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Tissue Engineering and Regenerative Medicine

Co-delivery of Synovium-derived Mesenchymal Stem Cells and TGF-# by a Hybrid Scaffold for Cartilage Regeneration Hongjie Huang, Xiaoqing Hu, Xin Zhang, Xiaoning Duan, Jiying Zhang, Xin Fu, Linghui Dai, Lan Yuan, Chunyan Zhou, and Yingfang Ao ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.8b00483 • Publication Date (Web): 20 Dec 2018 Downloaded from http://pubs.acs.org on December 25, 2018

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ACS Biomaterials Science & Engineering

Co-delivery of Synovium-derived Mesenchymal Stem Cells and TGF-β by a Hybrid Scaffold for Cartilage Regeneration

Hongjie Huang,† Xiaoqing Hu,† Xin Zhang,† Xiaoning Duan,† Jiying Zhang,† Xin Fu,† Linghui Dai,† Lan Yuan,‡ Chunyan Zhou,§ Yingfang Ao*,†

†

Institute of Sports Medicine, Peking University Third Hospital, Beijing Key Laboratory of

Sports Injuries, 49 North Garden Road, Haidian District, Beijing 100191, People’s Republic of China ‡

Medical and Healthy Analysis Centre, Peking University, 38 Xueyuan Road, Haidian

District, Beijing 100191, People’s Republic of China §

Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences,

Peking University, 38 Xueyuan Road, Haidian District, Beijing 100191, People’s Republic of China * Corresponding author. Tel.: +86 10 82267390; fax: +86 10 62010440. E-mail addresses: [email protected]

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ABSTRACT: Synovium-derived mesenchymal stem cells (SMSCs) are attractive tissue-specific cells for cartilage regeneration because of their easy availability, higher chondrogenic potential, and joint specificity. In the present study, we established a hybrid scaffold to co-deliver SMSCs and transforming growth factor beta (TGF-β), which can integrate the scaffolds, the growth factor and the autogenous cells within rabbit cartilage defects. A chitosan hydrogel and a decellularized bone matrix were used to fabricate the gel-solid duplex phase biomaterials, which were proven to retain more cells, promote tissue integration and provide mechanical support. In vitro experiments demonstrated that this hybrid scaffolds could release TGF-β in a controlled biphasic pattern, which can promote cell proliferation and chondrogenic differentiation of loaded rabbit SMSCs. For in vivo experiments, we filled cartilage defects with the hybrid materials, delivering autogenous SMSCs and TGF-β simultaneously via chitosan’s sol–gel transition. Histological analysis, magnetic resonance imaging, and nanoindentation assessment indicated superior cartilage regeneration using this co-delivery system compared with that from routine microfracture or control delivery scaffolds. Beyond cartilage regeneration, the easy preparation, favorable biophysical properties and controlled release ability make this co-delivery system applicable to transport other tissue-specific cells or biofactors for tissue engineering. KEYWORDS:

Cartilage

tissue

engineering;

Hybrid

scaffolds;

Synovium-derived

mesenchymal stem cells; TGF-β; Controlled release

2


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1. INTRODUCTION Articular cartilage injuries, especially large cartilaginous defects, remain an intractable challenge in the field of regenerative medicine because of their avascular histological characteristic and the paucity of resident cells.1-2 Partial-thickness cartilage injuries do not heal spontaneously, while full-thickness defects are repaired by fibrous tissue with low biomechanical properties, leading to articular degeneration or osteoarthritis.2 There is a substantially high incidence of cartilaginous injuries among the population aged 30 and older, as detected by magnetic resonance imaging (MRI) or arthroscopy exploration.3-4 The poor intrinsic repair capacity of articular cartilage, means that much effort has been devoted to repairing cartilage and preventing further degeneration of the injured cartilage, such as microfracture (MF), mosaicplasty, or autologous chondrocyte implantation (ACI), which can relieve some clinical symptoms, but fail to regenerate functional hyaline cartilage.5 Recently, tissue engineering strategies have been applied in the regeneration of articular cartilage, which involves the integration of cell sources, signaling molecules, and/or biological materials.6 As cell sources, mesenchymal stem cells (MSCs) show significant potential in tissue engineering research and clinical practice, and can partly overcome donor site morbidity and the limited availability of autologous chondrocytes. Synovium-derived mesenchymal stem cells (SMSCs) are a promising cell source for cartilage tissue engineering because of their easy availability and preparation, and high chondrogenic potential.7-8 Therefore, increasing attention has focused on SMSCs, thanks to their preferable chondrogenic differentiative capacity both in vitro and in vivo.9 In particular, synovial tissue is often discarded after partial excision of the synovium in a knee surgery. Furthermore, 3



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SMSCs may respond appropriately to mechanical or chemical stimulation of the joint cavity because of their intra-articular origination, which can facilitate cartilage tissue regeneration.9 During cartilage regeneration, signaling molecules or biofactors play important roles in cell proliferation, extracellular matrix (ECM) synthesis, induction of MSCs chondrogenesis and maintenance of the phenotype. The superfamily of transforming growth factor-β (TGF-β) has been proven to fulfill these biological functions.10 However, during tissue engineering, the biological functions of growth factors are largely dependent upon the scaffold structure or culture environment, especially when delivered via controlled release strategies.11-12 In addition, simulation of the biological and structural features of the native 3D microenvironment can facilitate MSC proliferation and differentiation.13 With many simulation to native microenvironment, hydrogels are attractive materials for the repair of cartilage defects.14 Chitosan hydrogel is a promising scaffold for biomedical applications because of its biocompatibility, biodegradability, nontoxicity and antimicrobial activity.15 A neutral chitosan solution displays a thermosensitive gelation property but low strength.16 DBM is a 3D collagen network with pliable, but strong mechanical properties. And DBM has favorable biocompatibility without biotoxicity and immunogenicity because of the absence of cellular antigens.17 In the present study, we used a hybrid scaffold to co-deliver SMSCs and TGF-β3, which can integrate the scaffolds, the growth factor, and the autogenous cells for cartilage tissue engineering (Figure 1). The SMSCs, which were harvested from knee synovium and proliferated in vitro, could be transported simultaneously with TGF-β via a chitosan hydrogel. The chitosan hydrogel and a decellularized bone matrix were integrated into one gel-solid 4


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duplex phase biomaterial mimicking the structure of native cartilage. The favorable biophysical properties, as well as the controlled release of TGF-β, facilitated the chondrogenesis of SMSCs and cartilage regeneration.

