Functionalized Scaffold for in Situ Efficient Gene Transfection of

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Biological and Medical Applications of Materials and Interfaces

Functionalized scaffold for in-situ efficient gene transfection of MSCs spheroids towards chondrogenesis Kunxi Zhang, Haowei Fang, Yechi Qin, Lili Zhang, and Jingbo Yin ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b12268 • Publication Date (Web): 12 Sep 2018 Downloaded from http://pubs.acs.org on September 13, 2018

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ACS Applied Materials & Interfaces

Functionalized scaffold for in-situ efficient gene transfection

of

MSCs

spheroids

towards

chondrogenesis Kunxi Zhang,* Haowei Fang, Yechi Qin, Lili Zhang, Jingbo Yin* Department of Polymer Materials, School of Materials Science and Engineering, Shanghai University, 99 Shangda Road, Shanghai 200444, PR China. KEYWORDS. cell adhesion, spheroid, reversible linkage, surface-mediated gene transfection, chondrogenesis.

ABSTRACT. Multicellular mesenchymal stem cell (MSC) spheroids possess enhanced chondrogenesis ability and limited fibrosis, exhibiting advantage towards hyaline-like cartilage regeneration. However, due to the limited cell surfaces in spheroid exposed to DNA/vector, it is difficult to realize efficient gene transfection, most of which highly rely on cell-substrate interaction. Here, we report a poly (L-glutamic acid) (PLGA)-based porous scaffold with tunable inner surfaces that can sequentially realize cell-scaffold attachment and detachment, as well as the followed in-situ spheroid formation. The attachment and detachment of cells from scaffold is achieved by the capture and release of fibronectin (Fn) via reversible imine linkage between aromatic aldehyde groups of scaffold and amino groups of Fn. Together with N,N,N-trimethyl

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chitosan chloride (TMC) condensing plasmid DNA encoding transforming growth factor-β1 (pDNA-TGF-β1), cell attachment realizes efficient surface-mediated gene transfection. Conversion of scaffold stiffness can affect the adhesion shape of cells. Stiffer scaffold reinforces the adhesion, leading to the amplification of peripheral focal adhesions and the promotion of cell spreading, as well as the promotion of gene transfection efficiency. After cellular detachment from the scaffold via lysine treatment, the subsequent spheroid formation with extensive cell-cell interaction up-regulates the corresponding protein expression with a prolonged term. With the induction effect of the expressed TGF-β1, significantly enhanced chondrogenesis of MSCs in spheroids is achieved at 10 d in vitro. Well-regenerated cartilage at 8 w in vivo indicates that the present gene transfection system is a platform that can be potentially applied towards cartilage tissue engineering.

1. INTRODUCTION Mesenchymal stem cells (MSCs) spheroid, the spherical micro-scale multicellular aggregates, has been found to enhance differentiation potentials of multiple lineages,1,2 stemness,3 matrix deposition,4,5 post-transplant survival

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and anti-inflammatory effects.7-9 In particular, there are

extensive cell-cell interactions and up-regulated N-cadherin expression in MSC spheroids, which are significant for cartilage regeneration.10,11 Thus, MSC spheroids possess the special advantage towards cartilage tissue engineering application. In our previous work, a poly (L-glutamic acid) (PLGA) based porous scaffold was developed to drive in-situ formation of adipose stem cells (ASCs) spheroids, so to promote chondrogenic differentiation and inhibit fibrous matrix deposition, leading to a hyaline-like cartilage regeneration.12


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Although spheroid has been often applied in MSC-based therapeutics, such as promoting tissue regenerative and reparative effects,13-15 due to the limited cell surfaces in dense spheroids that could expose to DNA/vector,16,17 there is a barrier to carry out the efficient gene transfection, which plays central roles in introduction of foreign DNA into host cells and regulation of cell behavior such as differentiation.18-20 It is thus valuable to create a platform that could realize efficient gene transfection towards spheroid, so to fabricate gene transfected spheroids for cartilage tissue engineering. To realize and promote successful gene transfection, surface-mediated gene transfer systems have been developed since 2000. Normally in this systems, cells adhere on the surfaces of substrates that pre-immobilize DNA or DNA/vector complexes, and take up the DNA on the substrates.21-23 Thus, to ensure adhesive interaction between cells and substrates is significant for surface-mediated gene transfection.24-26 However, most spheroid formation approaches, such as spinner flask and liquid overlay techniques, are based on the inhibition of cell-substrate interaction.27 Since cellular attachment to the substrate is minimized, cell-cell contact is maximized to drive aggregate formation.28 Obviously, surface-mediated gene transfection efficiency, which highly depends on the cell-substrate contact, would be limited during this process. Thus, it is a challenge to achieve efficient surface-mediated gene transfection before the spheroid formation that could amplify the corresponding protein expression and promote cartilage regeneration. The present study introduced a PLGA-based anti-cellular adhesive (non-fouling) porous scaffold, the inner surfaces of which were then functionalized with aromatic aldehyde groups via poly (ethylene glycol) (PEG). The present scaffold with tunable inner surfaces was to realize cell-scaffold attachment, detachment and in-situ spheroid formation in turn via reversible linkage


