Reconstruction of cartilage with clonal mesenchymal stem cell-acellular dermal matrix in cartilage defect model in nonhuman primates

International Immunopharmacology 16 (2013) 399–408

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Reconstruction of cartilage with clonal mesenchymal stem cell-acellular dermal matrix in cartilage defect model in nonhuman primates Anlun Ma a, Li Jiang a, b, Lijun Song a, Yanxin Hu a, Hao Dun a, Pierre Daloze a, Yonglin Yu b, Jianyuan Jiang b, Muhammad Zafarullah c,⁎, Huifang Chen a,⁎⁎ a b c

Department of Surgery, Research Center, CHUM (CRCHUM), Notre-Dame Hospital, University of Montreal, Montreal, Canada Department of Orthopedics, Huashan Hospital, Fudan University, Shanghai, China Department of Medicine, Notre-Dame Hospital, CRCHUM, University of Montreal, Montreal, Canada

a r t i c l e

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Article history: Received 7 January 2013 Accepted 1 February 2013 Available online 13 March 2013 Keywords: MSC therapy Immunoregulation Reconstruction of cartilage Monkey model

a b s t r a c t Objective: Articular cartilage defects are commonly associated with trauma, inflammation and osteoarthritis. Mesenchymal stem cell (MSC)-based therapy is a promising novel approach for repairing articular cartilage. Direct intra-articular injection of uncommitted MSCs does not regenerate high-quality cartilage. This study explored utilization of a new three-dimensional, selected chondrogenic clonal MSC-loaded monkey acellular dermal matrix (MSC-ADM) scaffold to repair damaged cartilage in an experimental model of knee joint cartilage defect in Cynomolgus monkeys. Methods: MSCs were characterized for cell size, cell yield, phenotypes, proliferation and chondrogenic differentiation capacity. Chondrogenic differentiation assays were performed at different MSC passages by sulfated glycosaminoglycans (sGAG), collagen, and fluorescence activated cell sorter (FACS) analysis. Selected chondrogenic clonal MSCs were seeded onto ADM scaffold with the sandwich model and MSC-loaded ADM grafts were analyzed by confocal microscopy and scanning electron microscopy. Cartilage defects were treated with normal saline, clonal MSCs and clonal MSC-ADM grafts, respectively. The clinical parameters, and histological and immunohistochemical examinations were evaluated at weeks 8, 16, 24 post-treatment, respectively. Results: Polyclonal and clonal MSCs could differentiate into the chondrogenic lineage after stimulation with suitable chondrogenic factors. They expressed mesenchymal markers and were negative for hematopoietic markers. Articular cartilage defects were considerably improved and repaired by selected chondrogenic clonal MSC-based treatment, particularly, in MSC-ADM-treated group. The histological scores in MSC-ADM-treated group were consistently higher than those of other groups. Conclusion: Our results suggest that selected chondrogenic clonal MSC-loaded ADM grafts could improve the cartilage lesions in Cynomolgus monkey model, which may be applicable for repairing similar human cartilage defects. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Articular cartilage lesions can result from a variety of causes such as many joint diseases or trauma including work- or sports-related injuries. They are frequently associated with joint pain, dysfunction and disability, and are believed to progress to severe forms of osteoarthritis (OA). Articular cartilage does not usually regenerate by forming original-quality tissue because of poor regenerative capacity ⁎ Correspondence to: M. Zafarullah, Department of Medicine, University of Montreal, K-5255 Mailloux, Notre-Dame Hospital of CHUM, 1560 Sherbrooke E, Montreal, Quebec, H2L 4M1, Canada. Tel.: +1 514 890 8000x25690; fax: +1 514 412 7612. ⁎⁎ Correspondence to: H. Chen, Laboratory of Experimental Surgery, Research Center, CHUM, Room Y1611, Notre-Dame Hospital, Department of Surgery, University of Montréal, 2099 Alexandre de Sève, Montréal, Québec, H2L 2W5, Canada. Tel.: +1 514 890 8000x27081; fax: +1 514 412 7581. E-mail addresses: [email protected] (M. Zafarullah), [email protected] (H. Chen). 1567-5769/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.intimp.2013.02.005

of chondrocytes, particularly, in elderly patients [1,2]. Despite applicability of total joint replacement surgery, osteotomy or arthrodesis to patients with OA, few methods of cartilage repair are available and effective [3]. Autologous chondrocyte implantation is thought to be a useful biomedical treatment that repairs articular cartilage damage. It provides pain relief, slows down the disease progression and considerably delays joint replacement surgery [4]. However, some severe patients are excluded from receiving chondrocyte transplantation due to non-availability of healthy chondrocytes. Recent studies have demonstrated that adult mesenchymal stem cells (MSCs) might represent an alternative source for cells with chondrogenic potential for cartilage regeneration [5–7]. MSCs have been known as non-hematopoietic progenitor cells found in various adult tissues. They are characterized by their ease of isolation and their extensive proliferative ability while retaining the potential to differentiate along various lineages of mesenchymal origin, including chondrocytes, osteoblasts, and adipocytes [8–10]. Additionally, MSCs

