Off-the-shelf Biomimetic Graphene Oxide-Collagen Hybrid Scaffolds

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Applications of Polymer, Composite, and Coating Materials

Off-the-shelf Biomimetic Graphene Oxide-Collagen Hybrid Scaffolds Wrapped with Osteoinductive ECM for the Repair of Cranial Defects in Rat Shaokai Liu, Shan Mou, Chuchao Zhou, Liang Guo, Aimei Zhong, Jie Yang, Quan Yuan, Jiecong Wang, Jiaming Sun, and Zhenxing Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b11071 • Publication Date (Web): 13 Nov 2018 Downloaded from http://pubs.acs.org on November 13, 2018

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ACS Applied Materials & Interfaces

Off-the-shelf Biomimetic Graphene Oxide-Collagen Hybrid Scaffolds Wrapped with Osteoinductive ECM for the Repair of Cranial Defects in Rat Shaokai Liu†, 1, Shan Mou†, 1, Chuchao Zhou†, Liang Guo†, Aimei Zhong†, Jie Yang†, Quan Yuan†, Jiecong Wang†, Jiaming Sun†, *, Zhenxing Wang†, *

†

Department of Plastic Surgery, Union Hospital, Tongji Medical College, Huazhong

University of Science and Technology, 1277 Jiefang Avenue, Wuhan, 430022, China.

1 These authors contributed equally to this work. * Corresponding authors

Corresponding authors’ E-mail addresses: *Zhenxing Wang: +86-027-85726114. E-mail: [email protected] *Jiaming Sun: +86-027-85726114. E-mail: [email protected]

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Abstract Hydrogels such as type I collagen have been widely studied in bone tissue repair, whereas their weak mechanical strength has limited their clinical application. By adding graphene oxide (GO) nanosheets, researchers have successfully improved the mechanical properties and biocompatibility of the hydrogels. However, for large bone defects, the osteoinductive and cell adhesion ability of the GO hybrid hydrogels need to be improved. Mesenchymal stem cells-secreted extracellular matrix (CDM), which is an intricate network, could provide a biomimetic microenvironment and functional molecules that enhance the cell proliferation and survival rate. In order to synergize the advantages of MSC-CDM with GO-collagen hybrid implants, we developed a novel CDM-scaffold construction method. Firstly, an osteoinductive extracellular matrix (OiECM) was created by culturing osteo-differentiated bone marrow mesenchymal stem cells (BMSC) for 21 days. Then the GO-collagen scaffold (GO-COL) was fully wrapped with the OiECM to construct the OiECM-GO-COL composite for implantation. The morphology, physical properties, biocompatibility, and osteogenic performance of the OiECM-GO-COL implants were assessed in vitro and in vivo (5 mm rat cranial defect model) respectively. Both gene expression and cell level assessments suggested that the BMSCs cultured on OiECM-GO-COL implants had a higher proliferation rate and osteogenic ability compared to the COL or GO-COL groups. In vivo results showed that the OiECM-GO-COL implants achieved better repair effects in rat critical cranial defect model, while bone formation in other groups


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was limited. This study provides a promising strategy which greatly improves the osteogenic ability and biocompatibility of the GO hydrogels without the procedure of seeding and culturing MSCs on scaffolds in vitro, demonstrating its potential as an off-the-shelf method for bone tissue engineering.

Keywords: graphene oxide; extracellular matrix; bone marrow mesenchymal stem cells; bone tissue engineering; rat cranial defect



1. Introduction Large bone defects resulting from tumor and accidents presents an increasing clinical burden globally. It has been recognized that over 10% of the bone fractures reported in the United States are complicated by non-union fractures1. However, the process of repairing large bone defects is faced with major challenges in the field of orthopedic and regenerative medicine2, 3.

Mesenchymal stem cells based BTE treatments have contributed significantly to clinical therapy, especially in for large-sized bone defects4. However, the therapeutic effect of BTE mainly relies on the properties of the biodegradable scaff old and the regulation on the cells’ behavior5. Generally, an ideal scaff old for BTE should have similar mechanical properties, biocompatibility, and osteo-inductivity to the bone extracellular matrix, promoting adhesion and migration of the seeded cells, and osteo-differentiation6, 7.

Compared with synthetic polymers that might be gradually degraded to acid products, natural hydrogels such as collagen, chitosan, and hyaluronic acid are preferred and have been widely studied and clinically used for BTE because of their favorable bioactivity and biocompatibility8-10. Nevertheless, these nature polymers could not satisfy the entire needs for the repair of segmental bone defects repair because of their insufficient mechanical properties, which makes them deformed when being inserted into the bone defect area11-13.


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In recent years, graphene oxide (GO) has been receiving growing research attention from biomaterial scientists due to its biocompatibility and biodegradation properties14, 15

. GO is chemically exfoliated from bulk graphite and it is the highly oxidized form of

graphene. Addition of GO nanosheets may improve the chemical functionality and mechanical strength of the natural hydrogels16. For instance, gelatin nanocomposite hydrogels incorporated with GO exhibited stronger mechanical properties of up to 288% in compressive strength17. However, when used as BTE scaffolds, simple GO hydrogels still possess drawbacks such as compromised osteoconductivity and biocompatibility which decreases its orthotopic reparative effects in vivo18-20.