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with fibronectin (Fn). Together with gene transfer vector, the present scaffold was evaluated for promoting surface-mediated gene transfection and the subsequent corresponding protein expression induced by spheroid formation, as well as in vitro chondrogenesis (Scheme 1). Moreover, the scaffold carrying gene transfected MSC spheroids was directly implanted into cartilage defect for the evaluation of in vivo cartilage regeneration. 2. EXPERIMENTAL SECTION 2.1. Preparation of non-fouling porous scaffold (S). N,N'-Carbonyldiimidazole (CDI) activated PLGA was obtained by add CDI to PLGA Dimethyl sulfoxide (DMSO) solution. Then, cysteamine (10% equivalent of carboxyl in PLGA) and cross-linking agent PEG400 (50% equivalent of carboxyl in PLGA) were added in turn to the solution. Meanwhile, NaCl with uniform diameter was poured into to the solution as pore-forming agents. After being incubated at 37 oC for 6-8 h, the gel was dialyzed in deionized water about 3 days. 2.2. Synthesis and characterization of APEGBA. Methylallyl poly (ethylene glycol) (Mw: 2000, APEG) and 4-Carboxybenzaldehyde (CBA) with molar rate 1:2 were dissolved in Tetrahydrofuran (THF) under nitrogen condition for 3 h. 4-dimethylaminopyridine (DMAP)/ Dicyclohexylcarbodiimide (DCC) was added to the solution as active agent and the solution was stirred at 37 OC for 24 h. The product was precipitated in an excess of diethyl ether and dried in vacuum drying oven. The precipitate was re-dissolved in dichloromethane, filtered and dried again. The product was characterized by 1H NMR/FTIR and stored by vacuum seal under 4 oC. 2.3. Preparation of APEGBA modified scaffold (S-PEGBA) and PEG modified scaffold (S-PEG). APEGBA was dissolved in DMSO, followed by adding α, α-Dimethoxy-αphenylacetophenone (DMPA). Then, S was immersed in the solution for 3h and exposed to UV


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light for 12 h. During this reaction, S was squeezed repeatedly to allow APEGBA permeate inside the S. The S-PEGBA was prepared and dialyzed in deionized water to remove residuum, freeze-dried at -20 oC and stored in 4 oC. S-PEG was prepared by the same procedure, only replacing the APEGBA with APEG. 2.4. Mechanical property test. The scaffolds with the size of 5mm×5mm×5mm was transferred to the clamp of DMA (Dynamic thermomechanical analysis) Q800. The stress-strain curve was recorded by compressing the scaffold to -50.00 % of its strain with velocity of 10.00 %/min. Rheological experiments were carried out on a rheometer (AR2000, TA instrument, USA) with parallel plate (diameter: 12 mm) at 37 oC in the oscillatory mode. Before testing, samples were placed on the parallel plate at 37 oC for 30 min. The storage moduli (G′) were obtained with respect to frequency. 2.5. Element measurement. XPS (X-ray photoelectron spectroscopy) from Thermo ESCALAB 250 (VG Scientiﬁc Co., UK) was employed to measure the element content of the surfaces of scaffolds. The scaffold was pressed into pallet, and transferred to the instrument to test at the condition of monochromatic Al Kα radiation excitation source. 2.6. Determination of thiol groups in scaffolds. The content of thiol groups in scaffold was determined by Ellman’s reagent (5, 5’-dithiobis (2-nitro benzoic acid), DTNB) and the establishment of standard curve was described in Supporting Information. Initially, the weight of 4 samples were recorded as M. Then, the samples were immersed into 250 µl reaction buffer (0.1 M sodium phosphate, pH 8.0, containing 1 mM Ethylenediaminetetraacetic acid). 50 µl Ellman’s agent (0.4 mg/ml of DTNB in reaction buffer) were added to the solution and incubated at room temperature for 15 minutes. After that, absorbance at 412 nm was measured, and the


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concentration was determined through the standard curve. The sulfhydryl content could be calculated by following formula: Sulfhydryl Content = c × 250 µL/M (mM/g). 2.7. Cytotoxicity studies. Fresh human lipoaspirates were obtained from 3 healthy patients in the Department of Cosmetic Plastic Surgery, Plastic Surgery Hospital of Chinese Academy Medical Sciences. All protocols for handing human tissue were approved by the patients and the Research Ethics Committee of the hospital. Human adipose derived stem cells (hASCs) were seeded in 96-well plates to estimate the cytotoxicity. The cell density was 40000 cells per well in 100µL LG-DMEM. After culturing for 24 h, the LG-DMEM was replaced with 200 µl 2 mg/mL lysine/LG-DMEM and 5 mg/mL lysine/LG-DMEM respectively to continuously culture for 1 h, 2h and 4h. LG-DMEM was employed as control. 10 µL of CCK-8 solution was added to each well of the plate to be incubated for 4 h, followed by measuring the absorbance (optical density (OD) value) at 450 nm using a microplate reader (SpectraMax M2, Molecular Devices) at 490nm. Cell viability % = (ODsample − ODcontrol)/ODcontrol × 100%. 2.8. Absorption and controlled release of Fn. Scaffolds were immersed in 0.1 mg/mL Fn solution for 4 h, followed by being washed with 1×PBS (phosphate buffer saline) for two times to remove unabsorbed Fn. Then, scaffolds were treated with 2 mg/mL and 5 mg/mL lysine respectively for 1 h, 2 h and 4h. Finally, the content of Fn was determined by immunofluorescence staining. Briefly, after being immersed in 5% BSA solution for 12 h, samples were transferred to Fn polyclonal antibody solution overnight under 4 oC, followed by being immersed into Alexa Fluor 647 labled goat polyclonal secondary antibody to human IgGH&L at 37 °C for 1 h. Confocal laser scanning microscope (CLSM) and Fn ELISA Kit were employed to measure the absorb and release of Fn.


