Book-Shaped Acellular Fibrocartilage Scaffold with Cell-loading

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Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Book-Shaped Acellular Fibrocartilage Scaffold with Cell-loading Capability and Chondrogenic Inducibility for Tissue-Engineered Fibrocartilage and Bone−Tendon Healing Can Chen,†,‡,§ Fei Liu,‡,§ Yifu Tang,†,‡,§ Jin Qu,†,‡,§ Yong Cao,‡,§,∥ Cheng Zheng,†,# Yang Chen,†,‡,§ Muzhi Li,†,‡,§ Chunfeng Zhao,¶ Lunquan Sun,□ Jianzhong Hu,*,‡,§,∥ and Hongbin Lu*,†,‡,§ Downloaded via UNIV OF NEW ENGLAND on January 10, 2019 at 08:39:03 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

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Department of Sports Medicine and Research Centre of Sports Medicine, ∥Department of Spine Surgery, □Center for Molecular Medicine, Xiangya Hospital, Central South University, Changsha, Hunan, China, 410008 ‡ Key Laboratory of Organ Injury, Aging and Regenerative Medicine of Hunan Province, Changsha, Hunan, China, 410008 § Xiangya Hospital-International Chinese Musculoskeletal Research Society Sports Medicine Research Center, Changsha, Hunan, China, 410008 # Department of Orthopedics, Hospital of Wuhan Sports University, Wuhan, Hubei, China, 430079 ¶ Division of Orthopedic Research and Department of Orthopedic Surgery, Mayo Clinic, Rochester, Minnesota 55905, United States S Supporting Information *

ABSTRACT: Functional fibrocartilage regeneration is a bottleneck during bone−tendon healing, and the currently available tissue-engineering strategies for fibrocartilage regeneration are insufficient because of a lack of appropriate scaffold that can load large seeding-cells and induce chondrogenesis of stem cells. The acellular fibrocartilage scaffold (AFS) contains active growth factors as well as tissue-specific epitopes for cellmatrix interactions, which make it a potential scaffold for tissue-engineered fibrocartilage. A limitation to this scaffold is that its low porosity inhibits cells loading and infiltration. Here, inspired by book appearance, we sectioned native fibrocartilage tissue (NFT) into book-shape to improve cells loading and infiltration, and then decellularized with four protocols: (1) 2% SDS for 6-h, (2) 2% SDS for 24-h, (3) 4 SDS for 6-h, (4) 4% SDS for 24-h, followed by nuclease digestion. The optimal protocol was screened with respect to microstructures, DNA residence, native ingredients reservation, and chondrogenic inducibility of the AFS. In vitro studies demonstrated that this screened scaffold is noncytotoxicity and lowimmunogenicity, allows adipose-derived stromal cells (ASCs) attachment and proliferation, shows superior chondrogenic inducibility, and stimulates collagen or glycosaminoglycans secretion. The underlying mechanism for this chondrogenic inducibility may be related to hedgehog pathway activating. Additionally, a novel pattern for fabricating tissue-engineered fibrocartilage was developed to enlarge seeding-cells loading, namely, cell-sheets sandwiched by book-shaped scaffold. In-vivo studies indicate that this screened scaffold alone could induce endogenous cells to satisfactorily regenerate fibrocartilage at 16week, as characterized by fibrocartilaginous extracellular matrix (ECM) deposition and good interface integration. Interleaving this book-shaped AFS with autologous ASCs-sheets significantly enhanced its ability to regenerate fibrocartilage. Cell tracking demonstrated that fibrochondrocytes, osteoblasts, and osteocytes in the healing interface at postoperative 8-week partly originated from the sandwiched ASCs-sheets. On that basis, we propose the use of this book-shaped AFS and cell sheet technique for fabricating tissue-engineered fibrocartilage to improve bone−tendon healing. KEYWORDS: fibrocartilage regeneration, tissue-engineering, adipose-derived stromal cells, book-shaped acellular scaffold, bone−tendon healing bone, calcified fibrocartilage, noncalcified fibrocartilage and tendon, which is considered to mediate and transfer of

1. INTRODUCTION Tendon normally insert into the bone through a transitional multitissue interface with spatial gradients in composition, structure, and mechanical properties.1 This multitissue interface, namely, bone−tendon interface (BTI), is histologically divided into 4 distinct yet continuous tissue layers: © XXXX American Chemical Society

Received: November 28, 2018 Accepted: December 21, 2018

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DOI: 10.1021/acsami.8b20563 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces myotility.1,2 Regrettably, BTI injuries are common in modern sports activities, and its functional healing still remain a formidable challenge in sports medicine.3−6 The cause for this complicated healing is partly due to the structural heterogeneity of BTI, more importantly because the native fibrocartilage regenerates slowly and incompletely.2,6,7 Therefore, how to promote functional fibrocartilage regeneration is pivotal to the healing of BTI injuries. Various approaches, such as biophysiotherapies and biotherapies, can effectively regenerate fibrocartilage during bone−tendon healing.2,3,5,8−11 However, these approaches are often inadequate at recapitulating functional fibrocartilage in a short time, thus the patients are in a state of injury recurrence for a long time.2,12,13 To date, tissue-engineering has emerged as a promising strategy for solving this problem, utilizing a combination of scaffolds, seed cells and bioactive molecules to replace or repair damaged tissues.6,14 Among the three factors, the scaffolds can be fundamentally critical, providing structural support, mimicking the in vivo microenvironment and potentially influencing cellular activities.15 Recent years, acellular tissue scaffolds have generated great interest in the field of tissue-engineering because of their low immunogenicity, high biocompatibility, good biodegradability, and high similarity in the architecture and composition of extracellular matrix (ECM) to the target tissue.15,16 Additionally, this scaffold has diverse physiological functions such as a reservoir of cytokines and growth factors, transmitting specific signals through interactions with cell surface receptors, and providing natural microenvironments to regulate the phenotype, attachment, migration and proliferation of cells.17,18 In musculoskeletal tissues, acellular scaffolds derived from articular cartilage were shown to stimulate chondrogenic differentiation of bone marrow stromal cells (BMSCs) and adipose-derived stromal cells (ASCs), and help differentiated cells maintain their phenotypes well, consequently proving them to be excellent in regenerating cartilage in vivo.15,16,19 Logically, fabricating an acellular scaffold derived from fibrocartilage tissue may be an ideal choice for tissue-engineered fibrocartilage. However, native fibrocartilage is a dense and compact tissue with low porosity, it is technically difficult to absolutely remove its antigenic cellular materials, while mostly conserving its native ECM and inherent bioactive ingredients, especially chondrogenic active growth factors (CAGFs).15,20 Furthermore, a whole block of acellular fibrocartilage scaffold (AFS) was not convenient for large number of seeding-cells loading and endogenous cells infiltration.21,22 Currently, the commonly utilized methods for tissue decellularization include a combination of physical and chemical treatments.21,23,24 Since fibrocartilage is dense, compact and low porosity in architecture, physically sectioning fibrocartilage tissue into book-shape was innovatively developed in this study, which can increase surface area for improving the permeation of chemical agents into tissue and help reduce the exposure time of tissue to chemical agents, thus reserving more native ECM ingredients.25 Moreover, this shape is convenient for graft handling during surgery and seeding-cells sandwiching, thus showed better capacity for cells loading. Toward chemical treatment, various approaches for cartilage decellularization have been reported with SDS, Triton X-100, EDTA and trypsin, but the method which can absolutely remove cells and mostly retain ECM and bioactive ingredients has not been reported.23,25 In this study, we aim to optimize the decellularization protocols for preparing AFS, thus can

effectively remove cellular components with only minimal disruption to other components, such as collagen, GAGs, and CAGFs. Seeding-cells is another critical component for tissueengineering.6,14 BMSCs and ASCs are normally considered as promising candidates for tissue-engineering owing to their rapid proliferation ability, multidifferentiation potential and ease to harvest.26 Both of them have strong chondrogenesis, thus have been widely used in combination with different scaffolds to repair articular cartilage in preclinical and clinical studies.27,28 Compared with BMSCs, the great advantages of ASCs are that they are convenient for harvesting, possesses multidifferentiation potential and easily expanded with genomic stability and lower immunogenicity.28 Thus, ASCs may be more suitable seeding-cells for tissue-engineered fibrocartilage. Toward seeding-cells transplantation, mesenchymal stromal cells (MSCs) have traditionally been injected as a suspension or combined with biomaterials, such as fibrin, silk, or polyglycolic acid for fibrocartilage regeneration during bone−tendon healing.8,29−31 However, these traditional methods have several limitations, including cell washout, insufficient cell retention, or an inadequate distribution of transplanted cells,32 which may hinder functional fibrocartilage regeneration due to the lack of fibrochondrocytes and ECMproducing cells. Currently, the emergence of cell-sheet technology addresses those shortcomings. Using temperature-responsive culture dishes, the acquired cell-sheet preserves cellular communication, junctions, endogenous ECM, and integrative adhesive agents.33 More importantly, this sheet can provide large cells to the scaffold and favors the retention of the transplanted cells into the scaffold, which may be favorable for ECM-production and the uniform distribution of seeding-cells, thus improving tissue formation.34 Therefore, ASCs rich cell-sheets were prepared for the convenience of being sandwiched by the book-shaped AFS. In this study, inspired by book appearance, AFS with bookshape was successfully prepared by our optimized decellularization protocol that can effectively remove cellular components with only minimal influence on native ECM architecture and internal biological ingredients, thus showed superior properties in chondrogenic induction and seedingcells loading. Additionally, we developed a novel pattern for fabricating tissue-engineered fibrocartilage, namely, cell-sheets sandwiched by book-shaped scaffold, which can significantly improve the number of loading cells in comparison with traditional pattern. In-vivo study indicated that this tissueengineered fibrocartilage can offer benefits to regenerate functional fibrocartilage during bone−tendon healing. It is believed that this book-shaped AFS will be ideal materials for tissue-engineered fibrocartilage and bone−tendon healing owing to its superior cell-loading capability and chondrogenic inducibility.