Figure 1. Schematic illustration of the proposed co-delivery strategy for the SMSCs and TGF-β3 using a hybrid scaffold for cartilage regeneration.

2. MATERIALS & METHODS 2.1 Cells isolation SMSCs were isolated according to a previous study.18 In brief, synovial membranes were obtained aseptically from the knees of 4~5 month-old New Zealand white rabbits (weight, 2.3~2.5 kg). The dissected synovial membrane was repeatedly rinsed in sterile 5



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phosphate-buffered saline (PBS) solution supplemented with 1% penicillin–streptomycin. The tissues were then trimmed into small fragments (1 mm3), which were enzymolysised with 0.2% collagenase I (GIBCO Invitrogen, Carlsbad, CA, USA) overnight at 37 °C. The enzymolysised tissues were transferred into a 10 cm2 flask containing Dulbecco's Minimum Eagle’s Medium (DMEM) containing 10% Fetal Bovine Serum (FBS). The SMSCs were then incubated in a humid atmosphere of 5% CO2 and 37 °C for 3–4 days and allowed to adhere to the flasks. Non-adherent cells were washed out by changing the medium. 2.2 Characteristics of SMSCs The differentiation potential of SMSCs was identified using a trilineage-induced differentiation experiment including adipogenesis, osteogenesis, and chondrogenesis. SMSCs at passage 4 were used for this experiment. For adipogenesis, the SMSCs were cultured in a 6-well plate (1 × 105 cells/well) with a commercial MSC adipogenic differentiation medium (Cyagen Biosciences Inc., Guangzhou, China). After 1-week of culture, the SMSCs were fixed and stained with oil red O to determine the extent of adipogenesis. The osteogenic induction culture of SMSCs was similar to the adipogenic differentiation, but the cells were cultured in osteogenic differentiation medium for 2 weeks. Alizarin red staining was performed to evaluate the osteogenic capacity of the SMSCs. For chondrogenesis, a micromass culture was used according to a routine protocol.19 In brief, passage 4 SMSCs were trypsinized and resuspended with growth media at a density of 1 × 107 cells/ml. Ten-microliter droplets were seeded in a 24-well plate and incubated for two hours at 37 °C to form a layer of cell aggregation. Then, 200 μl of a commercial chondrogenic medium (RASMX-90041; Cyagen Biosciences Inc., Guangzhou, China) was gently added and 6


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changed every three days without disturbing the cell aggregation. After 3 weeks of culture, the SMSC micromass was fixed with 4% paraformaldehyde and stained with Toluidine Blue to assess its glycosaminoglycan (GAG) formation. 2.3 Preparation of scaffolds Decellularized bone matrix (DBM) was made according to our previous study.13,

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Briefly, the dissected epiphyses of sheep femur epiphyseals were defatted by immersion in a 1:1 mixture solution of chloroform and methanol for 48 hours. The defatted samples were decellularized for 1 month at 4 °C in ethylene diamine tetraacetic acid solution (EDTA; 0.5 M, pH=8.3), which was changed every day and detected continuously by atomic absorption spectrophotometry to track demineralization. We also performed a radiographical test to ensure complete demineralization of the samples. The decellularized matrix was trimmed into a predetermined shape and stored at −80 °C. Before the in vitro or in vivo experiments, the prepared matrix was sterilized under cobalt−60 irradiation for 1 day. Chitosan hydrogel was prepared according a previously reported method.13, 21 In brief, 9 ml of chitosan solution (2.5 wt% in 0.1 M acetic acid solution; Aladdin, Shanghai, China) was stirred at 4 °C and 1 ml of β-glycerophosphate solution (60 wt% in ddH2O; Sigma, St. Louis, MO, USA) was added dropwise to form the final solution (pH = 7.2). This solution, with or without TGF-β3, was used to perfuse the DBM materials and incubated subsequently at body temperature (for about 15 min) to form the hybrid material with gel-solid duplex phase, which was used for SMSCs delivery in vitro or in vivo. 2.4 Release of the growth factor We performed an enzyme-linked immunosorbent assay (ELISA) to determine the 7



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release of the growth factor from the hybrid scaffolds. Briefly, the scaffolds loaded with 10 ng/μl of TGF-β3 were incubated with PBS at 37 °C.22 The cultured PBS was changed in 2 or 3 days and collected for analysis using a TGF-β3 ELISA kit (NO-KA4402, Abnova Corporation, Walnut, CA, USA) according to the manufacturer’s protocol. The pattern of growth factor release from the hybrid materials was profiled as the mass of the released TGF-β3 over time. 2.5 Co-delivery of SMSCs and TGF-β3 for in vitro 3D culture SMSCs at passage 4 were used for in vitro 3D culture. In brief, SMSCs were collected and resuspended in the prepared chitosan solution (3.0 × 107 cells ml−1). TGF-β3 was added to form a chitosan solution with 10 ng/μl TGF-β3. Then, about 20 μl of this solution (approximate 6.0 × 105 cells) were used to perfuse the DBM materials, which were then gelatinized at body temperature for 15 min to achieve a hybrid scaffold. This hybrid scaffold was transferred into a 24-well petri dish and washed three times (every 10 minutes) to remove residual β-glycerophosphate and untouched TGF-β3 using a basal medium (RASMX-90041; Cyagen Biosciences Inc., Guangzhou, China), which contained 0.1 μM dexamethasone, 50 μg/ml ascorbate, 100 μg/ml sodium pyruvate, 1× ITS+Supplement, and 40 μg/ml proline. We added 10 ng/ml TGF-β3 in basal medium to form complete chondrogenic medium. We then set four groups for in vitro SMSC culture: hybrid scaffolds without any TGF-β (control group), hybrid scaffolds without TGF-β cultured in complete chondrogenic medium (TGF in the culture medium), hybrid scaffolds loaded with TGF-β cultured in basal medium (TGF in the scaffolds) and hybrid scaffolds loaded with TGF-β cultured in complete chondrogenic medium (TGF in the scaffolds and medium). 8