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possess potent immuno-modulatory and anti-inflammatory effects through either direct cell–cell interaction or secretion of various factors. They exert a tremendous effect on local tissue repair through modulating local environment and activation of endogenous progenitor cells. These features make MSC-based cell therapy as a hotly pursued subject of investigation in cartilage regeneration. However, a crucial requirement for MSC-based cartilage repair therapy is the delivery of the cells to the defect site. Although direct intra-articular injection might be possible in early stages of articular cartilage lesions when the defects are restricted to the cartilage superficial layer, intra-articular injection of MSCs as a suspension led to their attachment to non-target areas. Such delivery of uncommitted MSCs to cartilaginous lesions does not lead to reproducible and satisfactory regenerated tissue, but rather induces fibro-cartilage formation [11,12]. To improve the quality of the treatment, different studies have reported the application of various synthetic scaffolds in tissue engineering, which enable MSC penetration, help in maintaining the MSCs, provide chondroinductive matrix, allow nutrient delivery and gas exchange, and mimic the natural tissue geometry [13–16]. However, many investigators are still concerned about the long-term safety and feasibility of synthetic scaffolds in the clinic. In this study, a novel strategy was explored via developing a three-dimensional selected chondrogenic clonal MSC-loaded acellular dermal matrix (ADM) in vitro, and replacing damaged cartilage by the transplantation of MSC-ADM grafts in an experimental, genetically close to human model of knee joint cartilage defect in Cynomolgus monkeys. All MSC samples were characterized for cell yield, phenotypes, proliferation and chondrogenic capacity. Chondrogenic clonal MSCs were selected and seeded to ADM. The repaired defects were evaluated by histological examination and immunohistochemistry on weeks 8, 16 and 24, respectively, during the 6-month period of this study. Our aim was to investigate whether transplantation of selected chondrogenic clonal MSC-loaded ADM grafts could improve cartilage lesions in Cynomolgus monkeys, so that feasibility and acceptability of MSC-based cell therapy could be applied precisely for clinical research in humans.

2. Materials and methods 2.1. Animals Male Cynomolgus monkeys (Macaca fascicularis), 3–5 years old, were provided by the Experimental Animals Center, Academy of Military Medical Sciences, Beijing, China. All animal experimental protocols were approved by the local Ethical Committee for Animal Experimentation. 2.2. Reagents and monoclonal antibodies Anti-human monoclonal antibodies (mAbs) cross-reacting with Cynomolgus monkeys were selected for this study. They were purchased from BD. Anti-human type I collagen and type II collagen mAbs were from Abcam. Recombinant human transforming growth factor-β (rh-TGF-β) was the product of R&D System. Insulin-like growth factor (IGF), fibroblast growth factor-2 (FGF-2), Alexa Fluor 546 and 488 antibodies were obtained from Invitrogen. Proteinase K, EnVision HRP and DAB solution were purchased from Dako. Dexamethasone, L-ascorbic acid, paraformaldehyde, Dispase II, Triton X-100, Alcian blue, Safranin O and Fast Green (SO/FG) stains were obtained from Sigma. Sircol collagen assay kit was from Biocolor.