2. MATERIALS AND METHODS A total of 211 mature male New Zealand rabbits (weight, 3.53 ± 0.24 kg) were used in this study. All animal experimental protocols complied with the regulations established by the Animal Ethics Committee of Central South University (Permit No. 2014-03-14). 2.1. Preparation of ASCs-Sheets. 2.1.1. Isolation and Identification of ASCs. After nuchal subcutaneous adipose tissue was harvested from 120 rabbits, they were returned to cages and numbered for the following in vivo experiment. The procedures for ASCs isolation are as follows: adipose tissues were extensively B


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ACS Applied Materials & Interfaces washed with PBS and then minced aseptically using sterilized surgical scissors, followed by digestion with type I collagenase solution (0.1 mg/mL, Gibco, USA) for 2-h in a 37 °C water bath shaker. After filtration with a sterile nylon mesh (F8210, Solarbio, China) and centrifugation at 1000 rpm for 5 min, and then the isolated cells were washed with PBS and resuspended in complete medium (DMEM/ F12 + 10% FBS+1% antibiotics, Gibco, USA), and incubated at 37 °C, 5% CO2. When reached 70−80% confluence, cells were passaged. ASCs identification was confirmed by flow cytometry analysis using antibodies against CD29, CD44, and CD90 (ASCs/stromal cells), CD31 (endothelial cell), CD45 (hematopoietic cells), and CD106 (VCAM-1). In-vitro osteogenic, chondrogenic, and adipogenic differentiations were also applied to identify ASCs (Figure S1). Passage 3 or 4 cells were used for further experiments. 2.1.2. Lentiviral Transduction of ASCs. ASCs were transduced with lentivirus containing enhanced mCherry protein genes (Genepharma, Shanghai, China) overnight at a multiplicity of infection (MOI) of 100 plaque forming units (PFU) per cell. Polybrene at 2 μg/mL was added to the medium to enhance the efficiency of transduction. The next day, the transduction medium was replaced with complete medium. After 4 days of incubation, transduced cells were selected using puromycin (2 μg/mL, Gibco, USA) to obtain purified mCherry-labeled ASCs (mCherry-ASCs). The stemness of ASCs and mCherry-ASCs was comparatively analyzed by trilineage differentiation potential (Figure S1). 2.1.3. ASCs-Sheets Preparation and Evaluation. ASCs were seeded at 400 000 cells/cm2 on temperature-responsive culture dishes (3.5 cm diameter, UpCell, ThermoFisher Scientific, USA), and cultured in complete medium with 20 μg/mL L-ascorbic acid (Sigma, USA) to form a coherent cell-sheet. After spontaneous detachment at room temperature, the ASCs-sheet was used to prepare tissue-engineered fibrocartilage or histologically evaluated with hematoxylin and eosin (H&E) staining. 2.2. Screening of Fibrocartilage Decellularization Efficacy. 2.2.1. Design and Preparation of AFS. To completely remove cellular components and mostly maintain native ECM as well as internal CAGFs of fibrocartilage tissue, four decellularization protocols were comparatively evaluated. Briefly, native fibrocartilage tissues (NFT) harvested from rabbit pubic symphysis were sectioned into cuboid (about 6 mm × 3 mm × 0.5 mm) or book shape with 5 pages (about 6 mm × 3 mm × 0.5 mm, page thickness = 100 μm, spine wide = 100 μm) using a freezing microtome (Leica CM1950; Nussloch, Germany). Then, the book-shaped NFT was disposed by SDS dissolved in 0.1% Triton X-100 with different concentrations and durations (2% SDS for 6 h, 2% SDS for 24 h, 4% SDS for 6 h, 4% SDS for 24 h) under vigorous agitation at 4 °C. After it was washed with PBS at 4 °C for three times (8 h per each time), it was digested in a nuclease solution (containing 500 U/mL DNase Type I and 1 mg/mL RNase) with agitation at 37 °C for 12 h. After they were washed with PBS three times (8 h per each time), the scaffolds were lyophilized in a vacuum freeze-drier (FD8−5T, SIM, USA) to get the 2%-6 book-shaped AFS, 2%-24 book-shaped AFS, 4%-6 bookshaped AFS, 4%-24 book-shaped AFS. During decellularization, 100 mg/mL streptomycin, 100 U/mL penicillin, and 2.5 mg/mL fungizone were included in all the above solutions. Aprotinin (10 KU/mL) was added into all solutions except the nuclease solution. 2.2.2. Histological Examination. Cuboid NFT and book-shaped AFS were embedded in paraffin, and sectioned into 5 μm slices (Leica RM 2125 Microtome; Reichert-Jung GmbH, Germany), which underwent staining with H&E, DAPI, toluidine blue (TB) and picrosirius red (PR) for histological observation using an Olympus CX31 microscope (Olympus, Japan). 2.2.3. Biochemical Assays for DNA Residence. After DNA was extracted from the cuboid NFS and the four kinds of book-shaped AFS (n = 6) using DNeasy Blood&Tissue Kit (Qiagen, USA), DNA content was quantified by PicoGreen DNA assay kit (Invitrogen, USA). 2.2.4. Porosity. Porosity of the cuboid NFT and the four kinds of book-shaped AFS (n = 6) was measured as follows: 3 mL ethanol was added to a 5 mL-volume cylinder, then scaffolds were immersed

in the cylinder for 10 min and the volumes were measured (V1). Scaffold was then taken out and the volume of the remaining ethanol measured (V2). Porosity was calculated according to the following equation: porosity (%) = (3 − V2)/(V1 − V2) × 100%. 2.2.5. Distribution and Content of GAGs and Collagen. The GAGs and collagen of the scaffolds (n = 6) was determined by synchrotron radiation-Fourier transform infrared spectroscopy (SRFTIR) at the BL01B beamline of National Facility for Protein Science Shanghai and Shanghai Synchrotron Radiation Facility, where synchrotron radiation from a bending magnet was collected, collimated and transported to a commercial FTIR interferometer bench. Sample preparation (slice thickness = 6 μ) and FTIR spectral analysis were preformed according to the published literature.35 The carbohydrate (1140−985 cm−1) and amide I (1720−1590 cm−1) peaks were respectively used to characterize the distribution and content of GAGs and collagen of the scaffolds. 2.2.6. Extraction of Growth Factors and Enzyme-Linked Immunosorbent Assays (ELISAs). To extract growth factors from the scaffolds, 10 cuboid NFT, 2%-6 book-shaped AFS, 2%-24 bookshaped AFS, 4%-6 book-shaped AFS, or 4%-24 book-shaped AFS were respectively were suspended in 1 mL extraction buffer (0.5 M acetic acid, 50 mM Tris-HCl, pH = 7.4, 0.1× protease inhibitors), followed by stirring at 4 °C for 3 days. The mixture was then centrifuged at 12 000g for 30 min at 4 °C, and then the supernatant was collected to measure the amount of TGF-β1, IGF-1, and BMP-2 in the extraction solution by ELISA kits (R&D Systems). 2.2.7. Inducibility Variation of the Scaffolds and Its Mechanism. Five cuboid NFT, 2%-6 book-shaped AFS, 2%-24 book-shaped AFS, 4%-6 book-shaped AFS, or 4%-24 book-shaped AFS were sterilized and immersed in DMEM/F-12 overnight, and then pasted on the surface of temperature-responsive culture plate (6-well, UpCell, ThermoFisher Scientific, USA) by polylysine. Each scaffold was respectively seeded with 105 ASCs and cultured in complete medium (AFS). In addition, 5 × 105 ASCs were seeded on tissue culture polystyrenes (TCPS) and cultured in complete medium (as control). Total cellular RNA was extracted with TRIzol reagent (Invitrogen) at postseeding 14 days. One μg total RNA were used as the template for reverse transcription with Prime-Script RT reagent kit (Takara Bio, Shiga, Japan). The expression of osteogenic (Runx-2), chondrogenic (Sox-9,) and tenogenic (Tnmd) genes was determined using a real-time PCR system (Bio-Rad) with SYBR GREEN PCR Master Mix. In addition, Sonic hedgehog pathway (Shh, Patched-1, and Gli-1) were investigated to explore the underlying mechanism of the book-shaped AFS on ASCs differentiation. In ASCs cultured on AFS, the smoothened antagonist (SANT-1, 5 μM) was used to inhibit SHH signaling to further confirm the mechanism. The primer sequences for these genes are listed in Table S1. Gene-specific primers were synthesized commercially (Shengong, Co., Ltd., Shanghai, China). The housekeeping gene (β-actin) was used for normalization. The results are shown as fold change related to the ASCs in the TCPS group. 2.3. In Vitro Bioactivity Analysis of the Screened BookShaped AFS. 2.3.1. Attachment and Viability Assay. The screened book-shaped AFS was sterilized and immersed in DMEM/F-12 overnight and then pasted on the bottom of the 24-well plate (Corning, USA) by polylysine (Corning, USA). 103 ASCs were respectively seeded onto AFS or TCPS (as control). After 1, 4, 7, and 10 days culture with complete medium (DMEM/F12 + 10% FBS + 1% antibiotics, Gibco, USA), ASCs proliferation on the AFS or TCPS were quantified by Cell Counting Kit-8 (7seabiotech, China), and the cell-scaffold composites were observed using scanning electron microscopy (SEM). In addition, to evaluate the cytotoxicity of AFS on ASCs, cell viability was evaluated with a Live/Dead Assay kit (Invitrogen) at postseeding day 4. The green- and red-stained cells were captured by a Leica TCS-SP8 confocal microscope (Leica, Germany) with excitation wavelength of 488/594 nm to quantify cell viability. 2.3.2. Inflammatory Responses of Macrophages to the Screened Book-Shaped AFS. To evaluate the influence of AFS on the proinflammatory cytokine release of macrophages, RAW 264.7 C