The extracellular matrix (ECM) is composed of numerous cell-secreted growth factors and basic structures similar to tissue-specific microenvironments21. These functional molecules and proteins (such as type I collagen, fibronectin, and glycosaminoglycan) could benefit cell proliferation, adhesion, and differentiation in vitro for the reason that it provides a biomimetic microenvironment22, 23. According to previous studies, mesenchymal stem cells(MSC) continuously secrete ECM and release many bioactive substances during in vitro proliferation and differentiation. These substances can be harvested and they were found to enhance cell persistence and implant vascularization in bone tissue engineering24,

25

. Therefore, research on the application of the

decellularized MSC-ECM in two and three dimensions has been performed to improve the biocompatibility and bioactivity of these biomaterials26.



In this study, to improve the osteo-inductivity and osteo-conductivity of the GO hybrid polymers, we developed a novel construction strategy based on the osteo-inductive extracellular matrix (OiECM) which was prepared from the osteo-differentiated BMSCs. As shown in Fig. 1, the OiECM was prepared and wrapped around the GO-collagen scaffold (GO-COL) in a petri dish to construct the OiECM-GO-COL biomimetic scaffolds. The morphology of composite scaffolds was analyzed by Micro-CT and SEM techniques. MSCs were seeded and cultured on the scaffolds to evaluate its biocompatibility and osteoinductivity. Finally, the constructed grafts were implanted into 5 mm critical-sized rat skull defects for orthotopic bone repair.

Figure 1. Experimental design. Osteoinductive extracellular matrix (OiECM) were prepared by decellularization of the BMSCs after in vitro osteogenic differentiation for 21 days. Then, GO nanosheets and type I collagen was mixed and crosslinked by EDC/NHS to form the GO-COL scaffolds. Thereafter, the OiECM membrane and the GO-COL scaffold were


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assembled in a petri dish. Finally, the orthotopic bone repair effect of this OiECM-GO-COL scaffold was evaluated in 5 mm rat skull defect model.

2. Materials and Methods 2.1. Isolation and characterization of rat BMSCs Fresh femurs of fetal SD rats were collected to isolate rat bone marrow mesenchymal stem cells (rBMSCs) according to the previously published protocol27. Briefly, the bone marrow cavities of femurs were washed repeatedly. Then the washed-out contents were collected, centrifuged (1500 rpm, 5 minutes) and washed twice. The obtained cells were seeded and cultured in 10 mm culture dishes supplemented with 10% FBS low glucose DMEM (Hyclone, USA). Cultured rBMSCs were passaged every three days. Chemicals and reagents were obtained from Sigma (Sigma Aldrich, China). The trilineage differentiation potential and the immunophenotype of the BMSCs was measured as previously described28. Osteogenic induction: passage 3 of rat BMSCs were seeded in 6 wells culture dishes supplemented with osteoinduced culture medium (L-DMEM with 10% FBS, 0.1 μM dexamethasone, 10 mM β-glycerophosphate, and 50 μg/ml L-ascorbic acid) for two weeks. And then the calcium deposition was evaluated by Alizarin Red S staining. Adipogenic induction: the adipogenic differentiation medium (H-DMEM with 10% FBS, 5 μg/mL insulin, 1 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, 0.2 mM indomethacin) was prepared for cultured BMSCs and changed every 3 days. The differentiation period lasts for 3 weeks. Then, Oil red staining was used to detect the lipid vacuoles. Chondrogenic differentiation: the chondrogenic induced medium (H-DMEM supplemented with 0.1 μM dexamethasone,



5.33 μg/mL linoleic acid, 0.17 mM ascorbic acid, 1 mM sodium pyruvate, 1% insulin-transferrin sodium-selenite, 1.25 mg/mL bovine serum albumin, 0.35 mM L-proline, and 0.01 μg/mL transforming growth factor-β). The differentiation period lasts for 1 month. And toluidine blue staining was used to determine the chondrogenic differentiation extent of BMSCs.

For the immunophenotype characterization, passage 3 of cultured BMSCs were seeded onto glass coverslips. Then the cells on coverslips were analyzed for MSC-markers (CD29, CD34, CD45, CD73, CD105) by immunocytochemistry.

2.2. Construction and characterization of the osteoinductive extracellular matrix (OiECM) Three passages of the BMSCs were initially cultured in 6-well plates (0.5 ~ 1×105 cells per well) with 10% FBS DMEM. The osteo-differentiation protocol was the same as above mentioned and the induction period lasted for 21 days. The differentiation period lasted for 21 days. Quantitative analysis of the calcium crystals secreted on the matrix was performed at 0, 7, 14, 21 days of osteogenic differentiation by calcium quantitative assay kit (Anaspec, America) flowing the manufacturer’s protocol. At day 21 of osteogenic differentiation, a decellularization procedure was performed as described in previous studies29 to obtain the osteoinductive extracellular matrix (OiECM). Briefly, cells were rinsed three times with sterile phosphate buffer solution (PBS), and treated with 0.1% SDS (Sigma, America) at 37°C for 60 min. After an additional mild agitation


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with PBS containing 0.05 mg/ml RNase A, 0.5 mg/ml DNase I, 50 mM Tris-HCl, and 10 mM MgCl2 for 2 h at 37°C, the cells were gently rinsed with sterile PBS. The OiECM was harvested and stored at -80°C for later use.

To examine the morphology of the OiECM, samples were fixed with 2.5% glutaraldehyde and subsequently dehydrated through increasing concentrations of tertiary butanol. After complete dehydration, the OiECM were spray-gold and observed by the scanning electron microscope (SEM, JSM-IT300, JEOL, Tokyo, Japan) at a voltage of 20kV.

DAPI staining was performed to evaluate whether the decellularization technique was successful. Samples were observed by Fluorescence Microscope (Nikon H600L, Tokyo, Japan), the nuclear material showed a blue fluorescence.