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2.6 Biochemical analysis 2.6.1 Total DNA quantification We analyzed the changes of DNA content quantitatively to evaluate SMSC proliferation within the hybrid scaffolds. In brief, after 1, 2, and 3 weeks of culture, scaffolds loaded SMSCs were washed with PBS to remove the medium. We determined the weight of the samples after removing redundant liquid with an absorbent paper. The samples were snap-frozen with liquid nitrogen and lyophilized by critical point drying for 12 hours. The samples were then digested at 60 °C for 2 days in a commercial papain solution (Sigma-Aldrich). The digest solution was stored at −80 °C until biochemical analysis. The DNA content of the SMSCs within the scaffolds was measured using fluorometric detection with a Hoechst-33258 reagent (Polysciences Inc., Warrington, PA, USA). In brief, 20 μl of the digest solution were added into 200 μl of Hoechst-33258 working solution (2 μg/ml) and incubated for 1 hour at 37 °C. Then, a multimode reader (Varioskan Flash, Thermo Scientific, Waltham, MA, USA) was used to detect the fluorescence intensities at 360 nm excitation and 460 nm emission. A standard curve of calf thymus DNA (sigma) was used to calculate the DNA content. 2.6.2 Analysis of the proteoglycan content The glycosaminoglycan (GAG) content was determined to evaluate the proteoglycan production

by

SMSCs

within

the

scaffolds

via

a

dye-binding

assay

with

1,9-dimethylmethylene blue (DMMB; Sigma). Briefly, 20 μl of the above-mentioned digestion was incubated with 200 μl of DMMB dye for 1 hour at 37 °C. Then, a plate reader was used to detect the absorbance at 525 nm. A standard curve was established using shark 9



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chondroitin 6-sulfate (Sigma) to determine the GAG content. 2.6.3 Estimation of collagen production The hydroxyproline content was quantified to determine the collagen production of SMSCs cultured within the hybrid materials. The above-mentioned digested solution was further hydrolyzed in 6 M HCl solution for 18 hours at 110 °C. A chloramine-T/Ehrlich’s spectrophotometry assay at 560 nm was then performed to detect the hydroxyproline content of the hydrolyzed sample. A standard curve using L-hydroxyproline (Sigma) was constructed to calculate the hydroxyproline content. 2.7 Analysis of gene expression Quantitative real-time polymerase chain reaction (qPCR) analysis was used to assess the effects of different TGF delivery patterns on gene expression during chondrogenic differentiation, cartilaginous hypertrophy, or osteogenesis. The qPCR primers for key genes are detailed in Table S1, including cartilaginous genes (Aggrecan, AGC; Collagen II, COL2), key osteogenesis markers (Collagen I, COL1; Osteocalcin, OCN), and a cell hypertrophy maker (Collagen X, COL10). After 1, 7, 14, and 21 days of culture, the scaffolds (n = 3) were rinsed with PBS and snap-frozen immediately in liquid nitrogen. The frozen samples were pulverized using a mortar pre-cooled in liquid nitrogen. Total RNA was extracted from the pulverized samples using a commercial TRIzol reagent (Invitrogen). Reverse transcription of the extracted RNA was then performed using a commercial kit (Promega, Madison, WI, USA) according to a routine method. A PikoReal Real-time PCR System (Thermo Fisher Scientific Inc., Rockford, IL, USA) with SYBR Green PCR Master Mix (Toyobo, Osaka, Japan) was used for the real-time PCR analysis. Finally, a dissociation stage was achieved following the 10


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amplification procedure, which did not show any nonspecific amplification from the dissolved curve. The expression changes of the target genes was evaluated in relative levels to GAPDH by the △△Ct method. Briefly, △Ct values of each gene at 1, 7, 14, or 21 days of culture were calculated by normalization to the expression of the GAPDH gene. In this analysis, △△Ct means the difference in △Ct values between 7, 14, 21 days and 1 day. We performed at least three independent assays for each value. 2.8 In vivo animal experiments The in vivo animal experiments were approved by the local Institutional Animal Care and Use Committee, and complied with the “Guide for the Care and Use of Laboratory Animals” (NIH Publication No. 85-23, revised 1996). Briefly, we used a trephine to drill a large full-thickness chondral defect (4 mm diameter, 2 mm deep) on the trochlear groove of New Zealand white rabbits (5–6 months-old, weight 2.7–3.0 kg, n = 14). A pre-trimmed DBM (4 mm diameter, 2 mm height) was implanted into the defect. The prepared chitosan solution was injected into the defect for the delivery of TGF-β (TGF delivery group), autologous SMSCs (SMSC delivery), and the TGF-β and autologous SMSCs (Co-delivery group) (Figure 1). The implanted scaffolds were then adjusted to the same level as the surrounding native cartilage. The operated knees were repositioned and protected with sterile gauze to allow the sol-gel transition of the chitosan solution (15 min) at body temperature. Finally, we performed several cycles of knee flexion and extension to ensure that the hybrid materials were retained within the defect before closure. As a control, a standard MF procedure alone was performed within the defects, using a G16 syringe needle to penetrate subchondral bone in 1-millimeter interval (MF group). The experimental animals were raised 11



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in cages postoperatively with no restriction of motion. 2.9 Histology At 4 and 12 weeks postoperatively, the repaired knees (n = 3) were harvested, and the surrounding soft tissues were dissected away. A general observation of the repaired tissues was performed immediately to evaluate tissue filling of the defects, integration with the surrounding cartilage and surface smoothness. For histological evaluation, the repaired knees were fixed for 2 days at 4 °C in 4% paraformaldehyde (pH 7.4), and decalcified for 2 weeks at 4 °C in 15% EDTA (pH 7.2 in PBS) containing 5% paraformaldehyde. After decalcification, the repaired sites were trimmed out, and dehydrated in a series of graded ethanol before paraffin embedding. Then the center of the repaired tissues was cut coronally into 7-μm slices, which were stained with a standard hematoxylin & eosin (HE) or safranin O staining.23 Moreover, an immunohistochemical assay for collagen II was conducted using a commercial antibody (Calbiochem Cat No. CP18-100UG; Novabiochem, Boston, MA, USA) according to the manufacturer’s protocol. We also assessed cartilage repair quantitatively using a common grading scale24 (Table S2) based on gross observation and histological performance. 2.10 Scanning electron microscopy At 12 weeks, the surface of the repaired tissues was scanned microscopically using a high-resolution scanning electron microscope (SEM). In brief, the repaired sites (n = 3) were drilled out with an empty Φ5 mm trephine in the interest of noncontact with the cartilage surface. The samples were fixed immediately in 4 ml of 25% v/v glutaraldehyde for 24 hours at 4 °C, dehydrated in a series of graded ethanol, and transformed into a state of absolute 12