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2.9. Dio labeled cell for observation of cell behavior. In order to observe the hASCs, 3,3’dioctadecyloxacarbocyanine perchlorate (Dio) was used for labeling cell. Firstly, hASCs were harvested and washed with DMEM. Then, hASCs were regulated into 1×107 cells/mL by DMEM, followed by adding 5µL/mL Dio to the suspension. After incubating for 20 min under 37 oC with 5% CO2, supernatant was removed. LG-DMEM was added to adjust cell density into 1×107 cells/mL. Finally, cells were seeded into scaffolds and observed by laser scanning confocal microscope. Statistics of spheroid size was collected and analyzed with the assistance of Nano Measurer 1.2 Software. 2.10. Cytoskeleton staining. Phalloidine was employed for cytoskeleton staining. hASCs were fixed by 4% formaldehyde/PBS and dehydrated by acetone about 5 min, followed by being treated with Phalloidin (FITC) for 30 min at room temperature and washed with PBS. After that, cells were stained with 4’,6-diamidino-2-phenylindole (DAPI) for 30 second and washed again with PBS. Fluorescence microscope or laser scanning confocal microscope was used for observation. 2.11. Gene transfection. N,N,N-trimethyl chitosan chloride (TMC) and DNA were dissolved in Milipore water to a concentration of 1 mg/mL and 0.5 mg/mL respectively. After that, they were sterilized by microporous membrane and mixed for 30 second, incubated at 37 oC with 5% CO2 for 30 min before use. Quantitative analysis of plasmid DNA encoding transforming growth factor-β1 (pDNA-TGF-β1) was achieved by ELISA Kit. Cell number was determined by DNA that marked with Hoechst 33258, using a fluorometer. After correlating the amount of DNA with the number of cells by a standard curve, the expression level of TGF-β1 per 104 cells was calculated.


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2.12. Evaluation of in vitro chondrogenic differentiation. Scaffolds and cells were fixed with

4%

paraformaldehyde

and

embedded

in

paraffin,

sectioned

(5

µm

thick).

Immunohistochemical staining of collagen II (COL II) were carried out to show the chondrogenesis of ASCs in different scaffolds. Furthermore, COL II gene expression was also detected. Total RNA was isolated using Trizol and determined from the optical absorbance at 260 nm. cDNA was then synthesized using iScript cDNA Synthesis Kit (Bio-Rad). RT-PCR was performed using SsoAdvanced SYBRGreen Supermix in each reaction. Relative expression levels for each gene were calculated by normalizing the quantified cDNA transcript level to that of the GAPDH. GAPDH: forward 5’-AACAGCCTCAAGATCATCAGC-3’, reverse 5’GGATGATGTTCTGGAGAGCC-3’; COL II: forward 5’-CAACACTGCCAACGTCCAGAT-3’ and reverse 5’-CTGCTTCGTCCAGATAGGCAAT-3’. 2.13. In vivo implantation of the scaffolds with gene transfected ASC spheroids. A full thickness articular cartilage defect with dimensions of 4 mm in diameter and 2 mm in depth was created to the depth of the subchondral bone at a non-weight bearing area of the femur trochlea on the femoropatellar groove of the knee joints. 2 defects were created in one rabbit. Scaffold with TGF-β1 gene transfected autogenous ASC spheroids was implanted into one defect. The other defect was treated with nothing. 5 adult New Zealand white rabbits aged 12 weeks were used to carry 5 samples. At the same time, animals were euthanized at 8 weeks post-surgery for sample collection. Experimental protocol in the present study was approved by the Animal Care and Experiment Committee of Shanghai Jiao Tong University School of Medicine. 2.14. Histological examination. The neo-cartilages were harvested. Every sample was divided into two parts. One part was for the histological examination. The other part was for the biomechanical and biochemical analyses. For histological examination, samples were fixed in


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neutral buffered formalin, decalcified in formic acid, embedded in paraffin and sectioned (5 µm thick). Histology of cartilage was observed by hematoxylin and eosin (H&E), toluidine blue staining, and COL II immunohistochemical staining. 2.15. Biomechanical and biochemical analysis of the neo-cartilage. Neo-cartilages and normal cartilages were collected via a trephine, followed by being tested on a material testing machine (Zwicki-line Z 2.5KN) at a speed of 1 mm/min. Compressive moduli were automatically given by the device. After the mechanical test, samples were collected and lyophilized for 12 h, followed by adding 1 mL cold H2O and incubated at 4 °C overnight. Then, repeated freeze thawing and sonication cycles were carried out for samples lysis. After centrifugation, the supernatant was collected for GAG determination. The precipitate was for COL II assay using a Native Type II Collagen Detection Kit (Chondrex, USA) according to the manufacturer’s instructions. 2.16. Statistical Analysis. All date were reported as mean ± standard deviation (SD). Oneway analysis of variance (ANOVA) was performed to reveal statistical differences. A p-value of less than 0.05 was considered statistically significant. For in vitro experiments, each measurement reported was based on duplicate analysis of at three independent experiments. For in vivo histological, biochemical and biomechanical analyses, five samples at 8 w were subjected. 3. RESULTS AND DISCUSSION 3.1. Fabrication of PLGA-based porous scaffolds (S, S-PEGBA and S-PEG). As a famous ECM molecular, Fn is related to focal adhesion via Arg-Gly-Asp-Ser sequence that binds integrins, promotes the adhesion of cells to matrix.29,30 Thus, in the present study, Fn was