For Western blot analysis, OiECM samples were washed with PBS and electrophoretically separated in the SDS−polyacrylamide gel (10%, Servicebio, China). The separated proteins were transferred to a polyvinylidene fluoride membrane (Servicebio, China). The membrane was then incubated with primary antibodies against Col I (Abcam, USA), FN (Abcam, USA), and BMP-2 (Abcam, USA) overnight at 4°C, then washed with TBST/5% skimmed milk and incubated again with secondary antibodies. Enhanced chemiluminescence (Servicebio, China) was used to detect the blots of the specific proteins. The luminescence was recorded on X-ray films



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(Servicebio, China).

2.3. Preparation of OiECM-GO-COL scaffolds 2.3.1. Synthesis of graphene oxide (GO) NaNO3, H2SO4, KMnO4, H2O2, acetic acid, and HCl were purchased from Sinopharm Chemical Reagent Co., Ltd, China. Graphite powder (10000 mesh) was purchased from

Qingdao

Huatai

Tech.

Co.,

Ltd,

China.

N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride crystalline (EDC) and N-hydroxysuccinimide (NHS) were purchased from Sigma Aldrich. GO was prepared by the modified Hummer’s method as in the previous studies30. Briefly, 2 g of graphite powder and 5 g NaNO3 were added into 50 ml concentrated H2SO4 and stirred for 30 min using an ice bath. After graphite was fully dispersed in the solution, 7.2 g of KMnO4 were slowly added into the system for 1 h and kept stirring for another 4 h at room temperature. Thereafter, 0.1 L of distilled water was added to the solution and the reaction temperature was raised to 95℃. After stirring for 15 min to complete the reaction, 2 L of iced distilled water and 10 ml of 30% H2O2 were added to terminate the reaction. To reached a neutral pH, the solution was centrifuged and washed with 5% HCl and pure water. The GO solution was sonicated for 4 h in the ultrasonic oscillator (Shenzhen Jato Science Technology. Co., Ltd, China) at the conditions of 40 kHz and 180 W to obtain thin GO flakes. After centrifugation at 4000 rpm for 30 min, the precipitate was discarded, and the solution was freeze-dried to obtain GO flakes.


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2.3.2. Preparation of OiECM-GO-COL scaffold GO-COL scaffold was prepared by chemical crosslinking. Firstly, GO dispersion (0.2 wt%) was prepared by dissolving GO powder into 0.1% acetic acid and sonicated using the ultrasonic processor (Branson, USA) for 30 min. Secondly, Type I collagen solution (4 wt%) (Chengdu Kele Science Technology. Co., Ltd, China) was prepared by dissolving collagen into 0.1% acetic acid and stirring to homogenize it in an ice bath. Then the collagen solution (4 wt%) and GO dispersion (0.2 wt%) were mixed at a 1:1 volume ratio and adjusted to a final concentration of 2 wt% COL and 0.1 wt% GO. The mixture was sonicated for 10 min to obtain a homogeneous GO-COL hydrogel. Then the hydrogels were poured into a mold made of polydimethylsiloxane, frozen at -20℃ overnight and subsequently lyophilized for 24h at -50℃ to form a porous scaffold. For the chemical crosslinking, the freeze-dried scaffolds were immersed in a 50 mM EDC with 20 mM NHS solution (H2O: ethanol = 5: 95) for 24 h, thoroughly washed in distilled water, and finally freeze-dried at -50℃ to obtain a cross-linked Graphene oxide-collagen (GO-COL) composite construct. The control COL scaffold (2 wt%) were prepared by the same methods.

The OiECM membrane and the GO-COL scaffold were assembled in a culture dish. Each GO-COL scaffold (d ≈ 6 mm, h ≈ 5 mm, surface area ≈ 250 mm2) was covered by OiECM harvested from one 6-well plate (surface area ≈ 960 mm2). After the OiECM membrane was fully spread in the culture dish, the sterilized GO-COL was put at the center of the ECM membrane and carefully wrapped using tweezers (Supplementary



Movie 1). The constructs were frozen at -20℃ and lyophilized at -50℃ to obtain OiECM-GO-COL scaffold.

2.4. Physiochemical and mechanical characterization of the scaffolds 2.4.1. Morphology of the scaffolds The COL/GO-COL/OiECM-GO-COL scaffolds (surface and cross-section) were coated with gold and observed by scanning electron microscopy (JSM-IT300, JEOL, Tokyo, Japan) at a voltage of 20kV. In addition, the scaffolds were also scanned by Micro-CT (SkyScan 1176, Burker, German) to evaluate the distribution of mineralization. The 3D reconstruction images and the volume quantification of the degree of mineralization were analyzed by the SkyScan software package.

2.4.2. FTIR, XRD, and Raman spectra analysis Fourier Transform Infrared Spectroscopy Microspectroscopy (FTIR, VERTEX 70, Bruker, German) was used to detect the functional groups of the scaffolds from 4000 cm-1 to 500 cm-1. Crystalline phases of scaffolds were evaluated by X-ray diffraction (XRD, Empyrean, PANalytical B.V. Netherlands) with Cu Kα radiation (scanning rate: 1.4583°/s, 2θ range from 5° to 40°). The composition of the scaffolds was examined by Dispersive Raman Microscope (FRA 106/s, Bruker, German) with a wavelength of 532 nm at room temperature.

2.4.3. Porosity, water absorption, and retention ratio


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The porosity of the scaffolds was measured using the liquid displacement method31. The diameter (d) and the thickness (h), and the dry weight (Wd) of the scaffold were measured. Then the scaffolds were immersed in ethanol for 5 min and the wet weight (Ww) was measured again. The porosity of the scaffold was calculated using the following formula: 𝑊 −𝑊

𝑤 𝑑 × 100% Porosity(%) = 𝜌𝜋ℎ(𝑑/2)

(1)

Where ρ refers to the density of ethanol (0.789 g/cm3), π is the π value 3.14159.