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dehydration by critical point drying. A 5-nm layer of gold was then used to paintcoat the surface of the repaired tissues in a high-vacuum gold spatter coater. After sample preparation, a high-resolution SEM (S-2500; Hitachi Ltd., Tokyo, Japan) was used to complete the microscopic scanning. 2.11 Cartilage magnetic resonance imaging Magnetic resonance imaging (MRI) scanning was performed to evaluate the imageology quality of the repaired knees at 12 weeks. After auricular vein anesthesia, the knees were put in a small animal-specific knee coil (Chenguang Medical Technologies Co., Ltd, Shanghai, China) and scanned using a Siemens TIM Trio 3T (T) MRI scanner (Siemens, Erlangen, Germany) according to our previous study.25 Five sequences of MR (35 minutes) were conducted for all samples to cover routine scanning and quantitative analysis (Table S3). In addition, we asked for the assistance of a senior musculoskeletal radiologist to calculate the T2 and T2* values using an inline processing package (SyngoMapIt; Siemens). 2.12 Nanoindentation of the repaired tissue We used a nanoindentation experiment to analyze the biomechanical properties of the repaired tissues according to previous studies.25-26 The samples (n = 5 for each group) were drilled out as in the SEM experiment, and normal control samples (n = 5) were prepared using the trochlea of untreated knees. The samples were put in a confocal dish and fixed via Vaseline fixation to ensure that the repaired tissue faced upwards vertically. During the experiments, the sample was maintained in a state of hydration with PBS at room temperature. A TriboIndenter System (Hysitron Inc., Minneapolis, MN, USA) was used for this experiment. A series of loading (10 s), holding (2 s), and unloading (10 s) forces were 13



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delivered using a 400-μm radius curvature conospherical diamond probe tip, which was performed on each indent site to form a trapezoidal load function. A depth of 550 nm was set as the maximum indentation for all experiments. Meanwhile, a snapshot using micro-scanning apparatus was taken to observe the nanoscale microstructure of the surface on the repaired tissues. Normal cartilage was tested as the positive control group. 2.13 Statistical analysis In this study, we repeated each experiment at least three times independently. The data are presented as mean ± standard deviation (SD). After homogeneous analysis of variance, we used a one-way analysis of variance (ANOVA) to determine the statistical differences between groups, and least significant difference (LSD) multiple tests for pairwise comparisons. Significance was set as a P-value < 0.05.

3. RESULTS 3.1 SMSC characterization We performed an aseptic partial synovectomy to harvest SMSCs. After in vitro passage and expansion, the SMSCs exhibited a homogeneous phenotype (Upper in Figure 2). At passage 4, SMSCs were assessed for differentiation into various lineage in vitro, which showed that the cells exhibited the potential to differentiate into lipocytes, osteocytes and chondrocytes (Bottom in Figure 2). The results indicated that the SMSCs harvested and cultured in this study were suitable for the experiments because of their homogeneity and identified tri-lineage differentiation capacity.

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Figure 2. Characterization of SMSCs differentiation capacity. SMSCs at passage 4 showed a homogeneous distribution (Upper). We performed oil red O, alizarin red or toluidine blue staining for adipogenesis, osteogenesis, or chondrogenesis, respectively (Bottom). Bar = 200 μm.

3.2 Controlled released of the growth factor from the hybrid materials During the building of the scaffolds, we noted that the chitosan hydrogel solution could be infused into the decellularized bone matrix (Left in Figure 3A) easily and uniformly, forming the hybrid scaffolds (Right in Figure 3A). The hybrid scaffolds showed an interconnected architecture macroscopically or microscopically, in which the chitosan thermogel was interspersed uniformly within the decellularized matrix (Right in Figure 3B). The addition of TGF-β did not influence the gelation time (12–15 min) of chitosan during the hybrid scaffold fabrication. The release kinetics of TGF-β from the hydrogel-DBM materials was determined using ELISA over 25 days (Figure 3C). The total growth factor loaded into 15



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the scaffolds was approximately 227 ± 32 ng (n = 27). TGF-β was released from the hybrid materials in a biphasic fashion, which was characterized by a fast rate of release over the first 2 weeks, followed by slower release for the rest of the test. Although at a much lower level than that at Day 1, the routine ELISA could still detect the released TGF-β at Day 25. Moreover, the experiment was terminated at Day 25 and was possible that more TGF could be released later.

Figure 3. Fabrication of the hydrogel-DBM scaffolds and TGF-β controlled released. (A) The hybrid scaffold (Right) comprised chitosan hydrogel and decellularized bone matrix (Left). (B) SEM images of DBM (Left) and the hybrid scaffold (Right). (C) Release of TGF- β from the hybrid scaffolds (n = 3 at each interval).

3.3 Chondrogenic differentiation of SMSCs encapsulated in the hybrid scaffolds Three different TGF delivery patterns and a negative control group based on the hybrid 16


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scaffolds were assessed for their influence on the expression of related genes. The expression level of cartilage-related genes (AGC and COL2) was significantly upregulated in the three groups involving TGF stimulation during the culture period, and the control group showed a significant lower expression of COL2 because of the lack of the growth factor (Figure 4A-i, ii). This indicated that supplementation with TGF-β through the medium or scaffolds could stimulate significantly chondrogenic differentiation of SMSCs compared with that of the pure DBM-hydrogel materials. For osteogenic differentiation, we did not detect any significant changes of related genes (COL1 and OCN) in all groups throughout in vitro culture (Figure 4A-iii, iv). For hypertrophy, the hybrid scaffolds loaded with TGF-β plus chondrogenic medium (10 ng/ml TGF-β3) showed significantly higher COL X expression (Figure 4A-v). Moreover, there was also a slight down-regulation of COL2 at Day 21 in the double TGF stimulation group, which might have been caused by the high-dose of TGF (Figure 4A-ii). The results suggested that TGF-β delivery by the hybrid materials could promote prolonged chondrogenic differentiation of the encapsulated SMSCs, without any undesired osteogenesis or hypertrophy.