2.4. Bone marrow harvesting and MSC culturing Monkey heparinized bone marrow (BM) cells were aspirated from the humerus with an 18-gauge needle. The mononuclear cells were layered over Ficoll solution and centrifuged at 1000 g for 25 min at room temperature. Cells were collected and resuspended in DMEM medium supplemented with 20% monkey serum, 0.1 M glutamine 10,000 mg/mL, penicillin–streptomycin 10,000 U/mL. Then, the cells were seeded at a concentration of 5 × 10 6 cells/T25 flask and incubated at 37 °C in 5% CO2. Twenty-four hours later, the non-adherent cells were removed by washing with PBS. The adherent cells were cultured with MSC expansion medium, 20% monkey serum-DMEM medium supplemented with rh-TGF-β (10 ng/mL), IGF (10 μg/mL), FGF-2 (10 μg/mL), 100 nM dexamethasone and L-ascorbic acid (50 μg/mL), and changed twice weekly with half medium volume. Once the cells reached about 80% confluence, the primary MSCs were passaged with 0.25% trypsin/0.001 M EDTA. Initial confluent culture cells were designated passage 0 polyclonal MSCs (P0). Fluorescence-activated cell sorter (FACS), growth kinetics and chondrogenic assays were performed at different passages. The amount of sulfated glycosaminoglycans (sGAG) (μg/pellet) was measured by Alcian blue staining [17]. The level of collagen protein was determined using FACS and Sircol collagen assays. 2.5. Colony forming unit (CFU) assay For CFU assay, 0.5 × 10 6 initial passage of polyclonal MSCs were seeded in triplicate wells of a 6-well cluster plate in MSC expansion medium and fed twice a week. On days 3, 7 and 14 after initial plating, the colonies were fixed in 1% paraformaldehyde and stained with 1% crystal violet. The number of colonies was counted. Colonies b 2 mm in diameter and faintly stained colonies were ignored. 2.6. Cell cloning Cell cloning was performed by limiting dilution of polyclonal MSCs (P0), and seeded at 2, 1 and 0 cells per well of 96-well plate. Microscopy was performed to ensure that clones were only taken from wells where a single cell had attached and proliferated. Colonies were passaged through a 25 cm2 flask as initial passage 0 of clonal MSCs. Chondrogenic assays were performed at different passages. When amounts of sGAG and collagen protein of colonies were higher than their mean levels in polyclonal MSCs, they were selected as positive selected clonal MSC lines for further studies. 2.7. FACS analysis Polyclonal and clonal MSCs were stained separately with CD3, CD11, CD14, CD29, CD34, CD45, CD73, CD90 and type II collagen antibodies. Matching isotype control mAbs were included in the experiments. Cell fluorescence was evaluated by FACSCalibur instrument (BD). The data were analyzed using CellQuest software (BD). 2.8. Collagen assay Level of total collagen protein was measured using the Sircol collagen assay kit. Absorbance at 540 nm was measured using a spectrophotometer. The concentration of collagen was interpolated from a standard curve that was established using collagen standard provided by the manufacturer [18]. 2.9. MSC-loaded ADM grafts with the sandwich model

2.3. Study design All monkeys were randomly distributed over three groups, normal saline (NS)-, MSCs-, and MSC-ADM graft-treated groups, three monkeys in each group.

Monkey skin (500 μm in thickness) was removed using a dermatome for preparation of allogenic ADM. The removed skin was washed three times in sterile PBS and preserved at −80 °C. After thawing, skin was treated with 2.5 U/mL Dispase II at 4 °C for 24 h with continuous

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shaking to remove the epidermis and other cellular components from the dermal matrix. Subsequently, the dermis was incubated in 0.5% Triton X-100 for 24 h with continuous shaking. Either sodium azide (0.02% wt./vol.) or a cocktail of antibiotics was present at all times during both of these steps to preclude microbial growth. The resulting ADM was then extensively washed and stored in PBS saline at 4 °C. Selected clonal MSCs (2 × 106, P5) were resuspended in 250 μL of MSC culture medium containing rh-TGF-β, IGF, FGF-2, dexamethasone and L-ascorbic acid, and seeded onto ADM scaffold (2.0 mm in diameter, 0.3–0.5 mm in thickness) in 96 well culture plates, and cultured on a rotating shaker at 37 °C, 5% CO2 for 21 days. The MSC-ADM grafts were evaluated by sGAG and collagen analyses, and analyzed by confocal microscopy and scanning electron microscopy.

immediately and for 2 days thereafter. Postoperatively, the animals were permitted cage activity without immobilization. Cartilage defect model was created in both knee joints by drilling three full-thickness cartilage defects (3.0 mm in diameter, 2.0 mm in depth) in Cynomolgus monkey (Fig. 1A). The areas of full-thickness cartilage defects were treated with NS solution which served as negative control. The others were treated by locally attaching the autologous selected clonal MSCs (2 × 106/defect) or transplanted with autologous selected clonal MSCloaded ADM grafts (2 grafts/defect) (Fig. 1B). Animals were sacrificed at weeks 8, 16 and 24, respectively after the operation for histological and immunohistochemical examinations.