The water absorption (WA) by the scaffolds was determined by immersing it in distilled water at room temperature overnight. After the scaffolds were equilibrated with distilled water, the wet constructs were blotted using a filter paper to remove the water adherent to its surface. The WA of the scaffolds were calculated using the following formula: WA (%) =

𝑊𝑞 −𝑊𝑑 𝑊𝑑

× 100%

(2)

Where Wq is the weight of scaffold filled with distilled water, Wd is the weight of the dry scaffold.

The water retention capacity (WR) was also tested. The scaffolds filled with water were centrifuged (1000 rpm, 5 min) in tubes and weighed again. The WR of the scaffolds was calculated using the following formula: WR (%) =

𝑊𝑐 −𝑊𝑑 𝑊𝑑

× 100%

(3)

Where Wd refers to the weight of a dry scaffold in Eq. (2), and Wc is the weight of wet



scaffold after centrifuging.

2.4.4. Mechanical property testing The scaffolds were cut into suitable sizes (height ≈ 5 mm, diameter ≈ 6 mm) and the stress-strain curve of the scaffolds was obtained by mechanical test using All-Electric Dynamic Test Instrument (Electro Puls E1000, INSTRON, British). The corresponding compressive modulus was calculated as previously discribed9.

2.5. Cell seeding, proliferation, viability and morphology of the BMSCs cultured on the scaffolds Passage 4 of the rat BMSCs were seeded on the scaffolds according to a previous study28. Briefly, BMSCs were washed with PBS, digested with trypsin, and centrifuged at 1500 rpm. Then they were re-suspended with a certain volume of growth medium. Each scaffold in the 12-well plates was seeded with 50 μm of a medium containing 1×104 cells. The scaffolds were incubated for 2 h for initial attachment. To evaluate the cell seeding efficiency, unattached rat BMSCs were carefully washed from scaffolds using PBS. Attached cells on scaffolds were quantitatively assessed by CCK-8 kit. Cell seeding efficiency was counted as the ratio between the initial cell seeding number and attached cells number. After initial cell attachment, the growth medium was added to submerge the scaffolds. Finally, the scaffolds were cultured in a humidified incubator at 37℃ with an atmosphere containing 5% CO2. The growth medium was replaced every


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day.

Cell viability and proliferation on the scaffolds was visualized with a LIVE/DEAD assay kit (Sigma Aldrich, USA) at 1, 7, and 14 days of culture. Live and dead cells were stained with FDA (green) and PI (red) for laser scanning confocal microscopy (Nikon A1Si, Japan). Live cells per unit volume were counted using Image J software (National Institutes of Health). Furthermore, the CCK-8 assay was performed to quantitatively evaluate the cell proliferation ability on scaffolds at 1, 4, 7, 10 and 14 days of culture. The cell-scaffold samples at 14 days of incubation were fixed with 2.5% glutaraldehyde and subsequently dehydrated through increasing concentrations of tertiary butanol. After dehydration was completed, the OiECM were coated with gold and observed by SEM.

2.6. In vivo craniofacial bone defect study 2.6.1. Surgery procedures Three groups (n = 6) of male SD mice (6 weeks old, average weight of 180-200 g, housed in the Experimental Animal Center of Tongji Medical College, Huazhong University of Science and Technology, China) were used as hosts. All the treatments and experiments of animals were performed according to the animal care guidelines and approved by the Ethics Committee of Tongji Medical College, HUST. The rats



were allowed to acclimatize in the animal facility for 14 days before the operation. Once the rats were anesthetized by inhaled isoflurane and subcutaneous buprenorphine injection, two critical-size defects (diameter = 5 mm) were created symmetrically in the parietal bone using an aseptic electric driller. Extreme care was taken to avoid injury to the dura mater beneath the calvarium. Animals were given penicillin for 3 days to prevent infection. At 12 weeks post-implantation, animals were sacrificed by an overdose of anesthetics for Micro-CT and histological analysis.

2.6.2. Micro-CT and histological analysis. The scaffold-implanted craniums were entirely and carefully collected from animals. The samples were fixed in 10% paraformaldehyde for 3 days and scanned by Micro-CT. Bone visualization was reconstructed using VGstudio software. Bone volume (BV) and bone volume relative to tissue volume (BV/TV) were calculated. Tissue volume (TV) is defined as the total tissue volume (including soft-tissue and bone) in the 5mm rat cranial bone defect area.

For histological analysis, samples were decalcified in 10% EDTA solution (Sigma Aldrich, USA). After decalcified samples were processed, H&E staining and Masson’s trichrome staining was performed separately. Moreover, immunohistochemical staining was performed. Briefly, the processed sections were incubated with rabbit anti-mouse OCN primary antibody (Proteintech Group, Wuhan, China) overnight at 4 °C, and then with the horseradish peroxidase-conjugated goat anti-rabbit secondary


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antibody (Proteintech Group, Wuhan, China). The images were observed and captured by an optical microscope (Nikon H600L, Tokyo, Japan)

2.7. Real-Time PCR analyses for BMSCs cultured on OiECM The mRNA expression levels of the OCN, COL I, BMP-2 in BMSCs incubated on OiECM-coated or uncoated petri dish for 14 days in regular DMEM were detected by real-time PCR. Total RNA was extracted from the cultured BMSCs using Trizol Reagent

(Thermos

Fisher

Scientific,

USA).