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employed to bind cells. The attachment and detachment of cells from scaffold was achieved by the capture and release of Fn via reversible imine linkage between aldehyde groups of scaffold and amino groups of Fn. During the present design, a well-structured scaffold that could prevent protein adsorption and cellular adhesion was fabricated at first. Previously we reported PLGA-based hydrogel scaffolds with hydration ability prevented cellular adhesion under conventional culture.31,32 Similarly, the present scaffold, which was defined as S, was fabricated from thiolated PLGA that cross-linked by PEG400 (polymer concentration 2%wt) as shown in Figure 1a. The well-organized porous structure with the pore size of 300-500 µm was achieved by salt particles (Figure 1b,c) (Supporting Information). Thiol groups that pre-grafted onto PLGA were used for further covalent modification through click chemistry of thiol-ene. Quantitative analysis of sulfhydryl content, as well as chemical structure of thiolated PLGA characterized through 1H NMR were in Supporting Information. XPS confirmed the present of thiol groups exposed on the inner surfaces of scaffold (Figure 1d). PLGA is a polyelectrolyte with hydrophilic nature. Such PLGA-based hydrogel substrates possess swollen networks do not adsorb any proteins, because the entropy changes due to a little more chain stretching become larger, the protein molecules become more strongly repelled from the surface.33-35 To realize the cell-scaffold adhesion on the anti-adhesion scaffold, APEGBA was synthesized by functionalizing APEG with carboxyl benzaldehyde as the reversible linkage to Fn. Imine bonds have been widely used in bio-conjugation and material science for their rapid formation under physiological conditions and their reversible nature.36-38 In the present study, for one thing, Fn capture was achieved through the formation of imine bonds. And aromatic aldehyde was used to stabilize the imine linkage between scaffold and Fn. For another, Fn release was realized


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through the break of the formed imine bonds. Moreover, APEG2000 with terminal double bonds was introduced to optimize the surface resistance to protein adsorption. Because benzene ring exhibits hydrophobic feature, which may form hydrophobic association with proteins. At the same time, PLGA-based scaffold possessed extra carboxyl groups, which may adsorb Fn or affect Fn conformation through electrostatic interaction.33,39,40 Thus, PEG segment of APEGBA was significant to disturb the electrostatic incorporation through hydration effect. Characterization of APEGBA synthesis was shown in Figure 1e,f. Then, APEGBA was grafted onto inner surfaces of PLGA-based scaffold through click chemistry of thiol-ene, to yield S-PEGBA. S-PEG was also prepared by grafting APEG to S as the control group. As shown in Figure 2a, structure of scaffold, as well as the inner surfaces with smooth appearance were not changed significantly. However, the content of thiol groups decreased significantly (over 78%) after the APEGBA or APEG grafting (Figure 2b). The content of thiol groups of each scaffold was listed in Supporting Information. The above results indicated the successful preparation of S-PEGBA. The function of S-PEGBA for Fn adsorption and cellular attachment needed further evaluation. 3.2. Fn adsorption and release of S, S-PEGBA and S-PEG. Fn adsorption and release was then carried out to evaluate the function of the present scaffold design. S, S-PEG and S-PEGBA were immersed in Fn solution for 4 h, followed by being dialyzed in PBS for 12 h. According to Figure 3a,b, The inner surfaces of S and S-PEG were still smooth. The representative fluorescence images of Fn staining showed weak red fluorescence presented around the S and SPEG scaffold, indicating the Fn resistance ability of S and S-PEG. Unlike S and S-PEG, SEM images showed that the smooth inner surfaces of S-PEGBA became rough with extensive matrix assembled on. Fluorescence presented around the S-PEGBA was significantly stronger with a


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wider range, indicating that Fn assembled on the inner surfaces of S-PEGBA. Quantitative analysis of Fn content showed that contents of Fn adsorbed in S and S-PEG were quite low, revealing the effective non-fouling ability. Besides, scaffold grafted with PEG (S-PEG) could repel Fn more effectively when compared with S. However, S-PEGBA exhibited efficient Fn combining ability. Fn adsorbed in S-PEGBA was significantly higher than that in S and S-PEG (Figure 3c). The above Fn adsorption results illustrated that the aromatic aldehyde covalently linked to S via PEG could efficiently form linkage with Fn. Furthermore, S-PEGBA adsorbed Fn was treated with lysine (Lys)/DMEM solution (Lys: 2 mg/ml) for 1 h, 2 h and 4 h, to evaluate the release of Fn from scaffold. Imine bond is a reversible linkage that sensitive to many biochemical stimuli including amino acids.41,42 Amine groups of Lys can react with benzaldehyde groups as the competitor of Fn protein, resulting in the release of Fn. As illustrated in Figure 3a, after being treated with Lys for 2 h, most Fn adsorbed on the inner surfaces of S-PEGBA was released, resulting in the restoration of smooth surfaces. Weak fluorescence of Fn staining also illustrated that there was little residual Fn around scaffold. Quantitative analysis of Fn content showed that over 60% Fn released from scaffold after 1 h treatment. Only 20% residual Fn was tested on scaffold after 2 h treatment (Figure 3d). Moreover, with the increase of Lys concentration (5 mg/ml), the efficiency of Fn release was promoted significantly. However, as shown in Supporting Information, Lys solution (5 mg/ml) exhibited more significant cytotoxicity. Thus, for cell detachment, Lys dilute solution was used to minimize its cytotoxicity effect. 3.3. Cell attachment, detachment and aggregation in S-PEGBA. ASCs from human were chosen as model cell to evaluate the function of scaffold for capturing and releasing cells. As shown in Figure 4, ASCs with rounded profile were observed to infiltrate the porous structure