2.10. Cartilage defect model in Cynomolgus monkeys

The dissected distal femurs were fixed in 10% neutral buffered formalin for 72 h, and decalcified with 0.5 M EDTA and embedded in paraffin blocks. The sections of 5 μm in thickness were stained with hematoxylin and eosin (HE), or subjected to SO/FG staining to assess proteoglycans and collagen in the matrix. For IHC, sections were adhered to Superfrost Plus Gold slides in order to enhance tissue adherence. They were treated with proteinase K to unmask the epitopes, and then incubated in 10% normal goat serum to prevent nonspecific bindings. The slides were then incubated

All surgical procedures were performed under general anesthesia and with the use of sterile techniques. Animals were anesthetized by intramuscular injection with ketamine (30 mg/kg body weight) and xylazine (5 mg/kg body weight). Anesthesia was maintained by means of a mixture of 2% forane and oxygen/nitrous oxide (1/0.4 L/min) delivered by an automatic ventilator. After surgical procedures, an antibiotic (Cephradine) and analgesic (Celecoxib) therapy was administered

2.11. Histological and immunohistochemical (IHC) analyses

Fig. 1. Cartilage defect model and characterization of MSCs. Cartilage defect model was created in Cynomolgus monkey knee joints by drilling the full-thickness defects (3 mm in diameter, 2 mm in depth) (A). The defect areas were treated with NS solution, or MSCs or transplanted by MSC-loaded ADM grafts (B). The growth kinetics curves of polyclonal (C) and clonal (D) MSCs during the period of 14-day culture are shown. Results are representative of 6 independent donors (n = 6, p b 0.05). Colony-forming number of MSCs (E) and cell morphologic features (F) are depicted. FACS analysis of cell size is presented by histogram in panel G. Expression of surface markers on MSCs was analyzed by FACS assay during the period of expansion. The data obtained from 6 independent experiments are shown in H (n = 6, p b 0.05). A representative example of FACS analysis of different markers is shown in I.

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Fig. 1 (continued).

with type I collagen (1:100) and type II collagen (1:200), respectively, for 2 h at room temperature. Slides were washed and incubated with rabbit anti-mouse biotinylated secondary antibody (1:200) at 37 °C for 30 min. The sections were bound with EnVision HRP and then incubated with DAB solution. Slides were counterstained with hematoxylin and mounted on glycerol gel. The slides were observed with a Carl Zeiss Axioskop 40. The sections were scored by using a grading scale modified from the International Cartilage Repair Society (ICRS) visual histological assessment scale [19,20]. The original ICRS criteria were: the surface, matrix, cell distribution, cell population viability, subchondral bone and mineralization of cartilage (Table 1). 2.12. Confocal microscopy and scanning electron microscopy (SEM) For confocal microscope examination, sections were incubated with 0.1% Triton-X for 5 min and unmasked the epitopes by treating with proteinase K at 37 °C for 15 min. After washing, samples were incubated at room temperature for 30 min with 10% normal goat serum to prevent nonspecific bindings. The slides were then incubated separately with CD29 and CD90 primary antibodies at room temperature for 2 h. After washing, Alexa Fluor 546 or Alexa Fluor 488 secondary

antibodies were incubated for 2 h with CD29 and CD90 staining, respectively. Fluorescently labeled cells were visualized and photographed using a laser scanning confocal microscope (MRC1024, Zeiss). For SEM examinations, samples were loaded onto aluminum studs and coated with gold for 3 min. Under the condition of vacuum pressure of 0.1 Torr at 8 mA, they were examined by a scanning electron microscope (JEOL model JSM-5610LV, Japan). 2.13. Statistical analysis Statistical analysis was performed using the SPSS v16.0 software (SPSS Inc., Chicago, Illinois, USA). Data were reported as Mean ± Standard Error. A p value of less than 0.05 was considered to be significant. 3. Results 3.1. Characterization of MSCs The curves of growth kinetics showed that polyclonal and clonal MSCs expanded rapidly after 4-day culture, particularly in polyclonal MSCs (Fig. 1C,D, n = 6, p b 0.05), then they grew smoothly up to day

A. Ma et al. / International Immunopharmacology 16 (2013) 399–408 Table 1 The modified ICRS visual histological assessment scale. Category A

B

C

D

E

F

G

H

The regularity of the surface Smooth/continuous Irregularities discontinuous Matrix morphology Hyaline Mixture: hyaline/fibrocartilage Fibrocartilage Fibrous tissue Cell distribution Columnar Mixed/columnar-clusters Clusters Individual cells/disorganized Cell population viability Predominantly viable Partially viable b10% viable Subchondral bone Normal Increased remodeling Bone necrosis/granulation tissue Detached/fracture/callus at base Cartilage mineralization (calcified cartilage) Normal Abnormal/inappropriate location Type I collagen staining of the matrix None Slight Moderate Abundant Type II collagen staining of the matrix Abundant Moderate Slight None Total score