Total

RNA (500

ng)

was

reverse-transcribed with the Revert Aid First Strand cDNA Synthesis Kit (Thermos Fisher Scientific, USA). Primers used in PCR were listed in Table S1. Real-time PCR was performed using KAPA SYBR FAST qPCR Kit Master Mix (KAPA Biosystems, USA) on a Real-Time PCR System (Prism 7900HT, ABI, USA) to quantify the PCR products. Amplification conditions were 95°C for 3 min, 95°C for 3 s, and 60°C for 30 s (40 cycles). Fold changes between experimental groups and the control were calculated with the 2-∆∆CT method.

2.8. Statistical analyses Parametric data were assessed with Student`s t-tests, and the multi-group comparisons were examined by one-way ANOVA tests. P-values which were less than 0.05 were considered statistically significant.

3. Results



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3.1. Characterization of the BMSCs derived osteoinductive extracellular matrix (OiECM) The BMSCs that were isolated from neonatal SD rats and cultured in a regular growth medium for 14 days exhibited a fibroblast-like morphology (Figure S1A). Alizarin-red-positive calcium nodules (Figure S1B) and cytoplasmic accumulation lipid vacuoles identified by oil red O staining (Figure S1C) were observed demonstrating that the BMSCs were active to osteogenic and adipogenic differentiation. BMSCs were also cultured in chondrogenic induction medium and stained with toluidine blue which turned purple thereby confirming the chondrogenic differentiation (Figure

S1D).

Typical

cell

markers

of

BMSCs

were

confirmed

by

immunofluorescence staining demonstrating that they were positive for CD73, CD105, CD44, CD29, and negative for CD34 (Figure S1E). In order to determine the favorable ECM cultivation time, the degree of mineralization of OiECM was detected as the Ca2+ content was gradually increased during the osteo-induction period of 21 days (Fig. 2D).

After decellularization, a gel-like osteoinductive extracellular matrix(OiECM) layer was gently separated from the culture dishes with cell scrapers. (Fig. 2A). SEM image also showed that the surface of OiECM was rough and the mineralization was observed (Fig. 2B and C, yellow arrows showed the mineralization on OiECM). To evaluate whether all the cells were removed, DAPI staining was performed before and after the decellularization procedure. Many cells (Fig. 2E) were observed on the ECM layer before decellularization procedure. After decellularization, there existed hardly any


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cells in the field of vision except the ECM (Fig. 2F). The osteogenic related protein of the OiECM was identified through western blot analysis. As shown in Fig. 2G, FN, COL I, and BMP-2 were expressed in the OiECM membrane.

Figure 2. Characterization of OiECM. (A) Optical image of OiECM in the culture dish (black arrow shows the OiECM, Scale bar: 1 cm) and (B) SEM image of the OiECM, Scale bar: 100 µm; (C) enlarged image (yellow arrows: calcium nodules on OiECM, Scale bar: 50 µm). (D) Quantitative analysis of calcium in the OiECM after 0, 7, 14, and 21 days of culture. (E) DAPI staining of the nuclei of OiECM before and (F) after decellularization. (G) Western blot analysis showing that OiECM contained protein such as BMP-2, collagen, and fibronectin. (***P < 0.001).

3.2. Characterization of the COL/GO-COL/OiECM-GO-COL scaffolds 3.2.1. Morphology of the COL/GO-COL/OiECM-GO-COL scaffolds. The COL scaffold was white, while the GO-COL and OiECM-GO-COL scaffold were brown in color. To demonstrate that OiECM and GO-COL are stably combined, OiECM-GO-COL scaffold was rubbed with a cotton swap. It turned out that OiECM could stably attach to GO-COL scaffold (Supplementary Movie 2). Highly



interconnected and porous structure was observed in all three groups of scaffolds (Fig. 3A-C). Due to the insertion of GO flakes, the GO-COL groups (Fig. 3B) presented a more irregular surface and a folded microstructure than that of the COL groups (Fig. 3A). Furthermore, the OiECM-GO-COL scaffolds also had a porous structure inside, but the pores on the surface were covered by a fibrous net-like structure demonstrating a unique rough topology (Fig. 3C, red arrows show the covered pores, yellow arrows show the spindle-like ECM fibers attached to the scaffold). The Micro-CT examination showed that only the OiECM-GO-COL scaffolds are surrounding by a mineralized layer (Fig. 3C, Micro-CT images), and the quantitative analysis verified that there was about 0.15 mm3 of mineralization on OiECM-GO-COL scaffold (Fig. 3D).


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Figure 3. Characterization of the COL/GO-COL/OiECM-GO-COL scaffolds. An optical image, SEM image, and Micro-CT 3D reconstruction of (A) COL scaffold, (B) GO-COL scaffold, and (C) OiECM-GO-COL scaffold (red arrows show the pores covered with freeze-dried ECM, yellow arrows show the ECM fibers attached to the scaffold). Scale bar: low magnification: 200 µm, high magnification: 100 µm, Micro-CT: 250 µm (D) Quantitative analysis of calcified nodule volume of the scaffolds.