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ACS Applied Materials & Interfaces cells (ATCC) were cocultured with AFS using 24-well transwell inserts (3464, Corning, USA) with 8 μm pore-sized filters. RAW 264.7 cells were cultured with complete medium (high glucose DMEM + 10% FBS + 1% antibiotics, Gibco, USA) in the lower compartment at a density of 5.0 × 104 cells/well. On the basis of the different medium in the upper compartment, the experiment was divided into three groups: only complete medium (TCPS, as negative control), complete medium with 10 μg/mL lipopolysaccharide (LPS) (Sigma-Aldrich, USA) (LPS, as positive control), and complete medium with AFS (AFS). The morphology of RAW 264.7 cells was captured on days 3 or 7. Meanwhile the culture supernatant was collected to determine its TNF-α, IL-6, and IL-1β levels using ELISA kits (R&D system, USA). 2.3.3. Collagen and GAGs Secretion. To test the collagen and GAG secretion of ASCs on AFS or TCPS, a temperature-responsive culture plate (6-well, UpCell, ThermoFisher Scientific, USA) was applied in this experiment. In the AFS group, five pages of scaffold (weight = 5.08 ± 0.21 mg) were torn off from the 2%-24 bookshaped AFS, and then sterilized and immersed in DMEM/F-12 overnight. After firmly being pasted on the temperature-responsive culture plate (6-well, UpCell, ThermoFisher Scientific, USA) with polylysine (Corning, USA), each page of the scaffold was seeded with 105 ASCs, and complete medium (DMEM/F12 + 10% FBS + 1% antibiotics, Gibco, USA) was exchanged every 3 days over the following 2 weeks. At postseeding days 3, 7, and 14, the culture supernatants were collected for quantifying collagen and GAGs in the medium. Then, the culture plates were placed at room temperature for harvesting the cell-scaffold composites for collagen and GAG quantifications. In the TCPS group, 5 × 105 ASCs were directly seeded on the temperature-responsive culture plate (6-well, UpCell, ThermoFisher Scientific, USA), and the medium was also exchanged every 3 days over the following 2 weeks. At postseeding days 3, 7, and 14, the culture supernatants were collected for quantifying collagen and GAGs in the medium. Then, the culture plates were placed at room temperature to harvest the cell sheets. In contrast, five pages of scaffold (weight = 5.11 ± 0.23 mg) were mixed together with the harvested cell sheets together for collagen and GAG quantifications. A hydroxyproline assay kit (GenMed, USA) was used to quantify collagen, and a 1,9-dimethylmethylene blue (DMMB) assay, and tissue GAG Total Content DMMB Colorimetry kit (GenMed, USA) were used for the quantification of GAGs. 2.4. In Vivo Evaluation of Tissue-Engineered Fibrocartilage Fabricated by the Screened AFS and ASCs-Sheets. 2.4.1. Fabrication and Evaluation of Tissue-Engineered Fibrocartilage. Two book-shaped AFS similar in shape and size were selected, and then a 0.1 mm scaffold was cut off from the free edge of the first page of the book-shaped AFS. After that, we reversed one book-shaped AFS and inserted it into the other book-shaped AFS to construct a bookshaped AFS implant using an interior extrapolation method (Figure S2). Then, 9 slices of ASCs-sheet were separately inserted into the pages of book-shaped AFS, and the other 2 slices of ASCs-sheet covered the top and bottom surfaces of the implant. Morphological characteristics of tissue-engineered fibrocartilage were evaluated by gross observation and subsequent H&E staining. 2.4.2. Surgical Procedure. The numbered rabbit was anesthetized with 3% sodium pentobarbital (0.8 mL/kg intravenous injection; Sigma), and then an anterolateral skin incision was longitudinally made to expose the patella, patellar tendon, and tibial tuberosity of right knee. A hacksaw (JX-0190A, Jerxun, China) was used to perform transverse osteotomy between the proximal 2/3 and the distal 1/3 of the patella. After the distal third of the patella and attached fibrocartilage were removed, two evenly spaced tunnels (0.8 mm diameter) were drilled longitudinally through the remaining patella. Similarly, two holes was also punched on the tissueengineered fibrocartilage (single AFS implant, as AFS group; ASCssheets/AFS implant, as ASCs/AFS group) with syringe needle, and then the implant was anatomically inserted into the remaining patella and patellar tendon. The nonabsorbable PDS II suture (No. 3-0, Ethicon, USA) was successively crossed the patella tunnel, the punched holes of implant and patellar tendon, then tied tightly at the

superior pole of patella. For animals allocated to control group, the remaining patella and patellar tendon were directly reattached together with suture. Additionally, a figure-of-8 tension band wire was drawn around the superior pole of the patella and the tibia to protect the surgical site. After closing the surgical incision, cast immobilization was performed at the knee resting position for the 4 weeks. 3-Day painkiller (tramadol; Grunenthal GmbH) was given after operation (intravenous injection, 1.5 mg/kg per 12 h). Rabbits were allowed free cage activities after operation. The number of rabbits required for PPT interface evaluations was determined by power analysis. 2.4.3. Macroscopic Observations. The quadriceps patella patellartendon tibia complexes (QPPTCs) were observed and photographed by a digital camera (60D, Cannon, Japan). 2.4.4. Radiographic Evaluation. After gross examination, samples were fixed in 4% formaldehyde, and then anteroposteriorly captured by a Faxitron MX-20 X-ray unit (Faxitron X-ray Corp., Lincolnshire, Illinois, USA) under exact same parameters (exposure time = 3 s; tube voltage = 32 keV) to measure the area of newly formed bone extending from the proximal patella. After X-ray examination, the microarchitecture of the newly formed bone was evaluated by synchrotron radiation−microcomputed tomography (SR-μCT) at the BL13W1 of Shanghai Synchrotron Radiation Facility in China, which has a higher resolution and precision in three-dimensional morphological evaluation of bone in comparison with desktop μCT.36 Briefly, those specimens were vertically scanned with an angular step of 0.25° over an angular range of 180°. The beam energy, exposure time and sample-to-detector distance were separately set to 18.0 keV, 0.5 s, and 5.0 cm. 720 radiographic projections were captured by the CCD detector with a pixel size of 3.25 μm. Dark-field and flat-field images were also captured to reduce the ring artifact during reconstruction. These projected radiographs were sequentially phase-retrieved and transformed into 8-bit slices by PITRE software written by BL13W1. According to our pervious protocol,37 bone was extracted from soft tissue or bone marrow by a fixed threshold segmentation, and then a median filter was used to reduce noise. Morphological parameters of the newly formed bone, such as bone volume to total volume ratio (BV/TV), trabecular thickness (Tb·Th), and trabecular number (Tb·N) were calculated. 2.4.5. Histological and Immunohistochemical Analysis. After radiographic examination, samples were decalcified in 10% EDTA and then embedded in paraffin and cut into 5 μm sections. Sections were then stained with H&E for general histology and with toluidine blue and fast green (TB&FG) to evaluate fibrocartilage regeneration and GAGs accumulation. Expression of type II collagen in the regenerated tissue was analyzed by immunohistochemical staining. Sections were dewaxed in xylene and hydrated through a series of alcohols. After blocking with 1% bovine serum albumin, sections were incubated with the primary antibodies (2 μg/mL, ab185430, Abcam) at 4 °C overnight. Following washing in PBS, the secondary Anti-Mouse IgG H&L (HRP) (diluted 1:2000, ab205719, Abcam) was added to the sections and incubated for 1 h at 37 °C. Staining was developed in diaminobenzidine solution, with hematoxylin counterstaining. PPT interface healing were semiquantitatively analyzed by one blinded observer (Y. Chen) using a histological score system adapted from Ide J et al. and Shah SA et al.38,39 (Table S2), and the area of cartilaginous metaplasia (CM) region was semiquantitatively assessed according previous method.40 2.4.6. Biomechanical Test. Biomechanical test was acted as the ultimate index to assess the overall quality of PPT interface healing, which was performed by a mechanical testing machine (MTS insight, MTS Systems Corp, USA) (Figure S3). Briefly, after the QPPTCs were thawed overnight at 4 °C, the periarticular connective soft tissues, the suture material and tension band wire were carefully dissected. Then, the QPPTC was loaded to failure at a rate of 20 mm/min after a preload of 1 N. Failure load (N) was obtained from the recorded load−displacement curve, and stiffness (N/mm) was calculated from the linear portion of this curve with Office Excel 2016 (Microsoft Corp). During testing, 0.9% saline was applied to avoid dehydration of the specimens. D


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Figure 1. Characteristics of the scaffolds. (A) Macroscopic features of the cuboid NFT and book-shaped AFS. (B) Histological analysis of the cuboid NFT and book-shaped AFS. Sections stained with hematoxylin and eosin (H&E), DAPI, toluidine blue (TB), and picrosirius red (PR). Scale bar = 100 μm. (C) Comparative graph showing the DNA content and porosity of the cuboid NFT and book-shaped AFS. “X%-Y” means “book-shaped scaffold decellularized with X% SDS for Y hours and followed by nuclease digestion”. All values are presented as means ± standard deviation (*P < 0.05, **P < 0.01, ***P < 0.001). 2.4.7. In Vivo ASCs Tracking. To investigate the fate of transplanted ASCs in book-shaped AFS, mCherry-ASCs-sheets were inserted into the book-shaped AFS to construct a tissueengineered implant, and then transplanted into 9 rabbits (mCherryASCs/AFS group). Another 18 rabbits were randomly allocated into two groups and, respectively, receive ASCs-sheet/AFS autologous implant (ASCs/AFS group) or no implant (control group). At postoperative week 0, 4 and 8, specimens were harvested and evaluated by in vivo imaging system (IVIS) to determine the survival of the transplanted cells. And then, specimens of 8 weeks were fixed, sectioned and incubated with a mouse monoclonal antibody directed against mCherry protein (diluted 1:200, ab125096, Abcam) overnight at 4 °C. The sections were then rinsed in PBS and treated with Goat Anti-Mouse IgG H&L (HRP) (diluted 1:2000, ab205719, Abcam) for 30 min at room temperature. After the visualization reaction was performed, sections that were immunohistochemically stained for mCherry were observed by light microscopy. 2.5. Statistical Analysis. All measurements were expressed as the mean ± standard deviation. For the in vitro experiments, we repeated it at least three times. Unpaired t test was used for the comparison between two groups, while one-way ANOVA with the Tukey’s post hoc test was used for the comparison above two groups. As for the in vivo experiments, the results were compared using a one-way ANOVA with Bonferroni post hoc test. A value of P < 0.05 was considered statistically significant. The analyses were performed using the SPSS 25.0 software (SPSS, USA).