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Figure 4. The proliferation and chondrogenic capacity of SMSCs during in vitro 3D culture. (A) The expression of related genes (n = 3, *p < 0.05, **p < 0.001): i-AGC, ii-COL2, iii-COL2, iv-OCN and v-COL10. (B) In vitro cell proliferation and cartilaginous matrix formation: i, DNA content of SMSCs within scaffolds in three TGF delivery patterns and in the negative control group (Wt, wet weight; n = 5, *p < 0.05); ii, glycosaminoglycan (GAG) contents of the different groups (n = 5, *p < 0.05, **p < 0.001); iii, hydroxyproline (HYP) changes of different groups (n = 5, *p < 0.05, **p < 0.001).

3.4 SMSCs proliferation and cartilage matrix production within the scaffolds We estimated the proliferation and chondrogenic behavior of SMSCs in three different TGF delivery patterns and in the negative control group (Figure 4B). The weight and DNA 18


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content of these four groups significantly increased during in vitro SMSCs culture without any difference among them (Figure 4B-i). We also perform a LIVE/DEAD assay to test the cell activity, which showed this hybrid scaffolds could support activity of SMSCs within a uniform distribution (Figure S1). We tested the glycosaminoglycan (GAG) and hydroxyproline (HYP) content to evaluate the cartilaginous matrix production by the encapsulated SMSCs during in vitro culture. GAG production was significantly increased during culture with the single TGF delivery (scaffolds or medium) groups (Figure 4B-ii). Moreover, significantly higher GAG production was observed in the single TGF stimulating groups than in control group at 2 and 3 weeks. Similar to the results of GAG, SMSCs within the single TGF-delivery scaffolds produced significantly more collagen content throughout 3 weeks of culture (Figure 4B-iii). Moreover, TGF released by the hybrid scaffolds stimulated more HYP production than duplex TGF simulation, which was consistent with the COL2 qPCR results. These results indicated the SMSCs could survive and proliferate within the hybrid materials. The co-delivery of SMSCs and TGF-β by the hybrid scaffolds could stimulate chondrogenic differentiation and promote more cartilaginous matrix production during in vitro culture. 3.5 Macroscopic and microscopic evaluation of the repaired knees The repaired knees were harvested for macroscopic and microscopic observations at 4 and 12 weeks post-operatively. The cartilage defects in the MF control group were scarcely filled at 4 weeks (Figure 5A-i), whereas tissue filling within the defects was revealed in the groups involving the hybrid scaffolds (Figure 5A-ii, iii, iv). However, the TGF delivery group showed tissue filling with a clear difference in color or tissue quality compared with the 19



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surrounding cartilage (Figure 5A-ii). The repaired filling of the co-delivery scaffolds was more complete and homogeneous (Figure 5A-iv). At 12 weeks, the knees repaired by the MF procedure or single TGF delivery displayed obvious degeneration (Figure 5A-v, vi), and the defects repaired by single SMSCs delivery via the hybrid materials showed complete tissue filling although there was some difference with the adjacent tissues (Figure 5A-vii). Although the repair tissue in the co-delivery group was a little whiter compared with the normal cartilage, it should be noted the neo-cartilage tissue was gradually remodeled and integrated with the surrounding cartilage (Figure 5A-viii). These results showed that chondral defects could be effectively filled using these gel-solid hybrid materials, which could facilitate the formation and remodeling of repaired cartilage tissues. Microscopic evaluation by SEM was performed to observe the changes in micromorphology on the surfaces of the repaired tissues (Figure 5B). At 12 weeks postoperatively, there were still obvious cracks within the repaired tissues in the MF group (Figure 5B-i). In the single TGF or SMSCs delivery groups, small cracks and fiber-like tissues were observed on the surface throughout the defects. The surface of the repaired cartilage in co-delivery group showed a smooth and homogeneous microscopic appearance (Figure 5B-iv).

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Figure 5. Macroscopic and microscopic observation of the repaired tissues. (A) We captured images of the repaired knees to evaluate their gross morphology at 4 and 12 weeks (n = 5; scale bar, 1 cm). (B) Scanning electron microscopy (SEM) was used to acquire the micromorphology of the surface of the repaired tissues at 12 weeks postoperatively (n = 3).

3.6 Histological assessment of cartilage repair At 1 and 3 months postoperatively, the MF group showed barren filling in the cartilage defects (Figure 6-i, v), and the MF controls showed extensive cartilage degeneration at 12 weeks (Figure 6-v). The single TGF or SMSCs delivery groups showed improved filling of the defect, with deep clefts (Figure 6A-ii, iii), and these groups showed obvious abrasion on the surface of the repaired cartilage at 12 weeks (Figure 6A-vi, vii). Moreover, we noted that 21



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there were more cells in the repaired site in the SMSCs delivery groups (Figure 6A-iii, iv). The co-delivery group showed continuous tissue repair within the defects. Although there were still small clefts on the surface between the repaired site and normal cartilage at 12 weeks, the major part of the regenerated tissue was integrated with the surrounding tissues (Figure 6A-viii). The SMSCs translated into a round or oval shape with an aggregation trend, especially in the magnified image of Fig.6-viii. These findings suggested that the co-delivery of TGF and SMSCs via this hybrid material could effectively enhance tissue filling within the cartilage defects and facilitate cartilage-like tissue formation.

Figure 6. Hematoxylin & eosin staining of repaired tissue (n = 3). The co-delivery of TGF and SMSCs via hybrid materials can more effectively facilitate tissue filling within the defects during cartilage repair compared with the other groups. The magnified images of each group show the cell morphology and distribution in the cartilage defects.

3.7 Cartilaginous matrix specific staining Safranin O staining and collagen II immunohistochemistry (IHC) were performed to evaluate the quality of the repaired tissues. Consistent with the gross observation and HE 22


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staining, the MF controls demonstrated lack of filling and progressive degeneration in the repaired sites (Figure 7-i, v). Although the single delivery groups showed improved cartilage matrix staining in the repaired tissues, there were still some deep clefts (Figure 7-ii, iii) or subsequent abrasions (Figure 7-vi, vii) within the repaired sites. SMSCs delivery showed more positive staining for cartilaginous ECM, which may reflect higher number of cells. However, the co-delivery scaffolds promoted the formation of cartilaginous matrix in the process of tissue filling and remodeling within the defects (Figure 7-iv, viii). At 12 weeks, the defects repaired by co-delivery of TGF and SMSCs via the hybrid material showed uniformly positive staining for the cartilage-like matrix. The results of IHC staining (Figure 8) corresponded with those of safranin O staining, and showed superior cartilage-like tissue formation in the co-delivery group without hypertrophy or calcification. These results indicated that the cells can be transformed into a chondrocyte-like phenotype with cartilaginous production, especially under stimulation by TGF.