3.2.2. FTIR, XRD, Raman spectra analysis of the COL/GO-COL/OiECM-GO-COL scaffolds. The chemical composition of the GO powder and scaffolds were characterized by



FTIR. The characteristic peaks of the GO powder at 3369, 1618, 1245, 1057 cm-1 were observed and are shown in Figure S2A. Typical collagen bands were observed in all three scaffold groups. The peak around 1600-1700 cm-1 belongs to the C=O stretch of amide I, while the peaks around 1500-1550 cm-1 and 1200-1300 cm-1 were dedicated to N–H deformation for amide II and N–H deformation for amide III separately. In OiECM-GO-COL scaffolds, was the peaks appeared at 1038.5, 604.5, 567.8 cm-1 mostly due to the mineralization which mainly consisted of hydroxyapatite (HA) in the OiECM (Figure S3A). As for the XRD analysis, the characteristic 11.9° diffraction peak of GO was observed (Figure S2B). The broad diffuse peak at about 22.5° can be attributed to collagen. In the OiECM-GO-COL, the peaks were detected around 32° which may correspond to the diffraction peaks of mineralization of OiECM (Figure S3B). The Raman spectrum was used to identify the carbon components in GO powder and three scaffold groups. As shown in Figure S2C, characteristic peak D band (around 1360 cm-1) and G band (around 1580 cm-1) were observed in the GO powder. In Figure S3C, there was no characteristic peak in the collagen scaffold. After GO was incorporated into COL and wrapped with OiECM, the D and G bands peaks were observed at 1355 cm-1 and 1593 cm-1, respectively.


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Figure 4. Characterization of the COL/GO-COL/OiECM-GO-COL scaffolds. (A) The porosity of the scaffolds. (B) Water absorption capacity and, (C) water retention capacity of the scaffolds. (D) The mechanical test indicated the GO-COL/OiECM-GO-COL exhibited a better mechanic strength than COL scaffold. (*P < 0.05, **P < 0.01, ***P < 0.001).

3.2.3. Porosity, water absorption and water retention of the OiECM-GO-COL scaffolds. The COL, GO-COL, and OiECM-GO-COL scaffolds were highly porous with a porosity of 76.4 ± 9.4 %, 81.5 ± 7.0 %, 70.0 ± 5.0 %, respectively (Fig. 4A). Scaffold with high water absorption and retention capacity could enhance the nutrients transfer and cell proliferation9. We found that the COL/GO-COL/OiECM-GO-COL scaffolds had high water absorption capacity of approximately 3700% (Fig. 4B). When compared to the other two groups of scaffolds, OiECM-GO-COL scaffold showed a higher water retention ability of approximately 82.66% (Fig. 4C).



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3.2.4. Mechanical properties of the COL/GO-COL/OiECM-GO-COL scaffolds. To determine whether GO flakes could enhance the mechanical strength of the pristine COL scaffolds, the stress-strain curves of the scaffolds were obtained from the compression test (Figure S3D). The results suggested that the GO-COL scaffolds increased Young’s modulus 2.5 folds compared to the pristine COL scaffolds. The mechanical strength of the OiECM-GO-COL scaffolds decreased slightly to 0.45 ± 0.07 MPa (p > 0.05) after a repeated freeze-drying process (Fig. 4D).

3.3.

Cell

adhesion,

viability,

and

proliferation

on

the

COL/GO-COL/OiECM-GO-COL scaffolds. Cell seeding efficiency test was carried out to evaluate whether the OiECM-GO-COL scaffolds could provide a more stable microenvironment for stem cells adhesion. As showed in Fig. 5A, the COL and GO-COL scaffold have a similar cell adhesion rate, but OiECM-GO-COL group has significantly higher cell adhesion rate than the COL and GO-COL. Cell proliferation curve also suggested OiECM-GO-COL scaffold exhibited higher proliferation rate of rat-BMSCs after day 7. In addition, GO-COL scaffold presented well cell biocompatibility comparing to COL scaffold, but the cell proliferation rate slightly slowed down after day 10 (Fig. 5B). The cell viability of the rat-BMSCs cells on scaffolds was evaluated with immunofluorescence staining. As shown by the live/dead staining (Fig. 5C) and live count cell (Fig. 5E), OiECM-GO-COL scaffolds demonstrated a significantly higher value of cell viability


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at 1, 7, 14 days of incubation compared to COL and GO-COL scaffolds. Although dead cells were observed in the early stage of culture in the three scaffold groups, the ratios of the living cells were consistently higher in OiECM-GO-COL scaffolds and the growth status was better on further cultivation. The cell morphology on the constructs at 14 days of incubation was observed by using SEM. On the OiECM-GO-COL scaffolds, there were more adherent cells exhibited spindle-shaped morphologies, and more fiber-like ECM compared to the GO-COL and COL scaffolds (Fig. 5D).



Figure

5.

Cell

viability

and

proliferation

of

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the

BMSCs

seeded

on

COL/GO-COL/OiECM-GO-COL scaffolds. (A) Cell seeding efficiency at 2 h. (B) Cell


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proliferation curve at 1, 4, 7, 10 and 14 days of culture. (C) Live/dead staining of cells on scaffolds were determined by FDA/PI assay at 1, 7 and 14 days of culture (live cells: green, dead cells: red). (D) SEM images of cells attached to the scaffold at14 days of culture (yellow arrows: seeded BMSCs). Scale bar: (A:250 µm, B: 50 µm); (E) Live cell density of BMSCs seeded on COL/GO-COL/OiECM-GO-COL scaffolds at 1, 7, 14 days of culture. (*P < 0.05, **P < 0.01).

3.4. In situ bone defect repair in rat cranial model. The general images of the COL/GO-COL/OiECM-GO-COL scaffold implanted into rat cranial defected areas and the skull samples 12 weeks post-implantation are demonstrated in Figure S4. Moreover, representative coronal and sagittal view of the 3D reconstructed bone tissue for each group is shown in Fig. 6A. Quantification of the volume of newly-formed bone and percentage of bone volume and bone volume relative to tissue volume (BV/TV) showed that they were significantly higher in the OiECM-GO-COL group compared to the COL and GO- COL group (P < 0.01). However, there was no significant difference (P > 0.05) between the COL and GO-COL group (Fig. 6B and C).