3. RESULTS 3.1. Decellularization Optimization Screening Analysis. 3.1.1. Gross Appearance and Histology. A gross photograph of the scaffolds was shown in Figure 1A, and the scaffold size was about 6.0 mm × 3.5 mm × 0.5 mm. The page thickness of the book-shaped scaffold about 100-μm, and the spine width is about 100-μm. H&E, DAPI, TB, and PR staining were used together to evaluate the acellular effects and the preservation of ECM components of fibrocartilage, such as GAGs and collagen (Figure 1A). Extensive staining for cell nuclei was observed in the H&E or DAPI staining of the cuboid NFT. In the book-shaped AFS, the cellular components cannot be absolutely removed under 2% or 4% SDS treatment for 6 h, while prolonging decellularization duration to 24 h can eliminate all nuclei from the scaffold. All decellularization treatments resulted in a reduction of staining for GAGs more or less. However, 24 h treatment reduced more GAGs than 6 h, and 2% SDS treatment was more suitable for the protection of GAGs with respect to 4% SDS. Finally, all scaffolds demonstrated extensive staining for collagen, but the book-shaped AFS prepared with 2% or 4% SDS for 6 h showed a little reduction of collagen staining, while the 24 h treatment with 2% or 4% SDS resulted in E


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Figure 2. (A) Distribution of collagen and GAGs in cuboid NFT and book-shaped AFS. Scale bar = 20 μm. (B) Characteristic FTIR spectrum for the cuboid NFT and book-shaped AFS, and note the amide I peak and the carbohydrate peak are respectively indicative of collagen and GAGs. (C) Collagen and GAGs contents of the cuboid NFT and book-shaped AFS. “X%-Y” means “book-shaped scaffold decellularized with X% SDS for Y hours and followed by nuclease digestion”. All values are presented as means ± standard deviation (*P < 0.05, **P < 0.01, ***P < 0.001).

obviously decreased staining. Moreover, treatment with 4% SDS for 24 h showed the lowest collagen among the four protocols. 3.1.2. DNA Residence and Porosity. The amount of DNA in the NFT was 0.95 ± 0.20 μg/mg (fibrocartilage dry weight) before decellularization. Treatment with 2% or 4% SDS for either amount of time resulted in a significant reduction of the DNA in the scaffold (Figure 1B). However, treatment with 2% or 4% SDS for 6 h only reduced part of the DNA on the scaffold, while treatment with 2% or 4% SDS for 24 h maximally removed the DNA on the scaffold. The porosity of each scaffold was also measured (Figure 1C): 86.69 ± 1.14% for the cuboid NFT, 87.52 ± 1.61% for the 2%-6 book-shaped AFS, 90.71 ± 0.88% for the 2%-24 book-shaped AFS, 88.22 ± 1.11% for the 4%-6 book-shaped AFS, and 91.28 ± 1.57% for 4%-24 the book-shaped AFS. 3.1.3. ECM Components and Chondrogenic Active Growth Factors (CAGFs). In the current study, we innovatively applied the SR-FTIR to quantitatively analyze the distribution and content of GAGs and collagen in the scaffolds. As shown in Figure 2, the collagen distribution at the scaffolds was mostly preserved following decellularization, and its content decreased about 10.36% in the 2%-6 bookshaped AFS, 21.97% in the 2%-24 book-shaped AFS, 15.55% in the 4%-6 book-shaped AFS, 24.80% in the 4%-24 bookshaped AFS compared with the cuboid NFT. For the GAGs at the scaffolds, its distributions at the NFT and AFS were similar, but the 2%-6 book-shaped AFS, the 2%-24 bookshaped AFS, the 4%-6 book-shaped AFS and the 4%-24 bookshaped AFS respectively lost about 21.46%, 26.09%, 23.17%,

and 29.51% of the GAGs content with respect to the cuboid NFT. To evaluate the influence of decellularization on the reservation of CAGFs in the scaffolds, TGF-β1, BMP-2, and IGF-1 levels were analyzed in extracts from AFS and NFT by ELISA (Figure 3A). The contents of TGF-β1, BMP-2, and IGF-1 in the 2%-6 book-shaped AFS, the 2%-24 book-shaped AFS, and the 4%-6 book-shaped AFS were increased a little with respect to those in cuboid NFT (P > 0.05 for all), while the contents of TGF-β1, BMP-2, and IGF-1 in the 4%-24 book-shaped AFS decreased about 30.77%, 30.77%, and 30.77% in comparison with the cuboid NFT (P > 0.05 for all). 3.1.4. Chondrogenic Inducibility of Scaffolds. To evaluate the effect of these scaffolds on ASCs differentiation, the expression of Runx-2, Sox-9, and Tnmd genes was evaluated by qRT-PCR (Figure 3B). In comparison with the ASCs cultured on TCPS, the ASCs cultured on the NFT, the 2%-6 book-shaped AFS, the 2%-24 book-shaped AFS, and the 4%-6 book-shaped AFS showed a significant expression in Sox-9 (P < 0.05 for all), while no significant increase was observed in the ASCs cultured on the 4%-24 book-shaped AFS (P > 0.05). Additionally, the 4%-24 book-shaped AFS showed a significant reduction in Sox-9 expression in comparison with the 2%-6 book-shaped AFS, and the 4%-6 book-shaped AFS (P < 0.05 for all). The expression of Runx-2 and Tnmd genes in ASCs showed no significant difference among the TCPS and the five scaffolds (P > 0.05 for all). The above results indicated that 2%-24 book-shaped AFS was cell free while containing most of fibrocartilage ECM and internal CAGFs. Moreover, this scaffold showed superior chondrogenic inducibility for ASCs. F


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Figure 3. (A) Comparison of growth factor content in the cuboid NFT and book-shaped AFS by ELISA. (B) Effects of the scaffolds on ASCs differentiation. qRT-PCR analysis shows that the expression of osteogenic (Runx-2), chondrogenic (Sox-9), and tenogenic (Tnmd) genes after ASCs cocultured on TCPS, NFT and AFS for 14 days. Here, it can be seen that the ASCs on the NFT, 2%-6 AFS, 2%-24 AFS, and 4%-6 AFS expressed chondrogenic genes more significantly on 14 days than the TCPS group. But the ASCs on the 4%-24 AFS showed a significant reduction in Sox-9 expression than the 2%-6 AFS and 4%-6 AFS. Additionally, the osteogenic or tenogenic genes showed no significant differences between the TCPS and the five scaffolds. (C) The stimulatory effect of 2%-24 AFS on hedgehog pathway (Shh, Patched-1, Gli-1) in ASCs. The ASCs cultured on the 2%-24 AFS were significantly increased the expression of Shh, Patched-1 and Gli-1 genes than the ASCs cultured on the TCPS, and this overexpression can be inhibited by a hedgehog signaling inhibitor (SANT-1, 50 μM). Gene expressions was standardized to the ASCs on the TCPS, the value of expression gene was the fold change relative to the ASCs on the TCPS. “X%-Y” means “book-shaped scaffold decellularized with X% SDS for Y hours and followed by nuclease digestion”. All values are presented as means ± standard deviation (*P < 0.05, **P < 0.01, ***P < 0.001).

3.2. In Vitro Biological Analysis of the 2%-24 BookShaped AFS. 3.2.1. ASCs Attachment and Viability on 2%24 Book-Shaped AFS. To investigate the effects of AFS on ASCs attachment and viability, SEM and CCK-8 assay were used to examine the morphology and proliferation of ASCs cultured on AFS. The SEM micrographs confirmed that decellularization with 2% SDS for 24-h following by nuclease digestion effectively removed the cellular components and well maintained the ECM structure of NFT (Figure 4A). In addition, SEM images indicated that ASCs were well attached to the AFS and proliferated well in the scaffolds. At 24-h after cell seeding, ASCs began to adhere to the scaffolds and exhibited typical polygonal morphology (Figure 4B). After 4 days, the seeded cells were spread out, integrated with the AFS and proliferated obviously. By the day 7 after seeding, cells had become more elongated, increased their proliferation, mutually and gradually become confluent. By day 10 after seeding, a large number of ASCs had fused on the surface of AFS with much ECM. The results of the CCK-8

assay confirmed that the AFS are nontoxic and suitable for ASCs growth (Figure 4C). ASCs viability on AFS and TCPS (as control) was evaluated by live/dead cell staining after 4 days of culture. For the TCPS and AFS, most cells were stained fluorescent green (living cells), with very few red (dead cells). The quantitative analysis (n = 4) suggested that cell viability on the AFS was slightly lower than that on the TCPS, but there were no significant differences (Figure 4D). 3.2.2. Inflammatory Activities of the 2%-24 Book-Shaped AFS. Pro-inflammatory cytokine (TNF-a, IL-6, and IL-1β) production was measured to investigate the inflammatory responses of monocytes/macrophages (RAW 264.7) to AFS. As shown in Figure 4E, the morphology of RAW 264.7 cultured on TCPS or AFS was similar without obvious cell tentacles, while most of the RAW 264.7 under lipopolysaccharide (LPS, as positive control) obviously showed many cell tentacles. In addition, RAW 264.7 cultured on AFS generated a little higher level of TNF-a, IL-6, and IL-1β than those cultured on TCPS at day 3 or 7 (P > 0.05 for all), but their G


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Figure 4. (A) Scanning electron micrographs of the cuboid NFT and 2%-24 book-shaped AFS. (B) Scanning electron micrographs of a 2%-24 book-shaped AFS on which rabbit ASCs had been cocultured for 1, 4, 7, and 10 days. (C) Comparative cell proliferation assay of ASCs seeded on the TCPS and 2%-24 book-shaped AFS (n = 4 for each group). (D) Live/dead cell analysis for the TCPS and 2%-24 book-shaped AFS on which ASCs had been seeded for 4 days. Representative 3D images show the live (green) and dead (red) ASCs in the TCPS and AFS, and the viability analysis for the cells on the TCPS and AFS. E) Representative RAW 264.7 morphology after 3 and 7 days of stimulation by TCPS, 2%-24 bookshaped AFS or LPSs. Pro-inflammatory cytokine (TNF-α, IL-6, and IL-1β) release by stimulation with TCPS, AFS, or LPS detected by ELISA assay. Scale bar = 10 μm. (F) Biochemical assay results showing collagen and GAG content estimated in TCPS and 2%-24 book-shaped AFS individually secreted by ASCs after days 3, 7, and 14 coculture (n = 3 for each group). “X%-Y” means “book-shaped scaffold decellularized with X % SDS for Y hours and followed by nuclease digestion”. All values are presented as means ± standard deviation (*P < 0.05, **P < 0.01, ***P < 0.001).