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and attached uniformly on the inner surfaces of S-PEGBA at 12 h post-seeding, and kept the attachment during the whole in vitro culture if there was no Lys treatment. No significant ECM deposition was observed. However, after being immersed in DMEM containing Lys (2 mg/ml) for 1 h, the uniform distribution of ASCs was disturbed. The linkages between cells and scaffold inner surfaces seemed to be broken. ASCs tended to pile up. Then, S-PEGBA/ASCs was transferred back to growth medium for further culture. And along with the in vitro culture, significant aggregation of ASCs was observed. The spherical cellular aggregates were clearly identified within the inner pores after 24 h. According to SEM images, spheroids covered with massive ECM did not show the distinguishable individual cells. 3.4. Effect of scaffold stiffness on the process of cellular attachment, detachment and aggregation. It is worth noting that during the cell attachment and detachment process, ASCs kept rounded profile. Even when attaching to the inner surfaces of scaffold, ASCs showed no significant cytoskeleton spreading. The limited spreading illustrated a relatively weak interaction between cells and scaffold, and might indicate a limited gene transfection efficiency.24-26 Stiffness of the substrate was proved to be an effective method to regulate cytoskeleton spreading.43,44 Thus, to enhance cell-scaffold interaction and promote the cell spreading area, SPEGBA with improved mechanical strength was prepared by adding 3% PLGA/PEG400 crosslinking networks to yield a polymer concentration of 5%. According to Supporting Information, the stress-strain curve and the storage moduli illustrated that the added PLGA/PEG400 turned the softer S-PEGBA to a relatively stiffer one, while with same content of thiol groups to the former scaffold. As shown in Figure 5, ASCs adhered on the inner surfaces of stiffer S-PEGBA presented wider spreading area with cytoskeleton being pulled into polygon, indicating the stronger


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interaction between ASCs and scaffold that mediated by Fn. Accordingly, matrix stiffness was reported to affect amount of Fn adsorption, which might cause the different cell spreading shape. However, the content of Fn adsorption in softer scaffold was detected to be similar with that in stiffer scaffold (Supporting Information). At the same time, the Fn adsorption on the two scaffolds, the inner surfaces of which were modified with PEG (without aromatic aldehyde), was also detected. The result showed that both the PEG modified softer PLGA porous hydrogel and the PEG modified stiffer PLGA porous hydrogel were typical non-fouling substances that can repel protein (Supporting Information). Thus, in the present study, Fn conjugated by Schiff base to scaffold was more dominant than physical adsorption that impacted scarcely. In fact, the matrices stiffness shows significant impact on cell adhesion through a traction-force-mediated inside-outside in the mechanotransduction pathway. Adherent cells exert traction forces to sense the mechanical responses of the matrices. According to the applied tension forces and the corresponding matrix deformation, adhesions reinforce or disassemble.45-47 On stiffer substrate, the adhesion was reinforced, leading to the amplification of peripheral focal adhesions and the promotion of cell spreading. At the same time, matrix stiffness was reported to affect Fn adsorption, which might also cause the different cell spreading shape. It was thus valuable to find out According to Figure 6a, ASCs adhered in stiffer S-PEGBA showed extensive and continuous distribution. After being treated with Lys for 1 h, the area of green fluorescence began to shrink. The continuous distribution became independent at 1.5 h. After being transferred back to growth medium, the separated cell populations kept shrinking and finally formed regular spheroids. Observation towards one single pore was further showed in Figure 6b to illustrate the release process of captured ASCs and the formation process of spheroid. Unlike the process in softer S-


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PEGBA, the release of cells and formation of spheroid in stiffer S-PEGBA exhibited population transformation. At first, ASCs adhered on the inner surface of the pore formed a close cell-cell connection and cell-scaffold connection. After the blocking of cell-scaffold connection, the cell population detached from scaffold and began to shrink. Along with the shrink, more and more cell-cell connects drove the significant aggregation, leading to the superposition of cells that can be identified from the higher contrast of cell population. Moreover, as shown in Figure 7a-d, almost all cells in stiffer scaffold participated in spheroid formation, and the spheroids formed in stiffer scaffold were more compact than those formed in softer scaffold. However, in softer scaffold, although most cells aggregated to form spheroids, many cells did not participate in spheroid formation after Lys treating. And it was worth noting that the number of adhered cells in the two scaffolds was detected to be similar at 12 h post-seeding, showing no significant difference. However, after the Lys treatment, the formed spheroids in the two scaffolds possessed different size. The diameter of most spheroids form in softer scaffold was larger than that of spheroids form in stiffer scaffold. The reason may be related to the phenomenon that the spheroids formed in stiffer scaffold were more compact than those formed in softer scaffold (Figure 7e,f). 3.5. Evaluation of the scaffold for surface-mediated gene transfection. Along with the attachment, detachment and spheroids formation, the cell-scaffold interaction and cell-cell interaction changed. During attachment period, ASCs interacted with Fn fibrils via integrin cellsubstrate receptors. The activation of integrin α5β1 is essential for initial cell-substrate adhesion.48 As Figure 8a illustrated, the intracellular protein expression of integrin α5 and β1 in stiffer S-PEGBA were significantly up-regulated when compared with those in softer S-PEGBA, illustrating the amplification of peripheral focal adhesions in stiffer S-PEGBA. Substrate