Figure 6. (A) Representative CT coronal and sagittal views of cranial defect area 12 weeks post-implantation. (B) Bone volume analysis, and (C) Bone volume to tissue volume (BV/TV) of bone formation in defect area. (**P < 0.01)

The HE staining showed that newly formed bony tissue was found in the defect areas in all groups 12 weeks post-implantation. As shown, the implantations were fully absorbed and its outline could not be distinguished, and much fibrous connective tissue and blood vessels were formed in the defect site. Compared to the control COL group and the GO-COL group, the newly-formed mature bone (NB) was clearly observed and it achieved a better bone repair effect in the OiECM-GO-COL group (Fig. 7A, NB indicate the newly-formed mature bone). Moreover, the NB was mostly


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observed at the edge of the defect, but there was a significant amount of new bone formation at the center of the defect site. Masson’s trichrome staining was conducted to show the collagen deposition of the repaired areas. New bone tissue and fibrous connective tissue were observed (Fig. 7B). These results were consistent with the Micro-CT images. The remaining graphene oxide debris in the defect area (labeled by *) was observed in GO-COL groups.

Figure 7. Histological analysis of the COL/GO-COL/OiECM-GO-COL scaffolds 12 weeks post-implantation. (A) H&E staining images and (B) Masson’s trichrome staining of explants at 12 weeks post-implantation; blue tissues represented the collagen fibers and brown structures (labeled by *) indicate the GO debris (Scale bars in lower magnification images:



500µm, scale bars in higher magnification images: 100 µm; NB: new bone). (C) Immunohistochemical staining of the OCN-positive cells on the newly-formed bone tissue (labeled by red triangles). Scale bars: 50 µm.

Furthermore, immunohistochemical staining was carried out to evaluate the expression of OCN in the bone tissue. It is known that OCN can only be secreted by mature osteoblasts32. We found that the OCN-positive cells were spread in the newly-formed bone and more OCN-positive cells were detected in the OiECM-GO-COL scaffold (Fig. 7C, OCN-positive cells were stained brown around the nuclei). These results exhibited that the OiECM-GO-COL scaffolds had a notably higher osteogenic capability in vivo compared with the GO-COL and COL scaffolds.

3.5. Effect of OiECM on the osteogenic differentiation Real-time PCR was performed to detect bone-specific gene expression in BMSCs seeded on the OiECM-coated or uncoated petri dish in a regular DMEM growth medium for 14 days. Results indicated that the BMSCs cultured on OiECM had a significantly higher gene expression of COL I (7.51 fold), BMP-2 (26.25 fold), and OCN (1.76 fold) compared to the control groups (Fig. 8) at 14 days. These outcomes demonstrated that OiECM could significantly influence the osteogenic gene expression of BMSCs in vitro.


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Figure 8. The osteogenic-related gene expression in BMSCs cells cultured on the OiECM for 14 days. BMSCs cultured on OiECM showed higher gene expression of COL I (A), BMP-2 (B) and OCN (C) comparing to that cultured on a petri dish. (*P < 0.05, ***P < 0.001).

4. Discussion The development of GO hydrogels is a growing technology for BTE . Current BTE strategies to modify scaffolds with ECM mainly rely on seeding cells on the surface directly, which were unsuitable for GO related materials or natural polymers due to unsatisfied cell seeding efficiency and proliferation rate33. Furthermore, application of these methods involves a long-term culture in dishes which leads to uneven distribution of extracellular matrix resulting from insufficient oxygen and nutrients supply25. Therefore, we developed an osteo-inductive ECM which was secreted by osteo-differentiated BMSCs, and demonstrated that this OiECM can be completely separated from the culture dishes and uniformly wrapped onto the GO-COL hybrid scaffold to form a tissue-engineered bone graft. We further demonstrated that this OiECM-wrapped GO-COL implants could greatly improve the proliferation and osteo-differentiation of BMSC in vitro and achieved completely critical-sized orthotopic defect repair effects in vivo.



So far, graphene and graphene oxide-based materials have been the spotlight of medical research34. Graphene oxide has excellent physical and chemical properties including abundant oxygen functional groups, well mechanical strength and high surface area ratio35, increasing the interfacial interaction between GO and nature polymer36. Furthermore, GO can lower the degradation rate of HAP-hyaluronic acid-chitosan scaffold37, making it possible to modulate the degradation time of natural polymers. In order to obtain better biocompatibility while constructing OiECM-GO-COL hybrid scaffolds in our study, we have tested different concentration of GO on cell proliferation rate. Rat-BMSCs were seeded in 0/0.05/0.1/0.2 wt% GO-COL scaffolds for 7 days and MTT assay was carried out. Results suggested that 0.1 wt% GO-COL showed better cell biocompatibility comparing to other groups of scaffolds (Figure S5). Hence, we choose 0.1 wt% GO-COL to prepare the OiECM-GO-COL scaffold.

Graphene oxide can be degraded in vivo and excreted out of the body through the kidneys, the liver, or other organs38. According to our in vivo experiments, GO debris was observed in the defect area repaired by GO-COL and OiECM-GO-COL scaffolds (Fig. 7 and Figure S4). GO sheets (according to their size) can be up-taken by macrophages or plasma membrane in vivo39. ECM coatings could not only reduce the M1 macrophage activation but also enhance the ratio of M2/M1 macrophages to polypropylene scaffold in vivo40, suggesting ECM might be able to influence the interaction between macrophages and GO. There is also evidence that GO can be degraded by myeloperoxidase (MPO) and hydrogen peroxide in vitro and in vivo41.