3.2.4. ECM Secretion of ASCs on 2%-24 Book-Shaped AFS. Quantification of total collagen and total GAGs was used to evaluate the effect of 2%-6 book-shaped AFS on the ECM secretion of ASCs (Figure 4F). The results indicated that the contents of collagen and GAGs increased over time in both TCPS and AFS groups (P < 0.05) (day 14 vs day 7; day 7 vs day 3). As collagen and GAGs can be secreted into the medium while ASCs are being cultured, total collagen and GAGs estimated both as that present in the media and that deposited in the substrata (AFS or TCPS). Our results

levels were remarkably lower than those of the LPS-treated group (Figure 4E). 3.2.3. Underlying Mechanism of 2%-24 Book-Shaped AFS on ASCs Differentiation. To further elucidate the mechanism of AFS on chondrogenic differentiation of ASCs, the expression of hedgehog signaling pathway related genes was evaluated (Figure 3C). SANT-1 is a hedgehog signaling inhibitor. AFS significantly up-regulated the expression of hedgehog pathway (Shh, Patched-1, Gli-1), which can be inhibited by SANT-1. H


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Figure 5. (A) Schematic diagram of assembly and the morphology of tissue-engineered fibrocartilage implants. (B) Macroscopic and microscopic morphology of ASCs-sheet. Scale bar = 100 μm. (C) H&E stained imaging of ASCs-sheets/AFS implant. Scale bar = 100 μm. (D) Partial patellectomy and tissue-engineered fibrocartilage implant transplantation. Proximal patella and residual patellar tendon after operation. A figureof-8 tension band wire was placed around the tibial tuberosity to the proximal pole of the patella to avoid separation of the junction reattachment. The yellow triangle labels the tissue-engineered implant. RP: Residual patella. PT: Patellar tendon. (E) Flowchart depicting the groups, time points for sacrifice, number of animals per group, and outcome assessments. (F) Macroscopic analyses of the regenerated PPT interface and the CM region. Blue dashed line indicates osteotomy site, and yellow dashed line circle indicates the area of CM region. All values are presented as means ± standard deviation.

indicated that the total contents of collagen and GAGs in AFS were significantly higher than those in the TCPS at day 7 and

14 (P < 0.05), but no significant difference was found at day 3 between TCPS and AFS group (P > 0.05). Those data I


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Figure 6. (A) Representative SR-μCT and X-ray images of the proximal patella in the control, AFS and ASCs/AFS groups at postoperative 8 weeks and 16 weeks. Blue quadrangle and dotted line indicate the osteotomy site. Red triangle shows the section position of tomography. Scale bar = 1 mm. (B) Comparison of the BA, BV/TV, Tb·Th, and Tb·N in newly formed bone among the 3 groups at different time points. All values are presented as means ± standard deviation.

indicate that the AFS can stimulate ASCs to secrete collagen and GAGs. 3.3. Tissue-Engineered Fibrocartilage Promoted Bone−Tendon Healing. 3.3.1. Morphology of the TissueEngineered Fibrocartilage. Two 2%-24 book-shaped AFS implant with/without 11 ASCs-sheets were, respectively, used to construct two types of tissue-engineered fibrocartilage implants using an interior extrapolation method (Figure S2): single AFS implant and ASCs-sheets/AFS implant (Figure 5A). The size of single AFS implant was about 6.0 mm × 3.5 mm × 1.0 mm, while the size of ASCs-sheets/AFS implant was about 6.0 mm × 3.5 mm × 1.2 mm. Histologically, 11 ASCs-sheets were sandwiched layer-by-layer by the bookshaped AFS implant, which was composed of a large number of cells and secreted ECM (Figure 5B and C). 3.3.2. Tissue-Engineered Fibrocartilage Transplantation and Evaluation Plan. To evaluate the effect of tissueengineered implant on fibrocartilage layer regeneration during bone−tendon healing, we respectively transplanted the single AFS implant and autologous ASCs-sheets/AFS implant into rabbits that had undergone partial patellectomy (Figure 5D).

According to Figure 5E, the specimens were harvested for the following evaluations. 3.3.3. Macroscopic Observation. Macroscopically, the PPT interface specimens at postoperative week 8 showed that a regenerated tissue was bridging the residual patella and patellar tendon in all groups, and a larger cartilaginous metaplasia (CM) region was formed in the scar tissue next to the articular cartilage of the remaining patella in the ASCs/ AFS group compared with the control and AFS groups (Figure 5F). Postoperative week 16, the specimens in the control, AFS and ASCs/AFS groups showed a similar gross morphology to the native PPT interface, while the specimens in the ASCs/AFS group were developed a more advanced CM region than those in the other groups (Figure 5F). 3.3.4. Radiological Evaluation. After evaluation by SRμCT together with X-ray, the morphological parameters of the newly formed patella in the PPT interface showed that the ASCs/AFS group had more new bone formation and remodeling at the healing interface than the control and AFS groups at 8 or 16 weeks postoperatively (Figure 6). At 8 weeks after operation, the bone area (BA), BV/TV, Tb·Th, J


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Figure 7. Histological analyses of regenerated patella−patellar tendon interface. (A) RPPTI sections stained with H&E, TB&FG, and immunostained for collagen II. One 4-month-old healthy rabbit was used as a native control. The yellow dotted line indicates the osteotomy site. CM: Cartilaginous metaplasia. RPPTI: Regenerated patella−patellar tendon interface. RF: Regenerated fibrocartilage. PPTI: Patella−patellar tendon interface. F: Fibrocartilage. RP: Residual patella. NB: New bone. PT: Patellar tendon. Yellow scale bar = 500 μm, black scale bar = 50 μm. (B) Histological scores for PPT junction healing. Black and blue dotted lines, respectively, indicate the perfect score (32 points) and worst score (8 points) in the histological scoring system. (C) Comparison of the area of CM at the PPT healing interface. Data are expressed as mean ± standard deviation.

difference in Tb·Th and Tb·N was not statistically significant among the 3 groups (p > 0.05 for all). 3.3.5. Histological Evaluation. Histological evaluations showed that the PPT healing interface in all groups at postoperative weeks 8 and 16 was characterized by newly formed bone enlarging from the proximal patella, regenerated fibrocartilage layer bridging the newly formed bone and

and Tb·N of the ASCs/AFS group were significantly larger than those of the control and AFS groups (p < 0.05 for all), but no significant difference was found between the control and AFS groups. At 16 weeks after surgery, the BA and BV/ TV of ASCs/AFS group was significantly higher than those of the control and AFS groups (p < 0.05 for all). Moreover, the K


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Figure 8. (A) Biomechanical properties of the PPT healing interface. Representative load−displacement curves for 8 and 16 weeks (A1). The failure load and stiffness of the regenerated PPT interface in different groups at postoperative week 8 or 16 (A2). (B) Dynamic monitoring of the transplanted ASCs survival in the PPT healing site by IVIS at postoperative day 0, week 4, and week 8. The fluorescence intensity is indicated by the color-coding scale bar at the bottom of IVIS images. RP: Residual patella. PT: Patellar tendon. (C) Representative images of immunohistochemical staining of mCherry at the PPT healing interface in different groups. Inset showed the high magnification of the line-boxed area of the image. Red arrow: mCherry-positive fibrochondrocytes, yellow arrow: mCherry-positive osteoblasts. Black arrow: mCherry-positive osteocytes. NB: Newly formed bone. RF: Regenerated fibrocartilage. PT: Patellar tendon. Scale bar = 10 μm. Data are expressed as mean ± standard deviation (n = 6).

residual patellar tendon, and CM region formation in the scar tissue next to the articular cartilage of the remaining patella (Figure 7). At 8 weeks after surgery, the implants in the AFS and ASCs/AFS groups were mostly absorbed, and some

histological differences of the PPT healing interface were observed among the three groups. In the ASCs/AFS group, there was more woven bone, larger CM region and more robust fibrocartilaginous junction with abundant paralleled L


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4. DISCUSSION Functional fibrocartilage regeneration during bone−tendon healing is a difficulty for clinicians and researchers in sports medicine due to the gradient structure of this tissue and its limited ability to regenerate once damaged.1,2 Clinically, no effective method can restore the functional fibrocartilage quickly and efficiently. Tissue-engineering is a promising strategy for functional fibrocartilage regeneration.6,30,41 However, the currently available tissue-engineering strategies are insufficient due to a lack of appropriate scaffolds that can loading large number of seeding-cells and induce chondrogenic differentiation of stem cells. In this study, we optimized the decellularization protocol, and then developed a novel book-shaped AFS, which showed no cell compositions and mostly retained the native ECM as well as internal CAGFs of fibrocartilage tissue. In addition, its shape was in favor of cells loading. In-vitro results indicated that this book-shaped AFS is noncytotoxic and nonantigenic, allows ASCs attachment and proliferation, stimulates the chondrogenic differentiation and fibrocartilaginous ECM synthesis of ASCs. The underlying mechanism for this chondrogenesis may be related to hedgehog pathway activating. Additionally, a novel assembly pattern for fabricating tissue-engineered fibrocartilage was developed to improve seeding-cells loading, namely, cell sheets sandwiched by book-shaped scaffold. In-vivo study confirmed that this scaffold sandwiching with or without autologous ASCs-sheets can result in improved mechanical and biological parameters of the PPT interface compared with those resulting from the control. Cell tracking results demonstrated that mCherry-labeled ASCs in the implants exhibited fibrochondrocytic morphology in the regenerated fibrocartilage layer or osteoblast/osteocyte appearance in the newly formed bone at 8-week after implantation. On the basis of these findings, we propose the use of book-shaped AFS and cell-sheet technique for fabricating tissue-engineered fibrocartilage to improve bone−tendon healing. In tissue-engineering, a scaffold is essentially an engineered replacement of the native ECM that provides a biomicroenvironment for cell adhesion, growth and even differentiation, thus guiding three-dimensional tissue formation.15,20 Theoretically, acellular scaffold derived from the same type of tissue is the ideal biomaterial for engineering new tissue, owing to their high levels of biomimicry, biocompatibility, and bioactivity.20,21 Recently, varieties of acellular scaffolds have been successfully developed for tissue-engineering.15,16,20,21,42,43 Regrettably, the porosity of acellular scaffolds derived from dense tissue is too low to be convenient for cell loading and infiltration, thus partly limiting their usage.15,20,21 Thus, many strategies have been developed to improve the porosity of the acellular scaffold derived from dense tissue, including the use of lasers, microwave radiation and tissue-cut slices.20 In the musculoskeletal system, cartilage, tendon and ligament belong to dense tissues. To improve the cellular infiltration property of acellular tendon scaffold and increase the surface area for seeding cells, Omae H et al. sectioned the acellular tendon into a multilayer structure and then seeded it with BMSCs to accelerate tendon repair.44,45 Later, for the sake of multilayer acellular tendons handling during surgery and improving their capacity for BMSCs loading, Omi R et al. established a novel tendon-book technique, by which tendon tissue was sliced to a 500-μm thickness and partially cut longitudinally into 5 layers, similar to a book shape.46 Inspired by this technique,46 we