Figure 7. Safranin O staining of repaired tissue (n = 3). The co-delivery scaffolds induced the formation of cartilaginous tissue with uniformly positive staining at 12 weeks (viii) although there was a thin layer of fibrous tissue on the surface of injury sites at 4 weeks (iv).

23



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Figure 8. Collagen II immunohistochemistry of the repaired tissue (n = 3). The quality and quantity of repaired cartilage in the co-delivery group were superior to that of the single delivery scaffolds or MF groups.

3.8 MRI examination and quantification High-resolution MRI showed an obvious degeneration of the joint in the MF group at 12 weeks postoperatively (Figure 9A-i). We also noted effusion and cartilage abrasion within the joints in the single delivery groups (Figure 9A-ii, iii). By contrast, uniform filling of the defects was revealed in the co-delivery group at 3 months, which was consistent with the histology results (Figure 9A-iv). We captured the T2 and T2* mapping to evaluate and quantitatively analyze the repaired tissues at 3 months. In Figure 9B, the MF or single delivery groups showed abnormally high signals and disorganized variations within the joints. The sites repaired by co-delivery displayed uniform cartilage mapping with lower values. For quantitative analysis, the co-delivery group showed significantly superior T2 values compared with those of the MF or single delivery groups (Left in Figure 9C). The T2 relaxation time of the SMSCs delivery group was also significantly lower than that in the MF 24


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group (Left in Figure 9C). In addition, the T2* value of the co-delivery group was significantly better than those of the MF or TGF delivery groups (Right in Figure 9C). These MR imaging results implied that the co-delivery materials could effectively facilitate the generation of cartilaginous tissue.

Figure 9. MRI observation and quantification of repaired knees. (A) Representative MR imaging of repaired tissues at 12 weeks after implantation (i-MF, ii-TGF delivery group, iii-SMSC delivery group; iv-co-delivery scaffolds; white bar, 1 cm). (B) we also captured the T2 mapping of different groups at 12 weeks (i-MF, ii-TGF delivery group, iii-SMSC delivery group, and iv-co-delivery scaffolds). The pseudo color scale indicates the relaxation time (millisecond, ms). (C) T2 relaxation time (Left in Fig. 9C) and T2 star relaxation time (Right in Fig. 9C) of the repaired tissues were quantified based on MR images (n = 3, *p < 0.05, **p 25



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< 0.01).

3.9 Biomechanical properties of repaired tissues and normal cartilage At 12 weeks, we tested the biomechanical properties of the repaired tissues and normal cartilage using nanoindentation. Snapshots images of the articular surface of the Microfracture control and single delivery materials were scraggy and rough (Figure 10A-i, ii). However, the micro-geomorphology of the surface on the neo-cartilage induced by co-delivery of TGF and SMSCs was integrated and smooth, which was comparable to that of normal cartilage (Figure 10A-iii, iv, v). Based on the load-displacement curves (Figure 10B), we calculated the biomechanical properties (reduced modulus and hardness) of normal cartilage and the different material groups. The reduced modulus accounts for the elastic deformation or resilience of the sample, which measures desponds on the initial unloading slope, if the contact area at peak load can be measured independently. The MF controls and TGF delivery scaffolds showed a significantly lower reduced modulus value than the other three groups, and the groups involving SMSCs delivery had a comparably reduced modulus value to that of native cartilage (Left in Figure 10C). The value of hardness is the mean pressure the repaired tissue will support under loading, and the MF controls and TGF delivery scaffolds also had a significantly lower hardness. Moreover, the hardness of the cartilage in the co-delivery group was significantly higher than that of the SMSCs delivery group, which may be caused by the stimulation of TGF (Right in Figure 10C). Therefore, the biomechanical experiments demonstrated that the co-delivery materials could induce well-organized cartilaginous tissue generation with a comparable mechanical strength to that 26


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of native cartilage.

Figure 10. Biomechanical experiments of repaired tissue and native cartilage. (A) Nanoscale snapshot of the repaired tissue captured at the beginning of the experiment (i-MF, ii-TGF delivery group, iii-SMSC delivery group; iv-co-delivery scaffolds, v-normal group). (B) Typical load-indentation curves in repaired tissues were compared with native cartilage. Reduced modulus (Left in Fig.10C) and hardness (Right in Fig.10C) of different material groups were also compared with the values of native cartilage, based on the biomechanical curves (n = 5, *p < 0.05, **p < 0.01) (i-MF; ii-TGF delivery group; iii-SMSC delivery group; iv-co-delivery scaffolds; v-normal group).

4. DISCUSSION Mesenchymal stem cells (MSCs), derived from many tissues, such as bone marrow, adipose, periosteum, or synovium, have become a promising cell source for cartilage regeneration. In 2001, synovium-derived mesenchymal stem cells (SMSCs) were first characterized, which show a favorable feature of chondrogenic differentiation both in vitro 27



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and in vivo.18 SMSCs may be a ‘‘tissue-specific’’ cell for articular cartilage regeneration because of their intimate origin relationship with the articular milieu interne.9 In the literature, tissue-specific stem cells are temporarily defined as a population of cells that originates from a specific tissue of one organ and can communicate with the organ-specific microenvironment.9, 27 In other words, the origin of stem cells influences their differentiation potential.28-29 He et al. used a decellularized extracellular matrix to perform in vitro 3D culture of SMSCs, and dramatically increased cell proliferation and greatly enhanced chondrogenic capacity were observed without concomitant adipogenesis or osteogenesis.30 In the present study, the SMSCs showed a significant chondrogenic differentiation and produced considerably more collagen content during in vitro 3D culture, especially under control stimulation of TGF (Figure 4). The low density of endogenous MSCs and uncertain targeting to damaged tissue impede endogenous MSC-based procedures, like microfracture (MF), especially in larger cartilage defects.25 In this study, autogenous SMSCs were expanded during in vitro culture and transported into the cartilage defect site via a hybrid scaffold platform. This hybrid scaffold could hold a sufficient quantity of cells in the cartilage defects via the sol-gel transition of the thermogel, and DBM could protect the neo-cartilage formation and maturation by its biomechanical properties, as revealed in our previous study.13, 25 Furthermore, SMSCs may respond most appropriately to mechanical or chemical stimulation of the joint cavity and potentially transform into a chondrocyte-like phenotype with cartilaginous production because of their intra-articular origin, which could facilitate cartilage tissue regeneration.9, 27-29