Score 3 0 3 2 1 0 3 2 1 0 3 1 0 3 2 1 0 3 0

403

those of polyclonal MSCs (42.9 ± 38.1 vs. 41.9% ± 11.1%, n = 6, p > 0.05), because clonal MSCs showed a broad positive range of 4.3%– 80.5% before selection. 3.2. MSC adhesion to cartilage defects in vitro To determine the length of optimum cell-attaching time and maximum number of cells adhering to cartilage defects, fresh monkey knee joint cartilage were created by drilling wells (3 mm in diameter, 2 mm in depth) into full-thickness cartilage. The side of cartilage defects was put upward (Fig. 2A,B). MSC suspension (1 × 106) was delivered into each well and kept for 5, 10, 15, 20 and 25 min at room temperature, respectively. Then, the side of cartilage defect was turned over (Fig. 2C), and cultured downward for another 10 min, so that non-adhered cells were resuspended into the culture medium. Finally, the number of cells adhered to cartilage defect was calculated by subtracting from the number of primary seeding cells. MSC kinetics results (Fig. 2D) showed that number of attached MSCs increased rapidly within 10 min and more than 69.00% ± 8.8% of the MSCs adhered into the cartilage defect, then, decreased slowly thereafter. These results indicated that MSCs have a strong adhesive capacity to cartilage defects within a short time and determined the appropriate time for MSCs' attachment to cartilage defects used in this study. 3.3. Chondrogenic potential of MSCs and MSC-ADM

3 2 1 0 3 2 1 0 24

14. Colony-forming capacity of MSCs of 6 independent donors was found in the early phase of the culture, and showed distinctive proliferation on day 7 and day 14 day cultures (25.8 ± 5.2 CFU/10 6 cells vs. 29.7 ± 3.7 CFU/106 cells, n = 6, p b 0.05, Fig. 1E). The cells in the colonies presented fibroblast-like morphology at day 14 (Fig. 1F). The results of FACS analysis (Fig. 1G) showed the size of the cultured cells increased with culture time extension. These results suggest the production of cartilage extracellular matrix during the culture. Phenotype analysis (Fig. 1H) at different passages showed that primary polyclonal MSC fraction (P0) contained mixed populations of MSCs and less round hematopoietic cells; they expressed negative or very low hematopoietic cell markers, such as CD45, CD34 (6.5% ± 2.1%, 2.1% ± 0.6%) and other lymphocyte markers CD3, CD11, and CD14 (1.2% ± 0.2%, 1.3% ± 0.3%, and 0.8% ± 0.1%). A representative example is shown in Fig. 1I. Clonal MSCs showed a similar profile at primary culture. As distinctive markers of MSCs, CD29, CD73 and CD90 displayed positive expression on polyclonal MSCs (25.7% ± 1.4%, 15.6% ± 5.7%, and 22.5% ± 6.2%), as well as on clonal MSC passages (25.7% ± 1.3%, 16.7% ± 5.9%, and 21.5% ± 6.1%), respectively. At the late passage cells (P5), their expression was increased in polyclonal MSCs (63.8% ± 18.1%, 75.7% ± 11.3%, and 43.4% ± 11.6%) and clonal MSCs (54.9% ± 38.3%, 62.1% ± 36.2%, and 49.5% ± 21.3%). There was a significant difference between early and late passage cells (n = 6, p b 0.05). The expression of type II collagen was also found to have increased at late passages of polyclonal MSCs compared with those of early primary cells (41.9% ± 11.1% vs. 2.0% ± 0.5%, n = 6, p b 0.05). However, in clonal MSCs, there was a significant difference between early and late-passages (42.4 ± 38.1 vs. 2.0 ± 0.5, n = 6, p b 0.05), but no statistical difference was found when compared with

