated silk fibers. S1 shows the best sample (sample7). Figure 1B shows the best mineralized fiber morphologies under optimized conditions. The morphology of S7, as the best sample, is presented in Figure 1B. The XRD patterns of the silk fiber and silk fiber/CaP are shown in Figure 1C. The XRD outline of the silk fiber/CaP showed two main diffraction peaks at 2ϴ = 26◦ and 32◦. It showed that the applied CaP ratio had a reflective effect on phase structure and crystallinity. Therefore, the XRD pattern confirmed the existence of the HA phase 38. Figure 1D shows the typical peaks of SF/F at 1630 cm−1 (amide I), 1527 cm−1 (amide II), and 1230 cm−1 (amide III)

39,

and split bands that appeared in the silk fiber/CaP at

approximately 560 and 603 cm−1, which was consistent with apatite formation. The results showed that the original silk structure was preserved despite the formation of HA.


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Figure 1. Optimized mineralization of natural silk fibers. Schematic presentation of calcium phosphate (CaP) coating process on the fibers (A). Scanning electron microscope (SEM) images of fibers with proper coverage without the growth of CaP sediment between the fibers (B). X-ray diffraction (XRD) (C). Fourier transforms infrared (FTIR) analysis of the mineralized fibers (D).



3.2. SF/F/CaP scaffold preparation We added the optimized CaP coated silk fibers with different concentrations of Ca2+/total weight of scaffold to a 5% (w/v) SF solution. The supplementary files (S3) show biocompatibility and bioactivity analyses of the SF/F/CaP scaffold. Based on the biocompatibility assay, we selected 30% SF/F/CaP scaffolds for doping with different concentrations of Co2+. 3.3. Characterization of the SF/F/CaP-Co scaffold Table 1 summaries the theoretical values of the elemental compositions of the coatings and experimental values detected by ICP. The results showed that increasing the Co2+ content caused a reduction in the amount of Co2+ doping. We evaluated the morphology of the freeze-dried SF/F/CaP-Co by SEM (Fig. 2A). The S/F/CaPCo microstructure was extremely porous, and considered to be suitable for nutrient and oxygen transport. Figure 2B shows the XRD patterns for SF/F/CaP-Co (2%), SF/F/CaP-Co (4%), and SF/F/CaP-Co (6%). All coatings were predominantly HA (indicated). The Co2+ incorporation led to a slight change in the phase compositions in the SF/F/CaP-Co (6%) group. However, no distinctive peaks indicating another Co2+ based compounds were detected in the whole obtained diffractograms38. Figure 2C shows the pore size distribution curve where the curved line over the histogram indicates normal distribution. The mean pore diameter was 92 µm with an approximate porosity value of 70%. Figure 2D shows the mechanical properties of the SF/F/CaP-Co, SF/F, and SF scaffolds. There was a significant difference in compressive Young’s modulus (E) of SF/F/CaP-Co scaffold and SF/F scaffold compared with SF scaffold. It seems that the presence of fibers in the structure of scaffolds caused increase the amount of E. There was no significant difference in the yield


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strengths (σy) of the samples. On the other hand, Ɛy (axial elastic deformation) and Ɛf (strain at fracture) meaningfully increased in the SF/F/CaP-Co scaffold compared with the other scaffolds. These results generally showed high elasticity in the SF/F/CaP-Co scaffold, which would be suitable for use in non-load bearing bone defects.

Figure 2. Macroscopic image and scanning electron microscope (SEM) micrographs of the SF/F/CaP-Co scaffold (A). X-ray diffraction (XRD) spectra of SF/F/CaP-Co scaffolds in different



concentrations of cobalt (Co2+) (B). Pore size distribution histogram of the SF/F/CaP-Co(2%) scaffold (C). Compressive strain/stress curve and mechanical properties of the SF/F/CaP-Co (2%), SF/F, and SF scaffolds (D).

3.4 Cell behavior study Figure 3A shows the results of the MTT assay for ADSCs in extract solutions of freeze-dried SF/F/CaP-Co scaffolds that contained 0%, 2%, 4%, and 6% Co2+. The ADSCs had higher proliferation rates in the extracts of the SF/F/CaP-Co scaffolds with 0%, 2%, and 4% Co2+ on the third day compared with the scaffold plus 6% Co2+ (P≤0.05). The scaffolds with 4% and 6% Co2+ significantly reduced cell proliferation after 7 days. According to these results, we selected the SF/F/CaP-Co scaffolds with 0% Co 2+ and 2% Co2+ for further analysis. Figure 4B-D show SEM images of the ADSCs grown on the SF/F/CaP-Co scaffolds with 2%, 4%, and 6% Co2+. Alignment of the cells along the fibers was apparent.


