2.1. Materials
2.1.1. Ethics statement
All methods were carried out in accordance with relevant guidelines and regulations. Research involving hulnan subjects was approved by the Ethics committee of Kyoto university and written informed consent was obtained from the donor. All animal experiments were approved by the ethics committee of Osaka University.
2.1.2. Cell culture
Human synovium MSC were isolated and expanded using serum-free culture medium STK1® and STK2® (Kanto Chemical Co., Inc., Tokyo, Japan). Briefly, synovium was obtained from a patient with anterior cruciate ligament injury (15-year-old female) at the time of arthroscopic surgery. The synovial specimens were minced and plated in an STK1 medium. Outgrown cells from tissue fragments were cultured in STK2 for cell expansion. The cells were confirmed to be CD13, CD44, CD34, CD73, and CD90 positive by flow cytometry analysis, and their potency in chondrogenic, osteogenic, and adipogenic differentiation was validated [23-26]. The cells were defined as synovial mesenchymal stromal cells (MSC) and used at passage 5 for subsequent experiments.
2.1.3. Development of the basic tissue engineered construct (TEC)
For the preparation of a basic TEC, MSC were plated on 24-well multi-well culture plates (BD Falcon science, NJ, USA) at a density of 4.0 x 105 cells/cm2 (8.0 x 105 cells/well) in growth medium containing 0.2mM ascorbate-2-phosphate (Asc-2P), an optimal concentration from earlier studies [16,17]. After an additional culture duration of 7 days, a monolayer complex of the cultured cells with an extracellular matrix was developed, and this complex formed a three-dimensional structure by detaching from the substratum of the plate. This construct was termed the basic TEC (Fig. 1A).
2.1.4. Development of a TEC containing dead MSC (fd-TEC)
Basic TECs were freeze-dried for 24 hours by vacuum lyophilization (Free Zone 2.5, Labconco, MO, USA). This process led to the death of the MSC and the resulting complex was termed an fd-TEC (Fig. 1A). Prior to use in experiments, the fd-TEC were rehydrated in sterile phosphate-buffered saline (PBS) at room temperature.
2.1.5. Development of iPS-CP by chondrogenic differentiation of hiPSC OHJ1 cells
The hiPSC line QHJl was generated by reprogramming human periphera1 blood monocytes derived from a healthy male individual with homozygosity for major HLA loci and was a gift from CiRA (Kyoto University; Kyoto, Japan). The cell line was generated by electroporating episomal plasmid vectors (pCXLE-hOCT3/ 4-shp53-F, hSK, hUL, EBNA1) into the cells of origin. The hiPS OHJ1 cells were chondrogenically differentiated to produce iPS-CP using a previously described method [14]. The iPSC-CPs were generated by chondrogenic differentiation of the cells for 10 weeks prior to being used in the current experiments (Fig. 1A). The cells in the iPSC-CP expressed collagen II as determined by qPCR (data not shown), a finding that is consistent with previous reports with similar iPSC-CP [27].
2.2. Characterization of materials
2.2.1 Cell viability
Cell viability in fd-TEC and basic TEC was assessed with the Live/Dead stain (Invitrogen, Carlsbad, CA, USA) and examined by epifluorescence microscopy.
2.2.2. Immunohistochemistry
Paraffin sections of tissues from in vitro experiments were prepared and subjected to immunohistochemistry. Cross-reactive monoclonal antibody to fibronectin (Fibronectin: 15613-1-AP, Proteintech, USA), vitronectin (Vitronectin: 15833-1-AP, Proteintech, USA), IL1-β (IL1-beta Antibody; R12-2204, Assay biotechnology Company, CA, USA), MMP-13 (Anti-MMP13; 18165-1-AP, Ptoteintech, Rosemont, IL, USA), and vascular endothelial growth factor (VEGF-A: ab1316, Abcom plc, UK) were used as the primary antibodies. The de-paraffinized sections were incubated in 3% H2O2 for 10 min to block endogenous peroxidase activity, followed by incubation in proteinase K (DAKO, Glostrup, Denmark) for 5 min for antigen retrieval, and then blocking one histo (Nakarai, Japan) for 30 min to avoid non-specific binding of primary antibodies. Primary antibody was applied to each section and incubated for 1 h. Detection was then performed using Histofine® Simple Stain™ MAX PO (MULTI; Nichirei Bioscience, Tokyo, Japan) and Simple Stain™ DAB Solution (Nichirei Bioscience).
2.3. In vivo studies
2.3.1. Implantation of the combined implants to osteochondral defects
Ten-week-old male athymic nude rats (F344 NJcl-rnu/rnu; CLEA Japan, Fujinomiya, Japan) were anesthetized by an intramuscular injection of a mixture of 0.3 mg/kg of medetomidine, 4.0 mg/kg of midazolam, and 5.0 mg/kg of butorphanol. After shaving, disinfection, and draping, a straight 1 cm long medial parapatellar incision was made at both knees of each animal. The patella was gently dislocated laterally and the femoral groove was exposed. Full-thickness articular osteochondral defects that were 3 mm in height, 2 mm in width, and 1.5 mm in depth, were created in the femoral groove of the distal femur using a drill with a sphere top of 1.5 mm diameter. The defects were generated at a moderate drill speed while irrigating the site with a room temperature saline solution to prevent thermal damage to the surrounding bone and cartilage.