To further investigate cartilage-forming potential of MSCs, the amount of sGAG was measured in clonal and polyclonal MSCs. At passage 5, the average sGAG content was higher in clonal cultured MSCs than that of polyclonal MSCs (8.7 ± 5.6 μg/pellet vs. 4.4 ± 1.5 μg/pellet, n = 6, p > 0.05), but no statistical difference was found because of a wide positive range in clonal MSCs (0.3–21.0 μg/pellet) (Fig. 3A). However, there was a significant difference in the level of collagen between both clonal and polyclonal MSCs (1.4 ± 0.28 μg/pellet vs. 0.9 ± 0.16 μg/pellet n = 6, p b 0.001, Fig. 3B). After positive selection, six autologous selected clonal lines were selected from 6 donors, three lines were continued culture for MSC treatment, and the other three lines were seeded on ADMs and cultured for 21 days for MSC-ADM treatment. The average amount of sGAG on day 21 was 4.6 ± 0.9 μg/mg MSC-ADM wet weight compared to that of 1.1 ± 0.2 μg/mg on day 3 (n = 6, p b 0.05, Fig. 3C). The level of type II collagen protein also increased on day 21 when compared to that of day 3 (6.2 ± 0.9 μg/mg vs. 2.4 ± 0.6 μg/mg, n = 6, p b 0.05, Fig. 3D). These data suggested chondrogenic potential in selected clonal MSC-ADM grafts. The results of scanning electron microscopy showed the structure of MSC-loaded ADM (Fig. 3E). The confocal fluorescence microscopy examination demonstrated that CD29- (Fig. 3G) and CD90- (Fig. 3H) expressing MSCs could be found inside the ADM structure compared with that of the no cell seeded ADM structure (Fig. 3F). 3.4. Macroscopic features of cartilage after the treatment Animals were sacrificed on weeks 8, 16 and 24 after the operation, respectively for clinical observation. At the early stage (8 weeks) of the treatment, the cartilage defects in the control groups were overlaid with blood clots and reddish tissue (Fig. 4A), and also in the MSC-treated group (Fig. 4B). In the MSC-ADM-transplanted group, the defects became whitish and glossy in some areas (Fig. 4C). In the middle stage of the treatment (16 weeks), cartilage defects of NS group were covered with whitish tissue in some areas, but the reddish tissue still remained in other areas (Fig. 4D). In the MSC-treated group, the reddish regions disappeared (Fig. 4E). In the MSC-ADM-transplanted group, the borders between graft and cartilage defect were indistinguishable (Fig. 4F). At the termination phase of this study (24 weeks), the cartilage defects still persisted in the control groups (Fig. 4G). In the MSC-treated group, the defects were covered with whitish tissue but the margins

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Fig. 2. Diagram of the engineering procedure of the sandwich model (upwards). Step A: suspension of MSCs was seeded on an acellular dermal matrix. Step B: another layer was stacked on top of the first layer. Step C: suspension of MSCs was seeded on each layer. Step D: a MSCs sandwich model was formed after stacking 2 layers. MSC attachment to cartilage defects in vitro (below). The full-thickness cartilage defects (3 mm in diameter, 2 mm in depth) were created on both knee joints in vitro (A). The cartilage defects were faced upward, filled with 106 MSCs, and held stationary for 5 to 25 min (B). The femurs were turned into defect side faced downward for 15 min (C). The non-adhered cells in the medium were counted. The number of adhered cells attached into cartilage defect was calculated by subtracting from the number of primary seeding cells (D).

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Fig. 3. Chondrogenic potential of MSCs. The concentrations of sGAG and collagen were measured in polyclonal and clonal MSCs (A, n = 6, p b 0.05, B n = 6, p b 0.001), the data obtained from 6 independent clones. The amounts of sGAG and collagen were evaluated in selected clonal MSC-seeded ADM (C, n = 6, p b 0.05, D n = 6, p b 0.05), the data obtained from 6 independent grafts. The results of scanning electron microscopy show the structure of MSC-loaded ADM (E). The confocal fluorescence microscopy examination demonstrated that CD29 (G) and CD90 (H) were positively expressed in MSCs inside ADM compared with those in no cell seeded ADM (F).

were still distinct (Fig. 4H). In the MSC-ADM-transplanted group, the peripheral lesion of the defect appeared to integrate into the surrounding native cartilage (Fig. 4I). 3.5. Histological and immunohistochemical observations For HE staining, the results showed that on week 8 after the treatment, formation of a new cartilage matrix was found in some defect areas of MSC-treated group (Fig. 5B). In MSC-ADM-treated group, the defects were filled with abundant cartilage matrix and chondrocytes, although they were rather small and flattened parallel to the surface (Fig. 5C), when compared to NS control defects, where no chondrocytes and poor cartilage matrix were found in the defect zones (Fig. 5A). In the middle stage of the treatment (16 weeks), the defect regions were filled with some connective tissue or fibrous cartilage tissue and were poorly healed in the control groups (Fig. 5D). In MSC-ADMtreated group, the cartilage matrix at the cartilage defects still remained and more chondrocytes were found in the defect region, and the border between regenerated cartilage and subchondral bone moved upward. Integration between native cartilage and regenerated tissue appeared