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Some reported that proteins in ECM can bind MPO and enhanced its activity42. As far as the present state of researches is concerned, whether the ECM could promote the GO degradation in vitro or in vivo still needs further study.

Many studies have proven that GO composites can promote tissue repair and reconstruction in cardiac43, neural44, cartilage45, and skin46 tissue, especially in bone tissue engineering47. Jing et al. developed a GO-carboxymethyl chitosan hydrogel and found that it can improve the mechanical strength of carboxymethyl chitosan and promote MSC osteo-differentiation as well as bone regeneration in vivo48. The role of GO in the osteogenic ability of OiECM-GO-COL scaffold was also tested in our previous animal experiments (DATA NOT SHOWN). We found that OiECM-GO-COL group exhibit better bone repair effect compared to OiECM-COL group, indicating GO might also play an important role in OiECM-GO-COL scaffold to repair bone defects in vivo. The high modulus of GO makes it possible for natural polymers with excellent and adjustable mechanical properties17,

49

. However, our results demonstrated that

simple GO hydrogels could not achieve complete repair in situ (Fig. 6A and 7A). In some studies, stimulation of the osteogenic ability of the GO hydrogels was performed by delivering high expensive recombinant growth factors which are often influenced by the inactivation and unstable releasing rate50, 51.

The ECM contains a complex 3D network which is consisted of polysaccharides and biomacromolecules26. Cell-derived extracellular matrix has tremendous advantages in



terms of the biophysical and biochemical properties, which makes it highly similar to the native extracellular matrix microenvironments52. It has been reported that various types of ECM can be harvested from different cell types and culture environment53, 54. For example, MSC-derived ECM can modulate osteogenicity and differentiation of MSCs as reported in many studies55,

56

. In this study, we cultured BMSC in an

osteogenic medium for 21 days to obtain OiECM because the level of mineralization of ECM was highest at 21 days (Fig 2D). The calcium nodules secreted by osteo-differentiated BMSCs promoted the osteoinductive ability because Ca2+ activates the CaMKII pathway and adenosine signaling to regulate cell proliferation and osteogenesis57,

58

. Ca2+ is also known to enhance the alkaline phosphatase activity,

stimulate the expression of osteogenic markers in osteoblastic cells, and to modulate the micro-environment for bone generation59.

Apart from the mineral substances, growth factors found in the ECM also play an essential role in regulating cellular behaviors. The OiECM presented in this study contains fibronectin (FN), COL I, and BMP-2, indicating that our chemical method was able to remove the cells and minimize the damage to the components of the ECM (Fig. 2G). In addition, a hydrogel scaffold wrapped on the ECM may provide more anchor sites on the scaffold for cell adhesion, thereby reducing the matrix deposition60. The interaction between the cell and the matrix is important61, 62. In this study, it was shown that the BMSC had higher proliferation rate and better cell morphology on OiECM-GO-COL scaffold compared to the GO-COL and COL scaffold (Fig. 5A and


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B). Furthermore, OiECM could promote the osteo-related gene expression of OCN, COL I, and BMP-2 in BMSCs without osteogenic differentiation medium (Fig. 8), demonstrating that the OiECM can alter the gene expression in MSCs and regulate their osteo-differentiation.

Traditional bone tissue engineering construction methods include at least three steps (cell seeding, expansion, and osteo-differentiation) which usually lasts for 14-21 days. As a result, these methods do not meet the desired time to treat acute injury and hence have limited application28. In addition, the labor-intensive manual operation procedures during the repeated change of culture medium might increase the risks of contamination63. Therefore, we developed an alternative TEBG construction strategy by wrapping OiECM on the GO hybrid scaffold. The OiECM can be prefabricated in different-sized Petri dishes and stored before clinical treatment without cell seeding and long-term culture procedures on scaffolds, thus dramatically shortening the process of constructing the TEBGs. Moreover, our results indicated that the OiECM was fabricated by only 0.5~1×105 BMSCs (cell amounts of one well in a 6-well plate) which is 1/2 ~ 1/3 of the cell number reported in other studies32, 64 to completely repair the 5 mm diameter rat cranial defect (Fig. 6A). For the scaffolds with high degradation rate in long-term culture in vitro, especially graphene oxide hybrid hydrogel, this study provides a promising TEBG construction strategy which not only ensures uniform deposition of the ECM on scaffold but also a high osteo-inductivity and biocompatibility.




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5. Conclusion In summary, we successfully developed a novel off-the-shelf TEBG construction strategy for GO hybrid hydrogels. Our findings revealed that the OiECM-GO-COL implants exhibited unique surface topography, good physiochemical and mechanical properties compared to collagen scaffolds. The coated OiECM not only promoted the proliferation of the seeded cells but also greatly enhanced osteo-related gene expression in vitro. The OiECM-GO-COL implants achieved better repair effects in rat critical cranial defect mode, demonstrating its promising application in bone defect repair. Due to the high osteo-conductivity and osteo-inductivity of the OiECM, this method of construction might greatly expand the scope of clinical application of the GO hydrogel in the field of bone tissue engineering.

6. Author Information Corresponding Author *Zhenxing Wang: E-mail: [email protected]. Tel. +86-027-85726114. *Jiaming Sun: E-mail: [email protected]. Tel. +86-027-85726114.

Author Contributions Shaokai Liu and Shan Mou contributed equally to this work.

Notes The authors declare no competing financial interest.



7. Acknowledgment This work was financially supported by the National Natural Science Foundation of China (No.81501688, No.81601701, No.81701922) and the Natural Science Foundation of Hubei Province (2017CFB263).


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TOC


Off-the-shelf Biomimetic Graphene Oxide-Collagen Hybrid Scaffolds

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