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responded the traction forces exerted from cell with stiff mechanical single, leading to the higher degrees of cell spreading. During spheroid formation period, N-cadherin,27 the molecule for intercellular adhesion showed significant expression when compared with ASCs attached to the scaffolds as shown in Figure 8b. Whether the higher degrees of cell spreading and the spheroids formation were benefit for gene transfection and the corresponding protein expression, needed to be further evaluation. Thus, TMC condensing pDNA-TGF-β1 was chosen to evaluate S-PEGBA as the effective gene transfection system.49 0.5 µg TMC with condensed 1 µg DNA could yield small particles with the average diameter of 110 nm, which were then bound to the inner surfaces of S-PEGBA along with Fn (Figure 8c,d). Similar to Fn, TMC with quaternization degree of 10% also possesses residual amino groups that can be immobilized on the inner surfaces of S-PEGBA. ASCs with same cell number (5 × 107 cells/ml) were seeded into softer S-PEGBA and stiffer S-PEGBA, both of which were associated with Fn and TMC/DNA. After cellular attachment and incubation for 8 h, the S-PEGBA/ASC complexes were immersed in DMEM containing Lys (2 mg/ml) for 1 h, followed by being transferred back in growth medium for further spheroids formation and continued TGF-β1 expression. The scaffolds with attached ASCs without Lys treatment were employed as the controls. For the control groups, after 5 d in vitro culture, content of TGF-β1 expression in stiffer S-PEGBA was higher than that in softer S-PEGBA. The stiffer scaffold promoted ASCs spreading, leading to more extensive contacts of ASCs with TMC/DNA, as well as more extensive contacts between cells. Remarkably, the TGF-β1 expression after spheroids formation exhibited significant up-regulation both in softer and stiffer scaffolds. And due to the promoted gene transfection efficiency in stiffer scaffold, the followed TGF-β1 expression in spheroids was higher. After 10 d culture, the TGF-β1 expression in


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spheroids kept at a high level. However, the TGF-β1 expression in control groups were downregulated significantly. Especially for ASCs attached in the stiffer scaffold, TGF-β1 expression reduced 56%, which may be attribute to the active cellular proliferation (Figure 8e). Furthermore, with the induction effect of the secreted TGF-β1, the chondrogenesis of the ASCs at 10 d in vitro culture was evaluated. A specific gene that related to cartilage, collagen II (COL LL), was quantified through RT-PCR in Figure 8f. The result was consist with the TGFβ1 expression. COL II gene expression in spheroids was significantly higher than that of ASCs attached inside the scaffolds. COL II gene expression of attached ASCs inside stiffer scaffold was higher than that of attached ASCs inside softer scaffold. COL II gene expression of ASC spheroids inside stiffer scaffold was higher than that of ASC spheroids inside softer scaffold. Moreover, COL II protein also expressed according to COL II immunohistochemical staining (Figure 8g, h). Positive expression of COL II was observed distributing along with the inner surfaces of scaffold. However, more significantly positive COL II expression was observed in spheroids. The enhanced chondrogenesis of ASC spheroids in stiffer S-PEGBA should attribute to the efficient gene transfection and the spheroid formation. In spheroids, there were extensive cell-cell interactions with up-regulated N-cadherin expression, which is involved in and significant for mesenchymal chondrogenesis.10,11 Accordingly, N-cadherin regulates the expression of Sox 9 and promotes MSCs chondrogenesis. Our previous work also illustrated that the N-cadherin expression up-regulated the chondrogenic differentiation of ASCs.12 3.6. Scaffold carrying gene transfected spheroids for in vivo cartilage regeneration. Above all, the porous scaffold realized the efficient gene transfection, as well as the in vitro chondrogenesis. In addition, the scaffold was based on PLGA, a synthetic polypeptide with wellperformed biological and physico-chemical properties, including non-toxic, hydrophilic and


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biodegradable. Scaffolds and hydrogels based on PLGA have been proved to support in vivo tissue regeneration, such as cartilage, bone and fat.12,31,50 Thus, the present scaffold was considered to be a well-performed implant. Then, the scaffolds carrying gene transfected autogenous rASC spheroids were implanted into full thickness articular cartilage defects for in vivo evaluation of cartilage regeneration (Figure 9a). As shown in Figure 9b, macroscopic observation showed that the defects were fulfilled with neo-tissue at 8 w post-implantation, showing similar appearances with host cartilage, including color and texture. Notably, the boundary between neo-tissue and surrounding normal cartilage was unobvious. No obvious immunological or infectious complications were observed. Histological analyses were then carried out to identify the neo-tissue. It was found that the neo-tissue possessed similar thickness with surrounding normal cartilage in experimental group (Figure 9c). Also, Cellular volume and density were similar to normal cartilage. More importantly, cells in neo-tissue exhibited similar arrangement features with those in normal cartilage, which were aligned perpendicularly to the articular surface. Cartilage lacuna structure was observed obviously (Figure 9d). While defects in control group with no-treatment were filled with fibrous tissue, showing no cartilage-like tissue regeneration (Figure 9e). Furthermore, toluidine blue staining and immunohistochemical staining were carried out to determine GAGs and COL II deposition, respectively. As shown in Figure 9f,g, deposition of GAGs and COL II were significant, indicating that the neo-tissue was cartilage-like tissue. While COL I immunohistochemical staining was negative as shown in Figure 9h, indicating limited fibrous matrix deposition. Meanwhile, quantitative analysis of GAGs and COL II in neo-cartilage and normal cartilage further confirmed the staining results. As shown in Figure 9i,j, GAGs and COL II contents of neo-cartilage at 8 weeks reached 83%, 82% of those of the normal cartilage,