to be improved (Fig. 5F). Although more cartilage matrices could be observed in the group of MSC treatment than in the control group, the height of the repaired tissue was lower than that of the surrounding cartilage (Fig. 5E). On week 24 of this study, the cartilage defects were still not healed and fibrous tissue was found in the control group (Fig. 5G). Although more chondrocytes could be observed in MSC-treated group (Fig. 5H), the treatment with MSC-ADM leads to further development of regenerated cartilage matrix and the cellular component was clearly increased. The thickness of the regenerated cartilage was similar to that of the neighboring cartilage. The borders between the native tissue and the regenerated tissue were well integrated and no gap was found (Fig. 5I). The immunohistochemical analysis revealed negative staining of type I collagen (Fig. 5K,L) and positive staining of the type II collagen (Fig. 5N,O) in the repaired areas of MSC-treated and MSC-ADM-treated groups compared to those in the control group (Fig. 5J,M) on week 24. SO/FG staining showed strong positive staining in repaired cartilages in MSC-ADM-treated group (Fig. 5R) and mild positive staining in MSC-treated group (Fig. 5Q) on week 24, compared to almost negative SO/FG staining in the control group (Fig. 5P).

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Fig. 4. Macroscopic observation of cartilage defects after the treatments. In NS, MSCs and MSC-ADM groups, macroscopic observation of cartilage defects was showed at week 8 (A, B, and C), week 16 (D, E, and F) and week 24 (G, H, and I) after the treatment.

3.6. Evaluation of histological scores The scores of MSC-treated and MSC-ADM-treated groups improved continuously through 24 weeks and were higher than those in other groups at each time point. The Kruskal–Wallis test revealed that there were significant differences between groups; MSC-treated group vs. control group, MSC-ADM-treated group vs. control group at each time point (n = 3, p b 0.01). A significant difference was obtained between MSC-treated group and MSC-ADM-treated group on week 24 (n = 3, p b 0.01). This analysis demonstrated that MSC-ADM-treated group got higher ICRS score when compared to those of the other two groups at the end point of this study (n = 3, p b 0.01, Fig. 5S). 4. Discussion MSCs have been an attractive cell source for regenerative medicine, particularly in the treatment of cartilage injuries [21,22]. A successful outcome from the use of MSCs for cartilage regeneration in joints requires sufficient quantity of MSCs and delivery of the MSCs to the defect site. Although direct intra-articular injection might be effective in the early stages of the disease when the defect is restricted to the cartilage layer, it was not suitable in advanced stages where the subchondral bone is exposed over large areas. It was found that only a small portion of these cells attached to the cartilage defects, and most of the intra-articular injected MSCs adhered to the synovial tissue, which would increase the risk of adverse effects, such as synovial proliferation [23]. With the development of biomedical technology, scaffold or matrix of some kind has been reported to be required to support the MCSs in functional performance [24,25]. Seeding MSCs into a natural or synthetic scaffold is useful for cell proliferation and matrix production. Our study used natural monkey acellular dermal matrix as a scaffold that presents a more natural microenvironment to support growth and proliferation of MSCs than synthetic