cartilage-like cells embedded by a characteristic matrix composed of GAGs and type II collagens in the PPT healing interface compared with the control and AFS groups. Statistically, using a modified histological score system based on Ide J et al. and Shah SA et al.38,39 (Table S2), the histological scores for the regenerated PPT interface in the ASCs/AFS group were better than those in the control and AFS groups (P < 0.05), and the CM region formed in the ASCs/AFS group was significantly larger than that of the control and AFS groups (P < 0.05). At 16 weeks after operation, the PPT healing interface in all groups displayed better remodeling with respect to postoperative week 8. Healing and remodeling at the PPT interface were almost complete, with a resemblance to a healthy BTI consisting of characteristic transitional layers in all groups. However, the newly formed bone of the ASCs/AFS group revealed better remodeling than that of the control and AFS groups, characterized by well-developed lamellar bone and formation of more marrow cavities. Additionally, the AFS and ASCs/ AFS groups showed a significantly larger histological score for the regenerated PPT interface in comparison with that of control group (P < 0.05 for all). Meanwhile, no significant difference was found between the AFS and ASCs/AFS group (P > 0.05). As for the CM region, the area in the ASCs/AFS group was the largest among the three groups, which was significantly higher than the control and AFS groups (P < 0.05 for all). Additionally, the CM region was significantly larger in the AFS group than in the control group (P < 0.05). 3.3.6. Biomechanical Evaluation. Using a mechanical testing machine (MTS insight, MTS Systems Corp, USA) (Figure S3), biomechanical testing results showed that the mechanical properties of the PPT interface was improved with time and greatly promoted with the influence of fibrocartilage tissue-engineering implants (Figure 8A). At postoperative week 8, the failure load and stiffness of the ASCs/AFS group were significantly greater than those of all other groups (P < 0.05 for all). However, no significant differences in the mechanical properties were found between the control and AFS groups. At postoperative week 16, the failure load and stiffness of the PPT interface in the ASCs/AFS and AFS groups improved significantly compared with the control group (P < 0.05 for all). There were no significant differences between the ASCs/AFS and AFS groups. After biomechanical testing, all specimens ruptured at the healing tissue, and no specimens were excluded. 3.3.7. Tracking the Fate of ASCs in Tissue-Engineered Fibrocartilage. Before implantation, the homogeneous population and differentiation potential of the mCherry-ASCs were identified, and the results indicated that the mCherryASCs applied in this study showed similar stemness as normal ASCs (Figure S1). An IVIS showed that mCherry-ASCs in the tissue-engineered implant remained observable at the PPT healing interface even at 8 weeks postoperation (Figure 8B). Importantly, immunohistochemistry (IHC) also demonstrated that the implanted mCherry-ASCs showed a round fibrochondrocytic-like appearance with typical lacuna structures at the regenerated fibrocartilage (Figure 8C) and osteoblasts or osteocytes at the newly formed bone (Figure 8C), indicating that the implanted ASCs have differentiated into fibrochondrocytes, osteoblasts or osteocytes respectively in their respective in vivo environments. M


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ACS Applied Materials & Interfaces designed a book-shaped model to section fibrocartilage tissue, thus can not only facilitate cell removal and seeding-cells loading, but also increase the porosity of scaffold. Toward chemical methods of decellularization, several chemical reagents like SDS, Triton X-100, EDTA, trypsin or nuclease were used in combination to decellularize cartilage tissue.23,25 Rahman S et al. compared different combination approaches with SDS and EDTA or trypsin to optimize the decellularization of auricular cartilage based on acellular effect and ECM preservation.25 Wu LC et al. decellularized the porcine annulus fibrosus using different freeze−thaw temperatures, SDS versus Triton X-100, and incubation times to determine the optimal protocols for cell removal.47 However, the screening index in these studies only focused on the acellular effects and ECM preservation, while the property of chondrogenic induction was not taken into consideration. In the current study, according to our preliminary research, we investigated the decellularization protocols for fibrocartilage tissue by setting the concentration (2% or 4%) and treatment duration (6-h or 24-h) of SDS as variable. After screening, we demonstrated that the book-shaped NFT was more convenient for decellularization (Figure S4), and the 2%-24 book-shaped AFS has few cellular components, retains most of the native ECM and GAGFs of fibrocartilage, is favorable for ASCs attachment and viability, shows a superior chondrogenic inducibility for ASCs, and stimulates ASCs secrete fibrocartilaginous matrix molecules. Using a rabbit partial patellectomy model, we found that the animal transplanted with 2%-24 book-shaped AFS implant did not significantly regenerate the junctional fibrocartilage at postoperative week 8, but a more robust fibrocartilage appeared at postoperative week 16 with respect to the case of no implant, as indicated by the histological and mechanical properties of regenerated tissue. Histologically, type II collagen and GAGs (the major components of fibrocartilaginous ECM) increased with time, and their content in the AFS group respectively improved in comparison with that in the control group. Biomechanically, the AFS group had significantly greater failure load and stiffness at the PPT healing interface than the control group. The reasons for this difference may be that PPT healing in the AFS group undergoes a more complex process, which successively includes endogenous cell infiltration, scaffold resorption and internalization, and formation of fibrocartilage layer-like structures. Thus, in the early stage, the implanted bookshaped AFS was more like a barrier for the integration of residual patella and patellar tendon. Consequently, the AFS group did not display good morphology and tensile property at postoperative week 8. Over time, the scaffold was gradually resorbed and internalized, and then provided a suitable situation for fibrocartilage regeneration, thus showing a better PPT healing quality at postoperative week 16. Therefore, we feel that the 2%-24 book-shaped AFS is an available scaffold for fibrocartilage regeneration. Even so, the curative effect of 2%-24 book-shaped AFS only is limited, especially in the early stage of PPT healing. To enhance their ability to repair fibrocartilage, we used temperature-responsive culture dishes to acquire autologous ASCs-sheets,48 and then inserted them layer-by-layer into the book-shape AFS implant to fabricate another tissueengineered fibrocartilage implant, namely, ASCs-sheet/AFS implant. Cell-sheets can not only achieve highly efficient cell delivery, but also effectively preserve cell−cell contact, the

ECM and various factors, thus exerting a significantly better effect on local tissue repair than administering cells via injection or fibrin glue.33,34,48,49 According to the literature, ASCs have the advantages of relative abundance, ease to harvest, rapid expansion and high proliferation potential, moreover they can maintain a stable phenotype over many culture passages.15,27 In addition, ASCs in combination with gradient mineral nanofibrous scaffold or fibrin glue carrier have been reported to accelerate fibrocartilage regeneration during bone−tendon healing.41,50 Furthermore, by cell pellet culture, we demonstrated that the ASCs isolated from rabbit nuchal subcutaneous adipose tissue have good trilineage differentiation potential. Therefore, autologous ASCs was selected as seeding-cells in the current study. Similar to previous studies using other scaffolds,41,50 the current study also showed that the radiological, histological, and biomechanical parameters of the PPT interface in the ASCs/ AFS group were significantly higher than those of the control and AFS groups at postoperative week 8. These findings demonstrated that autologous ASCs-sheets sandwiched by book-shaped AFS provide an abundance of seeding-cells for tissue-engineering approaches to regenerate fibrocartilage and new bone, which is a very important factor for the quality of early PPT healing. Despite this benefit, the contribution of implanted ASCs to the quality of early PPT healing is a critical issue whose elucidation may help us to better access the function and mechanism of ASCs in bone−tendon healing. Currently, several studies on cartilage tissue-engineering have determined that GFP-labeled stem cells implanted into an osteochondral defect site and differentiate into functional chondrocytes in sufficient quantity,51,52 which indicates that transplanted cells can play a major role in cartilage repair. However, there is still no similar research performed for bone−tendon healing, thus it is unclear whether ASCs contribute to fibrocartilage regeneration and new bone formation by partly differentiating into fibrochondrocytes or osteoblasts/osteocytes in the current study. By using lentivirus transduction, we labeled implanted ASCs with mCherry marker, which is similar to GFP and can steadily express in ASCs for months, thus allowing their tracking postimplantation.52 Our results indicated that implanted ASCs partly transformed into fibrochondrocytic cells in the regenerated fibrocartilage or osteoblast/osteocyte in the newly formed bone at 8 weeks after implantation. These findings helped us further understand how ASCs-sheets in the tissue-engineered implant contribute to fibrocartilage regeneration and new bone formation during bone−tendon healing. In the current study, the rabbits in the AFS and ASCs/AFS groups showed an accelerated various level of PPT healing, but the ASCs/AFS group exhibited much better results than the AFS group. Potential explanations for these marked differences may be that (1) additional ASCs in the implants could secrete a broad spectrum of bioactive molecules to optimize the local immune environment at the healing interface,53,54 thus guiding endogenous stem/progenitor cell infiltration, chondrogenic or osteogenic differentiation and promoting ECM synthesis, resulting in an improvement of fibrocartilage regeneration and new bone formation. (2) The inherent biomolecules, surface topography, and substrate stiffness of scaffolds had a certain promoting function on the differentiation of stem cells.55,56 Therefore, in the ASCs/ AFS group, additional exogenous ASCs were transplanted, N


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cell-sheets sandwiched by book-shaped scaffold. In-vivo studies indicated that this book-shaped AFS sandwiching with or without autologous ASCs-sheets can result in improved fibrocartilage regeneration and bone−tendon healing. This study provides a novel book-shaped AFS for tissue-engineered fibrocartilage and may have further broad clinical applications in promoting bone−tendon healing.

allowing more stem cells to be synthetically influenced by those properties of book-shaped AFS, and then differentiate into chondrocytes and fibrocytes. These cells gradually developed into the fibrocartilage layer and thus more effectively accelerated PPT healing. (3) Mechanical stimulation such as cyclic tensile strain can promote tenogenic, fibrogenic, chondrogenic, and osteogenic differentiation of MSCs in vitro.57−59 Therefore, we suspect that the mechanical environment at the PPT healing site may be another inductive factor for the differentiation of implanted ASCs, thus allowing the PPT interfaces in the ASC/AFS group to present better enhancement in healing quality, histologically characterized by greater mature fibrocartilage regeneration and woven bone penetrating into the tendon. (4) According to the assembly scheme of the ASCs-sheets/AFS implant, the surface of the implant was covered with a layer of ASCs-sheet, which may be the source of osteoblasts and osteocytes, thus more new bone was formed at the PPT healing interface in the ASCs/AFS group. To prove whether these interpretations are correct, further research is required. It is important to recognize the limitations of our study. First, logically speaking, the thinner the page of book-shape scaffold is, the easier it is to remove cells and host cells infiltration. However, book-shaped AFS with only a page thickness of 100 μm were studied currently, because 100 μm is the thinnest page of book-shaped scaffold that can be operated to sandwiching ASCs-sheets. A suitable way or instrument should be innovated to facilitate sandwiching ASCs-sheets by the book-shaped AFS. Second, only the response of RAW 264.7 to the AFS were investigated in vitro, and more animal studies should be performed to determine the biosafety and immunogenicity of the book-shaped AFS before clinical trials. Third, considering that the native fibrocartilage layer in the rabbit PPT interface is about 1 mm (Figure S5), we assembled two book-shaped AFS (0.5 mm in thickness) together to construct a tissue-engineered fibrocartilage implant. However, this matching method was not very precise. Fourth, striking a balance between maintenance of native ECM and clearing host cellular components is a primary challenge faced when decellularizing a tissue. In the current study, we only optimized the decellularization protocols for fibrocartilage tissue by setting the concentration and treatment duration of SDS as variable, while more works still should be performed to establish a best decellularization protocol for fibrocartilage tissue in future. Fifth, the SDS applied in decellularization process was reported to denature the original structure of protein, thus may influence the protein functions.60 Although the content of TGF-β1, BMP-2, and IGF-1 in AFS were measured by ELISA, the activities of these growth factors remain unclear. Thus, whether our decellularization protocol is able to preserve functional proteins are worth investigation. Despite these limitations, this study indicated that this book-shaped AFS is a favorable scaffold for tissue-engineered fibrocartilage and has great potential for future clinical application.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.8b20563.