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respectively. Along with the deposition of cartilage matrix, the neo-cartilage exhibited wellperformed mechanical property. The compressive modulus of neo-cartilage was 2.1±0.3 Mpa, reaching 80% of normal cartilage compressive modulus (Figure 9k). Together with histological analysis results, the functionalized scaffold carrying gene transfected ASC spheroids realized the reconstruction of cartilage morphology and function. Above all, the present gene transfection system can not only realize the efficient gene transfection, promoting the TGF-β1 expression, but also be implanted directly in vivo for successful cartilage regeneration. Besides its advantage towards chondrogenesis and great potential for cartilage regeneration in vivo, in fact, the present gene transfection system is a platform that can be applied towards other cell types and genes. The sequential cell attachmentdetachment-spheroids formation process support effective gene transfection with the enhanced subsequent related protein expression. At the same time, the biocompatible PLGA-based scaffold is quite suitable for in vivo application. 4. CONCLUSION Above all, the present study introduced an implantable biocompatible scaffold that could realize gene transfection towards MSC spheroids for cartilage regeneration. Aromatic aldehyde was introduced to the inner surfaces of PLGA-based scaffold with anti-cell adhesion property, endowing the scaffold with Fn capture and release ability via formation and interrupt of imine linkage. ASCs attachment was realized by Fn adsorption. Cell spreading area was promoted through the increase of scaffold stiffness. Together with TMC/pDNA-TGF-β1, surface-mediated gene transfection was achieved during cell-scaffold adhesion. Cell detachment was controlled by the treatment of Lys for further cellular aggregation to form spheroids in-situ. The multicellular spheroids in scaffold exhibited promoted expression of TGF-β1 as long as 10 d. Chondrogenic


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differentiation was also enhanced. Well-regenerated cartilage-like tissue in vivo showed the advanced potential of the present scaffold in cartilage tissue engineering.

ASSOCIATED CONTENT Supporting Information. Preparation of thiolated PLGA scaffold (S) and the 1H NMR of thiolated PLGA. Pore size of the porous scaffold (a) and quantitative analysis of sulfhydryl content. Cytotoxicity of Lys tested through CCK-8. Stress-strain curves of scaffold with polymer concentration of 2% and 5%. Cell distribution in the four scaffolds at 72 h post-seeding without Lys treating. The content of thiol groups. Method to establishment of standard curve.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (K.Z.). *E-mail: [email protected] (J.Y.). ORCID Kunxi Zhang: 0000-0002-6341-3182 Jingbo Yin: 0000-0001-7614-0331 Notes


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The authors declare no competing financial interest. ACKNOWLEDGMENT This research is supported by the National Natural Science Foundation of China (Nos. 51503119, 51773113), the Science and Technology Commission of Shanghai Municipality (No. 15JC1490400), and Shanghai University youth teacher training program. Also, we acknowledge the Instrumental Analysis Research Centre (Shanghai University) for the use and direction of 1H NMR, SEM. The Confocal Laser Scanning Microscope imaging of the cells by Xiaoyong Deng is acknowledged. REFERENCES (1) Sasai, Y. Next-Generation Regenerative Medicine: Organogenesis from Stem Cells in 3D Culture. Cell Stem Cell 2013, 12, 520−530. (2) Fatehullah, A.; Tan, S. H.; Barker, N. Organoids as an In Vitro Model of Human Development and Disease. Nat. Cell Biol. 2016, 18, 246−254. (3) Cheng, N. C.; Chen, S. Y.; Li, J. R.; Young, T. H. Short-Term Spheroid Formation Enhances the Regenerative Capacity of Adipose-Derived Stem Cells by Promoting Stemness, Angiogenesis, and Chemotaxis. Stem Cells Transl. Med. 2013, 2, 584−594. (4) Pampaloni, F.; Reynaud, E. G.; Stelzer, E. H. The Third Dimension Bridges the Gap between Cell Culture and Live Tissue. Nat. Rev. Mol. Cell Biol. 2007, 8, 839−845. (5) Abbott, A. Cell Culture: Biology’s New Dimension. Nature 2003, 424, 870−872. (6) Griffith, L. G.; Swartz, M. A. Capturing Complex 3D Tissue Physiology In Vitro. Nat. Rev. Mol. Cell Biol. 2006, 7, 211−224. (7) Uccelli, A.; Moretta, L.; Pistoia, V. Mesenchymal Stem Cells in Health and Disease. Nat. Rev. Immunol. 2008, 8, 726−736. (8) Ankrum, J. A.; Ong, J. F.; Karp, J. M. Mesenchymal Stem Cells: Immune Evasive, Not Immune Privileged. Nat. Biotechnol. 2014, 32, 252−260. (9) Cesarz, Z.; Tamama, K. Spheroid Culture of Mesenchymal Stem Cells. Stem Cells Int. 2016, 2016, 9176357. (10) Bian, L. M.; Guvendiren, M.; Mauck, R. L.; Burdick, J. A. Hydrogels that Mimic Developmentally Relevant Matrix and N-cadherin Interactions Enhance MSC Chondrogenesis. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 10117−10122.