Seventy-five rats were divided into five groups. For the untreated control group, the defects were left empty, while for the TEC group a basic TEC was implanted into the defect. For the iPSC-CP group an iPSCP was implanted into the defects. An iPSC-CP was wrapped with either a rehydrated fd-TEC (iPSC-CP/fd-TEC group) or a basic TEC (iPSC-CP/TEC group) immediately before implantation, and then these hybrid materials were implanted into the defects (Fig. 1B and 1C). Randomly selected rats in the five groups were euthanized at 4, 12, and 24 weeks postoperatively and the implants assessed.
Moreover, three animals were added for specimens in the iPSC-CP group at 4 weeks, two animals were added at 12 weeks, and one animal was added at 24 weeks, as the implanted iPSC-CP without a TEC detached from the defects in two of five implantation sites at 4 weeks.
2.3.2. Histological analyses and evaluation of repaired tissue
The isolated tissues were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) (pH 7.4) for a week, decalcified with EDTA, embedded in paraffin, and 3 µm sections were prepared from the center of the repair tissue. The sections were stained with hematoxylin and eosin (HE) and Safranin O staining.
Histological evaluation of tissue quality was performed using the modified O'Driscoll scoring system for cartilage and subchondral bone repair [20, 28].
2.3.3. Immunohistochemistry
Paraffin sections of tissues from in vivo experiments were prepared and subjected to immunohistochemistry. Sections derived from the intact knee from a 34-week-old male nude rat were used as the positive control. Cross-reactive monoclonal antibody to human vimentin (Anti-vimentin: ab16700, Abcom plc, UK), type II collagen (Col II: Kyowa Pharma Chemical, Japan), type X collagen (Col X: Abcom plc, UK), IL1-β (IL1-beta Antibody; R12-2204, Assay biotechnology Company, CA, USA), MMP-13 (Anti-MMP13; 18165-1-AP, Ptoteintech, Rosemont, IL, USA), vascular endothelial growth factor (VEGF-A: ab1316, Abcom plc, UK), rat/human CD31(CD31: ab182981, Abcam plc, UK), and human CD31 (CD31: ab76533, Abcom plc, UK) were used as the primary antibodies. The de-paraffinized sections were incubated in 3% H2O2 for 10 min to block endogenous peroxidase activity, followed by incubation in proteinase K (DAKO, Glostrup, Denmark) for 5 min for antigen retrieval, and then blocking one histo (Nakarai, Japan) for 30 min to avoid non-specific binding of primary antibodies. Primary antibody was applied to each section and incubated for 1 h. Detection was then performed using Histofine® Simple Stain™ MAX PO (MULTI; Nichirei Bioscience, Tokyo, Japan) and Simple Stain™ DAB Solution (Nichirei Bioscience). All procedures were performed at room temperature, and during each step, the sections were washed three times with 0.1% Tween 20 in PBS for 5 min. Human vimentin, IL-1β, MMP-13, and VEGF positive ratio were measured using Qupath software (https://qupath.github.io) [29, 30].
2.3.4. Radiological analysis using microfocus computed tomography (μCT)
The same five knees used for histological analysis were also used for radiological analysis (sham group: n=5). All the specimens, which were obtained at 4, 12, and 24 weeks postoperatively, were scanned using the Skyscan μ-CT (Skyscan 1272, Bruker, Belgium). Scanning was performed using the following parameters: Camera binning = 2 x 2, Source Voltage (kV) = 70, Source Current (μA) = 142, Image Pixel Size (μm) = 10, Rotation Step (degree) = 0.4, Filter = Al 0.5 mm. Image analysis was performed by CTAN software (Version 1.18.8.0+, Brucker). To evaluate the defect space, a rectangular interface region (2 mm × 1.5 mm × 3 mm) was defined as the Region of Interest (ROI; Fig. 1D, red rectangle). Bone volumes of new bone (BVnew bone), as well as the tissue volume (TV) inside the ROI, were calculated, and BV/TV% was defined as follow:
2.3.5. Biomechanical testing
Indentation testing was performed on the specimens using a self-made compression testing apparatus with an indentation probe consisted of a zirconia ceramic ball (Φ 1 mm). Each specimen was mounted on the sample stage of the compression tester and soaked in saline solution heated to 37℃. Indentation testing was performed on the specimens at an indentation rate of 30 μm/s and indentation depth was 100 μm (Fig. 1E). Five knees from intact male athymic nude rats (34 weeks of age) were subjected to biomechanical testing and served as a normal control group (n=5).
2.4. Statistical analysis
Statistical analysis was performed using analysis of variance followed by post-hoc testing for the postoperative changes of total histological scores and biomechanical testing. The comparison of results for O’Driscoll score between each group was analyzed by the Tukey-Krammer test. The comparison of results for BV/TV% and biomechanical parameters between the sham group and other groups was analyzed by the Bonferroni test. The results are presented as mean ± SD. The data were analyzed with JMP 16 (SAS Institute, Cary, NC), and significance was set at p < 0.05.
All procedures involving human participants were in accordance with the ethical standards of the institutional research committee in our institutions (the ethical committee of Osaka University Graduate School of Medicine, reference number: 14102-8, 16345-3) and the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Animal experiments were conducted after approval from the Ethics Committee of Osaka University (approval number: 01-074-001).