The animal experiments showed that the defect filling by the biomaterials (Figure 5, 6) 28


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was superior than that of the microfracture group, and the cell content in the defect was dramatically increased through the delivery of SMSCs using the scaffold (Figure 6-iii, iv; Figure S2). The remodeling and maturation of the repaired tissue was sufficient because of increased cell retention and growth factor stimulation (Figure 6-iv, viii). TGF-β, a 25 kDa polypeptide with a mature dimer structure, has been widely proposed to stimulate MSC chondrogenesis in tissue engineering studies thanks to its potent and pleiotropic regulation of cell behavior, especially when delivered via controlled release strategies.31 Although there is no consensus about the optimal dose (10 ng/mL is used frequently) of TGF-β for chondrogenic differentiation, maintaining a threshold concentration for a specific period is vital to initiate and maintain chondrogenic differentiation.22,

31

By

contrast, high dose release may not be beneficial practically for chondrogenic stimulation. In the literature, continuous high concentration induction by TGF-β may cause the accumulation of aggrecan cleavage products during in vitro culture or hypertrophic tissue in vivo.12, 32 In this study, hypertrophy or aggrecan down-regulated expression was caused by dual TGF-β stimulation via scaffolds delivery plus culture medium (Figure 4A-ii, v). Similarly, arbitrary high doses of TGF-β in the joint cavity led to pathological changes, such as synovial fibrosis, synovitis, synovial vascularization and cartilage degeneration. The bio-modulation effects of TGF-β therefore necessitate its delivery in a controlled manner. Many compounds and scaffolds have been generated to achieve a controlled, localized release of TGF-β in vitro or in vivo.31 They can be categorized into hydrogels, solid and hybrid scaffolds. Hydrogels are polymeric networks consisting of cross-linked hydrophilic polymers such as hyaluronic acid (HA), chitosan, collagen, fibrinogen, fibrin, and agarose.13, 29



33

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In general, hydrogels are characterized by good biocompatibility, high permeability for

nutrient substances, and availability of cell delivery in a homogeneous pattern.34 One of the most attractive features for cartilage regeneration is that cells encapsulated in hydrogels can maintain a spherical chondrocyte phenotype without dedifferentiation.13,

33

Otherwise,

hydrogels can be delivered to the site of interest via simple injection, providing a more stable interface between the scaffolds and the surrounding natural cartilage. In this study, the biomaterials promoted the integration between the repaired tissue and the surrounding cartilage (Figure 6-8), which is consistent with our previous reports.25 By contrast, hydrogels also showed some disadvantages, such as a high degradation rate and low mechanical properties, which might influence the release pattern of internal factors, implant maturation, and ECM remodeling.13, 25 For controlled release, the rapid degradation of the carrier often causes a burst release of the encapsulated growth factors.31 However, solid scaffolds can provide sufficient mechanical support immediately after implantation, but their highly porous nature results in inhomogeneous cell distribution with possible cell dedifferentiation and loss of ECM components over time.13 To circumvent these shortcomings, combinations of both hybrid hydrogels and matrix materials are a highly attractive approach. We used a chitosan hydrogel (30-70 μm) and a decellularized bone matrix (300-600 μm) to fabricate the gel-solid hybrid materials with an intertwined micro-framework of two different pore sizes, mimicking the structure of proteoglycans and the collagen network within native cartilage. From our previous report, the degradation rate of the hybrid materials was somewhere between that of the hydrogels and solid scaffolds.13 In the present study, this hybrid material showed controlled release of TGF-β, characterized by a rapid release rate in the first 2 weeks, 30


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followed by slower release for the rest of the test (Figure 3C), which could promote the chondrogenic differentiation of SMSCs (Figure 4). The hybrid scaffolds can hold more repairing cells homogeneously within the thermosensitive hydrogel and provide mechanical protection for neo-cartilaginous tissue. Moreover, the animal experiments indicated that co-delivery of autogenous SMSCs and the growth factor could effectively facilitate the repaire of cartilage. Furthermore, the mechanical strength of the repaired tissue and the integration with adjacent healthy cartilage was improved using this co-delivery scaffold system. The current study has a limitation that need to be discussed. We concluded the difference of in vivo cartilage repairing between groups based on a rabbit model (4 mm diameter, 2 mm deep), which has not been systematically studied for its spontaneous repair potential. It has reported that 3-mm defects of rabbit knees may heal spontaneously with repair tissue composed of hyaline or fibrocartilage.35-37 A larger defects in rabbit trochlear (>6 mm) or large animal models (mini pig or goat) are recommendable for cartilage regeneration study.38 And systematically researches are needed to determine the spontaneous repairing of cartilage defects in various sizes for different animal models.

5. CONCLUSIONS In the present study, we demonstrated that a gel-solid duplex phase biomaterial, composed of chitosan hydrogel and a decellularized bone matrix, could be used to deliver SMSCs and growth factors simultaneously. In vitro experiments demonstrated that this hybrid scaffold could release TGF-β in a controlled, bi-phasic manner, which could promote 31



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cell proliferation and chondrogenic differentiation of loaded SMSCs. For the in vivo experiments, we filled cartilage defects with chitosan-matrix hybrid materials, which can deliver autogenous SMSCs and TGF-β simultaneously via chitosan’s sol–gel transition. Consistent with our previous study, the hybrid scaffolds could retain more cells, promote tissue integration and provide strength support. This was indicated a superior cartilage regeneration by the co-delivery system compared with routine microfracture or control delivery scaffolds. Beyond cartilage regeneration, the easy preparation, favorable biophysical properties, and controlled release ability make this co-delivery system applicable to transport other tissue-specific cells or biofactors for tissue engineering.