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Rabbit ASC identification by trilineage differentiation potential and cell surface marker, schematic diagram for the fabrication of single AFS implant, book-shaped AFS implants, mechanical testing of the QPPTC using a custom-made jig, failure mode of the patella-patellar tendon interface in tensile testing, cellularity reservation of the AFS prepared with different decellularization protocols, thickness of fibrocartilage layer of mature rabbit PPT interface, and primer sequences utilized for qRT-PCR gene expression analysis (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel.: +86-731-89753059. Fax: +86-731-84327332. *E-mail: [email protected]. Tel.: +86-731-89753001. Fax: +86-731-84327332. ORCID

Can Chen: 0000-0003-0261-245X Fei Liu: 0000-0002-8122-2902 Yifu Tang: 0000-0003-3742-4942 Cheng Zheng: 0000-0003-1726-0258 Yang Chen: 0000-0002-1555-1871 Muzhi Li: 0000-0001-7575-4585 Hongbin Lu: 0000-0001-7749-3593 Author Contributions

All authors contributed to the work. H.L. and J.H. planned the project and guided the work. Y.C., M.L., Y.T., and F.L. analyzed the data. C.C. finished the experiment and wrote the initial manuscript, which was revised by C.Z., J.Q., Y.C., and L.S.. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Nos. 81730068, 81472072, and 81171699). The authors would like to thank the staffs at BL01B station of National Facility for Protein Science Shanghai (NFPS) and the BL13W1 station of the Shanghai Synchrotron Radiation Facility (SSRF), Shanghai, China, for their kind assistance during the experiments. At last, sincere appreciation for my wife’s (Dr. Cui Li) full support.

5. CONCLUSION In the current study, we found an appropriate decellularization protocols for fibrocartilage tissue, and prepared a novel bookshaped AFS that showed a superior property in seeding-cells loading and chondrogenic inducibility. Additionally, a novel assembly pattern for fabricating tissue-engineered fibrocartilage was developed to enlarge seeding-cells loading, namely,

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REFERENCES

(1) Lu, H. H.; Thomopoulos, S. Functional attachment of soft tissues to bone: development, healing, and tissue engineering. Annu. Rev. Biomed. Eng. 2013, 15, 201−226.

O


Research Article

ACS Applied Materials & Interfaces (2) Atesok, K.; Fu, F. H.; Wolf, M. R.; Ochi, M.; Jazrawi, L. M.; Doral, M. N.; Lubowitz, J. H.; Rodeo, S. A. Augmentation of tendonto-bone healing. J. Bone Joint Surg Am. 2014, 96 (6), 513−521. (3) Xu, D.; Zhang, T.; Qu, J.; Hu, J.; Lu, H. Enhanced patellapatellar tendon healing using combined magnetic fields in a rabbit model. Am. J. Sports Med. 2014, 42 (10), 2495−2501. (4) Lu, H.; Chen, C.; Qu, J.; Chen, H.; Chen, Y.; Zheng, C.; Wang, Z.; Xu, D.; Zhou, J.; Zhang, T.; Qin, L.; Hu, J. Initiation Timing of Low-Intensity Pulsed Ultrasound Stimulation for Tendon-Bone Healing in a Rabbit Model. Am. J. Sports Med. 2016, 44 (10), 2706−2715. (5) Lu, H.; Qin, L.; Fok, P.; Cheung, W.; Lee, K.; Guo, X.; Wong, W.; Leung, K. Low-intensity pulsed ultrasound accelerates bone− tendon junction healing: a partial patellectomy model in rabbits. Am. J. Sports Med. 2006, 34 (8), 1287−1296. (6) Font Tellado, S.; Balmayor, E. R.; Van Griensven, M. Strategies to engineer tendon/ligament-to-bone interface: Biomaterials, cells and growth factors. Adv. Drug Delivery Rev. 2015, 94, 126−140. (7) Nebelung, W.; Becker, R.; Urbach, D.; Ropke, M.; Roessner, A. Histological findings of tendon-bone healing following anterior cruciate ligament reconstruction with hamstring grafts. Arch Orthop Trauma Surg 2003, 123 (4), 158−163. (8) Li, F.; Jia, H.; Yu, C. ACL reconstruction in a rabbit model using irradiated Achilles allograft seeded with mesenchymal stem cells or PDGF-B gene-transfected mesenchymal stem cells. Knee Surg Sports Traumatol Arthrosc 2007, 15 (10), 1219−1227. (9) Kawakami, Y.; Takayama, K.; Matsumoto, T.; Tang, Y.; Wang, B.; Mifune, Y.; Cummins, J. H.; Warth, R. J.; Kuroda, R.; Kurosaka, M.; Fu, F. H.; Huard, J. Anterior Cruciate Ligament-Derived Stem Cells Transduced With BMP2 Accelerate Graft-Bone Integration After ACL Reconstruction. Am. J. Sports Med. 2017, 45 (3), 584− 597. (10) Lui, P. P.; Wong, O. T.; Lee, Y. W. Application of tendonderived stem cell sheet for the promotion of graft healing in anterior cruciate ligament reconstruction. Am. J. Sports Med. 2014, 42 (3), 681−689. (11) Hu, J.; Qu, J.; Xu, D.; Zhang, T.; Qin, L.; Lu, H. Combined application of low-intensity pulsed ultrasound and functional electrical stimulation accelerates bone−tendon junction healing in a rabbit model. J. Orthop. Res. 2014, 32 (2), 204−209. (12) Walsh, W. R.; Harrison, J. A.; Van Sickle, D.; Alvis, M.; Gillies, R. M. Patellar tendon-to-bone healing using high-density collagen bone anchor at 4 years in a sheep model. Am. J. Sports Med. 2004, 32 (1), 91−95. (13) Leung, K. S.; Chong, W. S.; Chow, D. H.; Zhang, P.; Cheung, W. H.; Wong, M. W.; Qin, L. A Comparative Study on the Biomechanical and Histological Properties of Bone-to-Bone, Bone-toTendon, and Tendon-to-Tendon Healing: An Achilles TendonCalcaneus Model in Goats. Am. J. Sports Med. 2015, 43 (6), 1413− 1421. (14) Yin, Z.; Chen, X.; Zhu, T.; Hu, J. J.; Song, H. X.; Shen, W. L.; Jiang, L. Y.; Heng, B. C.; Ji, J. F.; Ouyang, H. W. The effect of decellularized matrices on human tendon stem/progenitor cell differentiation and tendon repair. Acta Biomater. 2013, 9 (12), 9317−9329. (15) Kang, H.; Peng, J.; Lu, S.; Liu, S.; Zhang, L.; Huang, J.; Sui, X.; Zhao, B.; Wang, A.; Xu, W.; Luo, Z.; Guo, Q. In vivo cartilage repair using adipose-derived stem cell-loaded decellularized cartilage ECM scaffolds. J. Tissue Eng. Regener. Med. 2014, 8 (6), 442−453. (16) Xue, J. X.; Gong, Y. Y.; Zhou, G. D.; Liu, W.; Cao, Y.; Zhang, W. J. Chondrogenic differentiation of bone marrow-derived mesenchymal stem cells induced by acellular cartilage sheets. Biomaterials 2012, 33 (24), 5832−5840. (17) Woods, T.; Gratzer, P. F. Effectiveness of three extraction techniques in the development of a decellularized bone-anterior cruciate ligament-bone graft. Biomaterials 2005, 26 (35), 7339−7349. (18) Reddi, A. H. Morphogenesis and tissue engineering of bone and cartilage: inductive signals, stem cells, and biomimetic biomaterials. Tissue Eng. 2000, 6 (4), 351−359.