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Figure 3. Cytocompatibility evaluation of the SF/F/CaP-Co scaffolds with different concentrations of cobalt (Co2+) using isolated adipose-derived mesenchymal stem cells (ADSCs). Graph of the cytotoxicity assay (MTT assay) (A). Scanning electron microscope (SEM) micrographs of the scaffolds that contained 2% (B), 4% (C), and 6% Co2+ (D) seeded with ADSCs for 7 days. . 3.5. Osteogenic induction in vitro

3.5.1. Quantitative real-time PCR (qRT-PCR) analysis We cultured ADSCs on SF/F and SF/F/CaP scaffolds, in addition to the SF/F/CaP-Co scaffold with 2% Co2+ in order to evaluate the expressions of the osteogenic and angiogenic associated genes. qRT-PCR revealed that the early phase of the osteogenesis-related gene (RUNX2) overexpressed in the cells cultured in all of the scaffolds in comparison with the control groups after 7 days. On the other hand, the late phase osteogenesis-related genes (OPN and OCN) over-expressed in cells cultured on the SF/F/CaP and SF/F/CaP-Co scaffolds in comparison with the other groups after 21 days (Fig. 4A).

3.5.2. Immunofluorescence analysis Figure 4B shows the immunostaining results of osteoblast markers (OCN and COL1) expressed by ADSCs cultured on SF/F/CaP scaffolds with 2% Co2+ and without Co2+ (0%).



The red color indicates expressions of the OCN and COL1 proteins, whereas the blue color is related to the cell nucleus. The osteoblast target marker over-expressed in both the SF/F/CaP and SF/F/CaP-Co groups after 14 days. Figure 4C, D show graphs that represent quantitative analysis of immunostaining at days 7 and 14 post-incubation times.

3.5.3. Alkaline phosphatase (ALP) activity and calcium (Ca2+) content We measured ALP activity, as an early indicator for osteogenic differentiation, after 7 and 14 days of incubation. Figure 4E shows ALP production of ADSCs grown on the freeze-dried SF/F, SF/F/CaP, and SF/F/CaP-Co (2%) scaffolds. The ADSCs produced more ALP in all samples compared with the control group 7 days after culture. It showed the cells on SF/F scaffold significantly produced the most ALP in day 7. However, ALP activity decreased in all test groups on day 14 compared with day 7. It shows that all groups (except TCP) enter the osteogenic differentiation after two weeks. We also estimated the Ca2+ deposition of ADSCs seeded on freeze-dried SF/F, SF/F/CaP, and SF/F/CaP-Co scaffolds on days 7 and 14 (Fig. 4F). The ADSCs cultured on scaffolds showed a higher level of Ca 2+ after 7 days in comparison with ADSCs cultured on tissue culture plates. After 14 days, the cells that grew on the scaffold with CaP had more released Ca2+ in comparison with the SF/F scaffold and tissue culture plate. The presence of Co2+ did not significantly affect deposition in the cells (P≤0.05).


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Figure 4. Osteogenic activity in vitro. Osteogenic gene [RUNX2, osteocalcin (OCN), OPN] expressions of adipose-derived mesenchymal stem cells (ADSCs) grown in tissue culture polystyrene (TPS) and the SF/F, SF/F/CaP and SF/F/CaP-Co (2%) scaffolds. Data represent the fold-change in comparison with the expression levels of un-induced ADSCs (A). Immunofluorescence analysis for 2 protein markers, OCN and collagen type 1 (Col1), at 7- and 14-days post-incubation of ADSCs seeded on SF/F/CaP (- Co) and SF/F/CaP-Co (2%) (+ Co) in vitro (B). Expressions of OCN and Col1 were detected through FITC-conjugated polyclonal antibodies (red color). The total cell nuclei present on the scaffold were identified by DAPI staining (blue color). Scale bar: 200 µm. The graphs represent quantitative analysis of the immunofluorescence-stained slides at 7 days (C) and 14 days (D) after incubation. Histogram shows alkaline phosphatase (ALP) activity (E) and calcium (Ca2+) content (F) in the test groups. (*) Shows there is a statistically significant difference between the marked bar and the control (cells cultured with standard cell culture media, with no scaffold) in the same time period. (#) Shows there is a statistically significant difference between marked group and other test groups in the same time period. Values are mean ±SD (n = 3). P

Endothelial and Osteoblast Differentiation of Adipose-Derived

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