ASSOCIATED CONTENT Supporting Information MATERIALS & METHODS: LIVE/DEAD assay Table S1. Real-time PCR primers Table S2. Histological scoring system Table S3. MRI examination imaging sequences Figure S1. Viability of SMSCs in scaffolds. According to a LIVE/DEAD assay, this hybrid scaffolds can support activity of SMSCs within a uniform distribution. (A, optical microscope image; B, green fluorescence labelling live cells; C, red fluorescence labelling dead cells; D, merge image). Figure S2. Histological score for repaired knees during in vivo cartilage repairing. There was a significant improvement from 4 to 12 weeks in the co-delivery group (#n = 5, 32


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p < 0.05). Moreover, the scores of co-delivery group were superior to MF control or TGF delivery groups at 12 weeks (*n = 5, p < 0.01).

AUTHOR INFORMATION Corresponding Author: *E-mail: [email protected]. ORCID: 0000-0002-8909-2022 Notes: The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by grants from the Beijing Municipal Natural Science Foundation (No. 7174361). We are also thankful for the help from Youth project of National Natural Science Foundation of China (No. 81601927). We are thankful to Prof. Ming Qiang, who provided the expertise and facility needed in nanoindentation analysis, and Dr. Ziyan Zhao and Dr. Huimin Yin for help in Magnetic Resonance Imaging of animal knee cartilage.

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For Table of Contents Use Only.

Co-delivery of Synovium-derived Mesenchymal Stem Cells and TGF-β by a Hybrid Scaffold for Cartilage Regeneration Hongjie Huang, Xiaoqing Hu, Xin Zhang, Xiaoning Duan, Jiying Zhang, Xin Fu, Linghui Dai, Lan Yuan, Chunyan Zhou, Yingfang Ao

36


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Figure 1. Schematic illustration of the proposed co-delivery strategy for the SMSCs and TGF-β3 using a hybrid scaffold for cartilage regeneration. 132x117mm (300 x 300 DPI)



Figure 2. Characterization of SMSCs differentiation capacity. SMSCs at passage 4 showed a homogeneous distribution (Upper). We performed oil red O, alizarin red or toluidine blue staining for adipogenesis, osteogenesis, or chondrogenesis, respectively (Bottom). Bar = 200 μm. 199x119mm (300 x 300 DPI)


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Figure 3. Fabrication of the hydrogel-DBM scaffolds and TGF-β controlled released. (A) The hybrid scaffold (Right) comprised chitosan hydrogel and decellularized bone matrix (Left). (B) SEM images of DBM (Left) and the hybrid scaffold (Right). (C) Release of TGF- β from the hybrid scaffolds (n = 3 at each interval). 101x68mm (300 x 300 DPI)



Figure 4. The proliferation and chondrogenic capacity of SMSCs during in vitro 3D culture. (A) The expression of related genes (n = 3, *p < 0.05, **p < 0.001): i-AGC, ii-COL2, iii-COL2, iv-OCN and v-COL10. (B) In vitro cell proliferation and cartilaginous matrix formation: i, DNA content of SMSCs within scaffolds in three TGF delivery patterns and in the negative control group (Wt, wet weight; n = 5, *p < 0.05); ii, glycosaminoglycan (GAG) contents of the different groups (n = 5, *p < 0.05, **p < 0.001); iii, hydroxyproline (HYP) changes of different groups (n = 5, *p < 0.05, **p < 0.001). 150x124mm (300 x 300 DPI)


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Figure 5. Macroscopic and microscopic observation of the repaired tissues. (A) We captured images of the repaired knees to evaluate their gross morphology at 4 and 12 weeks (n = 5; scale bar, 1 cm). (B) Scanning electron microscopy (SEM) was used to acquire the micromorphology of the surface of the repaired tissues at 12 weeks postoperatively (n = 3). 122x100mm (300 x 300 DPI)



Figure 6. Hematoxylin & eosin staining of repaired tissue (n = 3). The co-delivery of TGF and SMSCs via hybrid materials can more effectively facilitate tissue filling within the defects during cartilage repair compared with the other groups. The magnified images of each group show the cell morphology and distribution in the cartilage defects. 74x27mm (300 x 300 DPI)


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Figure 7. Safranin O staining of repaired tissue (n = 3). The co-delivery scaffolds induced the formation of cartilaginous tissue with uniformly positive staining at 12 weeks (viii) although there was a thin layer of fibrous tissue on the surface of injury sites at 4 weeks (iv). 78x31mm (300 x 300 DPI)



Figure 8. Collagen II immunohistochemistry of the repaired tissue (n = 3). The quality and quantity of repaired cartilage in the co-delivery group were superior to that of the single delivery scaffolds or MF groups. 89x40mm (300 x 300 DPI)


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Figure 9. MRI observation and quantification of repaired knees. (A) Representative MR imaging of repaired tissues at 12 weeks after implantation (i¬-MF, ii-TGF delivery group, iii-SMSC delivery group; iv-co-delivery scaffolds; white bar, 1 cm). (B) we also captured the T2 mapping of different groups at 12 weeks (i¬-MF, iiTGF delivery group, iii-SMSC delivery group, and iv-co-delivery scaffolds). The pseudo color scale indicates the relaxation time (millisecond, ms). (C) T2 relaxation time (Left in Fig. 9C) and T2 star relaxation time (Right in Fig. 9C) of the repaired tissues were quantified based on MR images (n = 3, *p < 0.05, **p < 0.01). 128x109mm (300 x 300 DPI)



Figure 10. Biomechanical experiments of repaired tissue and native cartilage. (A) Nanoscale snapshot of the repaired tissue captured at the beginning of the experiment (i¬-MF, ii-TGF delivery group, iii-SMSC delivery group; iv-co-delivery scaffolds, v-normal group). (B) Typical load-indentation curves in repaired tissues were compared with native cartilage. Reduced modulus (Left in Fig.10C) and hardness (Right in Fig.10C) of different material groups were also compared with the values of native cartilage, based on the biomechanical curves (n = 5, *p < 0.05, **p < 0.01) (i-MF; ii-TGF delivery group; iii-SMSC delivery group; iv-co-delivery scaffolds; v-normal group). 76x38mm (300 x 300 DPI)


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Codelivery of Synovium-Derived Mesenchymal ... - ACS Publications

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