(19) Xue, J.; He, A.; Zhu, Y.; Liu, Y.; Li, D.; Yin, Z.; Zhang, W.; Liu, W.; Cao, Y.; Zhou, G. Repair of articular cartilage defects with acellular cartilage sheets in a swine model. Biomed Mater. 2018, 13 (2), No. 025016. (20) Gong, Y. Y.; Xue, J. X.; Zhang, W. J.; Zhou, G. D.; Liu, W.; Cao, Y. A sandwich model for engineering cartilage with acellular cartilage sheets and chondrocytes. Biomaterials 2011, 32 (9), 2265− 2273. (21) Cheng, C. W.; Solorio, L. D.; Alsberg, E. Decellularized tissue and cell-derived extracellular matrices as scaffolds for orthopaedic tissue engineering. Biotechnol. Adv. 2014, 32 (2), 462−484. (22) Guo, L.; Qu, J.; Zheng, C.; Cao, Y.; Zhang, T.; Lu, H.; Hu, J. Preparation and Characterization of a Novel Decellularized Fibrocartilage ″Book″ Scaffold for Use in Tissue Engineering. PLoS One 2015, 10 (12), No. e0144240. (23) Sun, Y.; Yan, L.; Chen, S.; Pei, M. Functionality of decellularized matrix in cartilage regeneration: A comparison of tissue versus cell sources. Acta Biomater. 2018, 74, 56−73. (24) Huang, Z.; Godkin, O.; Schulze-Tanzil, G. The Challenge in Using Mesenchymal Stromal Cells for Recellularization of Decellularized Cartilage. Stem Cell Reviews and Reports 2017, 13 (1), 50−67. (25) Rahman, S.; Griffin, M.; Naik, A.; Szarko, M.; Butler, P. E. M. Optimising the decellularization of human elastic cartilage with trypsin for future use in ear reconstruction. Sci. Rep. 2018, 8 (1), 3097. (26) Koga, H.; Muneta, T.; Nagase, T.; Nimura, A.; Ju, Y. J.; Mochizuki, T.; Sekiya, I. Comparison of mesenchymal tissues-derived stem cells for in vivo chondrogenesis: suitable conditions for cell therapy of cartilage defects in rabbit. Cell Tissue Res. 2008, 333 (2), 207−215. (27) Perdisa, F.; Gostynska, N.; Roffi, A.; Filardo, G.; Marcacci, M.; Kon, E. Adipose-Derived Mesenchymal Stem Cells for the Treatment of Articular Cartilage: A Systematic Review on Preclinical and Clinical Evidence. Stem Cells Int. 2015, 2015, 597652. (28) Gupta, P. K.; Das, A. K.; Chullikana, A.; Majumdar, A. S. Mesenchymal stem cells for cartilage repair in osteoarthritis. Stem Cell Res. Ther. 2012, 3 (4), 25. (29) Ju, Y. J.; Muneta, T.; Yoshimura, H.; Koga, H.; Sekiya, I. Synovial mesenchymal stem cells accelerate early remodeling of tendon-bone healing. Cell Tissue Res. 2008, 332 (3), 469−478. (30) Fan, H.; Liu, H.; Wong, E. J.; Toh, S. L.; Goh, J. C. In vivo study of anterior cruciate ligament regeneration using mesenchymal stem cells and silk scaffold. Biomaterials 2008, 29 (23), 3324−3337. (31) Yokoya, S.; Mochizuki, Y.; Natsu, K.; Omae, H.; Nagata, Y.; Ochi, M. Rotator cuff regeneration using a bioabsorbable material with bone marrow-derived mesenchymal stem cells in a rabbit model. Am. J. Sports Med. 2012, 40 (6), 1259−1268. (32) Sukho, P.; Boersema, G. S. A.; Cohen, A.; Kops, N.; Lange, J. F.; Kirpensteijn, J.; Hesselink, J. W.; Bastiaansen-Jenniskens, Y. M.; Verseijden, F. Effects of adipose stem cell sheets on colon anastomotic leakage in an experimental model: Proof of principle. Biomaterials 2017, 140, 69−78. (33) Harada, Y.; Mifune, Y.; Inui, A.; Sakata, R.; Muto, T.; Takase, F.; Ueda, Y.; Kataoka, T.; Kokubu, T.; Kuroda, R.; Kurosaka, M. Rotator cuff repair using cell sheets derived from human rotator cuff in a rat model. J. Orthop. Res. 2017, 35 (2), 289−296. (34) Qi, Y.; Du, Y.; Li, W.; Dai, X.; Zhao, T.; Yan, W. Cartilage repair using mesenchymal stem cell (MSC) sheet and MSCs-loaded bilayer PLGA scaffold in a rabbit model. Knee Surg Sports Traumatol Arthrosc 2014, 22 (6), 1424−1433. (35) Khanarian, N. T.; Boushell, M. K.; Spalazzi, J. P.; Pleshko, N.; Boskey, A. L.; Lu, H. H. FTIR-I compositional mapping of the cartilage-to-bone interface as a function of tissue region and age. J. Bone Miner. Res. 2014, 29 (12), 2643−2652. (36) Kazakia, G. J.; Burghardt, A. J.; Cheung, S.; Majumdar, S. Assessment of bone tissue mineralization by conventional x-ray microcomputed tomography: comparison with synchrotron radiation microcomputed tomography and ash measurements. Med. Phys. 2008, 35 (7), 3170−3179. P


Research Article

ACS Applied Materials & Interfaces (37) Lu, H.; Zheng, C.; Wang, Z.; Chen, C.; Chen, H.; Hu, J. Effects of low-intensity pulsed ultrasound on new trabecular bone during bone−tendon junction healing in a rabbit model: a synchrotron radiation micro-CT study. PLoS One 2015, 10 (4), No. e0124724. (38) Ide, J.; Kikukawa, K.; Hirose, J.; Iyama, K.; Sakamoto, H.; Fujimoto, T.; Mizuta, H. The effect of a local application of fibroblast growth factor-2 on tendon-to-bone remodeling in rats with acute injury and repair of the supraspinatus tendon. J. Shoulder Elbow Surg 2009, 18 (3), 391−398. (39) Shah, S. A.; Kormpakis, I.; Havlioglu, N.; Ominsky, M. S.; Galatz, L. M.; Thomopoulos, S. Sclerostin Antibody Treatment Enhances Rotator Cuff Tendon-to-Bone Healing in an Animal Model. Journal of Bone and Joint Surgery 2017, 99 (10), 855−864. (40) Lu, H.; Qin, L.; Cheung, W.; Lee, K.; Wong, W.; Leung, K. Low-intensity pulsed ultrasound accelerated bone−tendon junction healing through regulation of vascular endothelial growth factor expression and cartilage formation. Ultrasound Med. Biol. 2008, 34 (8), 1248−1260. (41) Lipner, J.; Shen, H.; Cavinatto, L.; Liu, W.; Havlioglu, N.; Xia, Y.; Galatz, L. M.; Thomopoulos, S. In Vivo Evaluation of AdiposeDerived Stromal Cells Delivered with a Nanofiber Scaffold for Tendon-to-Bone Repair. Tissue Eng., Part A 2015, 21 (21−22), 2766−2274. (42) Choi, K. H.; Choi, B. H.; Park, S. R.; Kim, B. J.; Min, B. H. The chondrogenic differentiation of mesenchymal stem cells on an extracellular matrix scaffold derived from porcine chondrocytes. Biomaterials 2010, 31 (20), 5355−5365. (43) Jakus, A. E.; Laronda, M. M.; Rashedi, A. S.; Robinson, C. M.; Lee, C.; Jordan, S. W.; Orwig, K. E.; Woodruff, T. K.; Shah, R. N. ″Tissue Papers″ from Organ-Specific Decellularized Extracellular Matrices. Adv. Funct. Mater. 2017, DOI: 10.1002/adfm.201770203. (44) Omae, H.; Sun, Y. L.; An, K. N.; Amadio, P. C.; Zhao, C. Engineered tendon with decellularized xenotendon slices and bone marrow stromal cells: an in vivo animal study. J. Tissue Eng. Regener. Med. 2012, 6 (3), 238−244. (45) Omae, H.; Zhao, C.; Sun, Y. L.; An, K. N.; Amadio, P. C. Multilayer tendon slices seeded with bone marrow stromal cells: a novel composite for tendon engineering. J. Orthop. Res. 2009, 27 (7), 937−942. (46) Omi, R.; Gingery, A.; Steinmann, S. P.; Amadio, P. C.; An, K.N.; Zhao, C. Rotator cuff repair augmentation in a rat model that combines a multilayer xenograft tendon scaffold with bone marrow stromal cells. Journal of Shoulder and Elbow Surgery 2016, 25 (3), 469−477. (47) Wu, L. C.; Kuo, Y. J.; Sun, F. W.; Chen, C. H.; Chiang, C. J.; Weng, P. W.; Tsuang, Y. H.; Huang, Y. Y. Optimized decellularization protocol including alpha-Gal epitope reduction for fabrication of an acellular porcine annulus fibrosus scaffold. Cell Tissue Banking 2017, 18 (3), 383−396. (48) Neo, P. Y.; Teh, T. K.; Tay, A. S.; Asuncion, M. C.; Png, S. N.; Toh, S. L.; Goh, J. C. Stem cell-derived cell-sheets for connective tissue engineering. Connect. Tissue Res. 2016, 57 (6), 428−442. (49) Yang, J.; Yamato, M.; Kohno, C.; Nishimoto, A.; Sekine, H.; Fukai, F.; Okano, T. Cell sheet engineering: recreating tissues without biodegradable scaffolds. Biomaterials 2005, 26 (33), 6415− 6422. (50) Kosaka, M.; Nakase, J.; Hayashi, K.; Tsuchiya, H. AdiposeDerived Regenerative Cells Promote Tendon-Bone Healing in a Rabbit Model. Arthroscopy 2016, 32 (5), 851−859. (51) Zhou, G.; Liu, W.; Cui, L.; Wang, X.; Liu, T.; Cao, Y. Repair of porcine articular osteochondral defects in non-weightbearing areas with autologous bone marrow stromal cells. Tissue Eng. 2006, 12 (11), 3209−3221. (52) Xie, X.; Wang, Y.; Zhao, C.; Guo, S.; Liu, S.; Jia, W.; Tuan, R. S.; Zhang, C. Comparative evaluation of MSCs from bone marrow and adipose tissue seeded in PRP-derived scaffold for cartilage regeneration. Biomaterials 2012, 33 (29), 7008−7018.

(53) Shen, H.; Kormpakis, I.; Havlioglu, N.; Linderman, S. W.; Sakiyama-Elbert, S. E.; Erickson, I. E.; Zarembinski, T.; Silva, M. J.; Gelberman, R. H.; Thomopoulos, S. The effect of mesenchymal stromal cell sheets on the inflammatory stage of flexor tendon healing. Stem Cell Res. Ther. 2016, 7 (1), 144. (54) Sukho, P.; Hesselink, J. W.; Kops, N.; Kirpensteijn, J.; Verseijden, F.; Bastiaansen-Jenniskens, Y. M. Human Mesenchymal Stromal Cell Sheets Induce Macrophages Predominantly to an AntiInflammatory Phenotype. Stem Cells Dev. 2018, 27 (13), 922−934. (55) Bao, M.; Xie, J.; Huck, W. T. S. Recent Advances in Engineering the Stem Cell Microniche in 3D. Advanced Science 2018, 5 (8), 1800448. (56) Abagnale, G.; Steger, M.; Nguyen, V. H.; Hersch, N.; Sechi, A.; Joussen, S.; Denecke, B.; Merkel, R.; Hoffmann, B.; Dreser, A.; Schnakenberg, U.; Gillner, A.; Wagner, W. Surface topography enhances differentiation of mesenchymal stem cells towards osteogenic and adipogenic lineages. Biomaterials 2015, 61, 316−326. (57) Carroll, S. F.; Buckley, C. T.; Kelly, D. J. Cyclic Tensile Strain Can Play a Role in Directing both Intramembranous and Endochondral Ossification of Mesenchymal Stem Cells. Front. Bioeng. Biotechnol. 2017, 5, 73. (58) Fahy, N.; Alini, M.; Stoddart, M. J. Mechanical stimulation of mesenchymal stem cells: Implications for cartilage tissue engineering. J. Orthop. Res. 2017, 36 (1), 52−63. (59) Meier, E. M.; Wu, B.; Siddiqui, A.; Tepper, D. G.; Longaker, M. T.; Lam, M. T. Mechanical Stimulation Increases Knee Meniscus Gene RNA-level Expression in Adipose-derived Stromal Cells. Plastic and reconstructive surgery. Global open 2016, 4 (9), No. e864. (60) Moriyama, Y.; Watanabe, E.; Kobayashi, K.; Harano, H.; Inui, E.; Takeda, K. Secondary structural change of bovine serum albumin in thermal denaturation up to 130 degrees C and protective effect of sodium dodecyl sulfate on the change. J. Phys. Chem. B 2008, 112 (51), 16585−16589.

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Book-Shaped Acellular Fibrocartilage Scaffold with Cell-loading

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