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Scheme 1. Design and function of the porous scaffold.


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Figure 1. Preparation of the scaffold (S) and synthesis of APEGBA. a) gross observation of S, and the polymer network of S; b) stereomicroscope observation of S; c) SEM image of S; d) XPS detection of the surface of S; e,f) 1H NMR and FT-IR of APEGBA. δ=4.96, 4.89, 3.93-3.50, 1.74


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ppm belong to APEG (a, e, b). New peaks at δ=10.12, 8.24, 8.22, 7.98, 7.96, 4.53, 4.52, 4.51 ppm correspond to APEGBA (d, c’, c, f). In FT-IR, peak at 1697 cm-1 was corresponding to the stretching vibration of carbonyl on ester bond and aldehyde group. (Bar scale: 200 µm for b; 100 µm for c)

Figure 2. Preparation and characterization of S, S-PEGBA and S-PEG. a) SEM images of S, SPEGBA and S-PEG; b) quantitative analysis of thiol groups. The more the yellow, the more thiol groups. (# and * mean these data was significant different from others) (Bar scale: 200 µm for left column images of a; 20 µm for right column images of a)


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Figure 3. Fn adsorption and release. a) SEM images and b) Fn staining images of S, S-PEG and S-PEGBA after adsorbing Fn, as well as S-PEGBA (+Fn) treated with Lys solution; c) Fn capture on S, S-PEG and S-PEGBA at 2, 4 and 12 h; d) Fn released from S-PEGBA at 1, 2 and 4 h through Lys solution treatment. Lys concentration in DMEM was 5 and 2 mg/ml. (* p＜0.05, ** p＜0.01, # means the data was significant different from others) (Bar scale: 100 µm for top line images of a; 5 µm for bottom line images of a; 2 µm for b)


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Figure 4. ASCs response to S-PEGBA. a) stereomicroscope observation of attached ASCs in SPEGBA, and ASC spheroids formed after Lys treatment; b) CLSM and SEM images to show the Dio-labled ASCs attachment-detachment-aggregation process in S-PEGBA. After being treated with Lys for 1 h, attached ASCs detached from scaffold, followed by the aggregation. (Bar scale: 100 µm for a, top line images of b; 50 µm for bottom line images of b)


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Figure 5. ASCs shape and cytoskeleton responded to softer (a) and stiffer (b) scaffold. The stiffer S-PEGBA forced ASCs attached with polygon shape and wider attached area. (Bar scale: 100 µm for left column images of a and b; 20 µm for the right three column images of a and b)


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Figure 6. ASCs response in the stiffer S-PEGBA. a) CLSM monitored the response process of Dio-labled ASCs in the stiffer S-PEGBA; b) digital/fluorescence merged images showed one ASC spheroid formation in an inner pore of the stiffer S-PEGBA. (Bar scale: 100 µm for a; 50 µm for b)


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Figure 7. Phase contrast microscope showed the cell aggregation efficiency. a,b) most cells aggregated to form spheroids (blue arrows), but many cells in soft scaffold did not participate in spheroid formation after Lys treating, as indicated by red arrows; c,d) the spheroids (blue arrows) formed in stiffer scaffold were more compact than those formed in softer scaffold. Almost all cells in stiffer scaffold participated in spheroid formation. e) cell number in softer scaffold and stiffer scaffold at 12 h post-seeding; f) diameter range of spheroids. (Bar scale: 100 µm)


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Figure 8. Evaluation of gene transfection. a) Integrin α5β1 gene expression; b) N-cadherin gene expression; c) TMC/DNA complexes; d) TMC/DNA adsorbed on the inner surfaces of S-


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PEGBA; e) TGF-β1 expression tested via ELISA; f) COL II gene expressions; g) and h) COL II immunohistochemical staining of attached ASCs and aggregated ASCs in scaffolds. (* p＜0.05, data in f was significantly different with each other) (Bar scale: 500 nm for c, d; 100 µm for g, h; 30 µm for g1)

Figure 9. In vivo evaluation cartilage regeneration. a) full thickness articular cartilage defect was created in rabbit knee joints. Scaffold carrying gene transfected ASC spheroids was implanted into the defect; b) gross observation of the repaired cartilage at 8 w; c) H&E staining of cartilage in experimental group. Black arrow indicated the boundary between neo-tissue and surrounding normal cartilage; d) higher-magnification images selected from the normal cartilage areas and neo-cartilage areas; e) H&E staining of no-treatment defect at 8 w in control group; f) toluidine blue staining; g) COL II immunohistochemical staining; h) COL I immunohistochemical staining; i) GAGs content; j) COL II content; k) compressive moduli. (* p＜0.05) (Bar scale: 200 nm for c, d, f, g; 50 µm for e) Graphical Abstract


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Functionalized Scaffold for in Situ Efficient Gene Transfection of

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