scaffolds. The results of scanning electron microscopy and confocal fluorescence microscopy showed that MSCs can survive in ADM grafts. This study served to develop surrogate tools for mimicking human joint diseases in an experimental cartilage defect model of nonhuman primates. This genetically closest to human model was used to compare the efficacy of two different cell delivery approaches, suspension and ADM scaffolds, to investigate the safety and efficacy of MSCs, and evaluated the utility of autologous selected clonal MSC-ADM grafts and selected clonal MSCs alone for delaying the progression of cartilage lesions and repairing cartilage defects in Cynomolgus monkeys. For the present study we developed monkey acellular dermal matrix as a new type of MSCs scaffold, which is natural and super-thin, super-thin, and a simple manipulation. Our results revealed that articular cartilage defects were considerably improved and repaired by selected chondrogenic clonal MSC-based treatment, particularly, in MSC-ADM-treated group. On week 24 of this study, cartilage defects of knee joints were repaired with newly formed hyaline-like cartilage after transplantation of MSC-ADM grafts. Guo et al. reported that using MSCs- loaded bioceramic scaffold repaired articular cartilage defects in a sheep model, and the mean histological score was18 ± 3.38 after 24-week treatment [26]. A similar study was demonstrated by Necas A. et al., in which the transplantation of MSCs in a composite scaffold was of benefit to the repaired cartilage in miniature pigs with a histological score of 14.7 ± 3.82 [27]. The morphological, histological, and immunohistochemical scores were consistently higher in MSC-ADMtreated group than that in other groups. The tissue regeneration with MSC-ADM was better than those with MSCs alone. Immunohistochemical staining showed absence of fibroplasia in the selected clonal MSC-ADM-treated defects, but it would need to be verified in a longer-term study. Some studies have demonstrated that MSCs can secrete a broad spectrum of bioactive molecules that have immunoregulatory and regenerative activities. Bioactive factors secreted by MSCs have been shown to inhibit tissue scarring, suppress apoptosis, stimulate angiogenesis, and enhance mitosis of tissue-intrinsic stem or progenitor cells [28–30]. Another related study by us showed that MSCs could up-regulate T regulatory (Treg) cells and secrete IL-10 cytokine, but not pro-inflammatory cytokines (our unpublished data). It confirmed that MSCs have immunoregulatory effects, which not only suppress activation of lymphocytes, but also enhance the function of Treg cells. These results support that the use of ADM scaffold delivery of autologous clonal MSCs to the defect site was more effective than the direct injection of MSC suspension. Ultimately, it will be directed towards developing a clinically feasible strategy for use of MSC-ADM in the reconstruction of the articular cartilage. In vitro results demonstrated that bone marrow-derived MSCs can differentiate into the chondrogenic lineage in this study, and after positive clone selection, monoclonal MSCs retained better chondrogenic capacity than those in polyclonal MSCs, particularly, in higher passages. When selected clonal MSCs were seeded on ADM and cultured for 21 days, the average amounts of sGAG and type II collagen protein were increased. Thus, prolonged MSC-ADM scaffold interactions favored enhanced chondrogenic potential. Nagase et al. reported that BM derived MSCs can undergo a time-dependent transition from small cells to large cells and that the culture time can affect the chondrogenic differentiation potential [31,32]. Our data were similar to their findings, with extending culture period, cell size, amount of sGAG and collagen that were found to have increased in selected monoclonal MSC lines. These

Fig. 5. Pathological and immunohistochemical examinations of cartilage groups after the treatment, and histological score. In NS, MSCs and MSC-ADM graft treated groups, the microscopic observation of HE staining is presented at week 8 (A, B, and C), week 16 (D, E and F) and week 24 (G, H, and I) after the treatment. At week 24 after the treatment, the results of type I collagen staining in groups of NS, MSCs and MSC-ADM treatment are shown in J, K, and L (magnification ×40). The results of type II collagen staining for groups of NS, MSCs and MSC-ADM treatment are presented in M, N, and O (magnification ×40). SO/FG staining results of groups of NS, MSC and MSC-ADM treatments are revealed in P, Q, and R (magnification ×40). The histological scores for the cartilage defect repair after the treatment (S). The Kruskal–Wallis test revealed that significant differences were observed between MSCs and MSC-ADM treatment at week 24 (n = 3, p b 0.01).

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results suggested that the increased cell size can be attributed to the production of extracellular matrix which can be quantitative indicators for the ability of MSCs to produce chondrogenesis in vitro. As distinctive markers of MSCs, CD73 and CD90 are considered whose positive expression represents enhanced chondrogenic capacity [33–35]. Our results as well as those from other studies clearly showed a trend of increased expression with extended culture time. Of these detected surface markers, CD29 was demonstrated to mediate cell– cell and cell-extracellular matrix interactions as adhesion molecules [36,37]. This study showed that the expression of CD29 in MSCs was dependent upon time of the cell culture which suggested that its higher expression might be critically important for MSCs to mediate adhesion of the transplanted MSCs to the cartilage lesion tissue of the recipients. In conclusion, our results suggest that selected chondrogenic clonal MSC-loaded ADM grafts could significantly improve the cartilage lesions in Cynomolgus monkey model. Due to genetic closeness of this model to humans, these results may be potentially applicable to repair cartilage defects in patients. Funding source Funding for this study was provided in part by the Canadian Institutes of Health Research (CIHR) grant #MOP-57848 (MZ), the Research Center of CHUM (CRCHUM), University of Montreal, Canada, and Fondation de la recherche en transplantation, Québec, Canada. Competing interests The authors declare that they have no competing interests. References [1] Yoshimura H, Muneta T, Nimura A, Yokoyama A, Koga H, Sekiya I. Comparison of rat mesenchymal stem cells derived from bone marrow, synovium, periosteum, adipose tissue, and muscle. Cell Tissue Res 2007;327:449–62. [2] Horwitz E, Le Blanc K, Dominici M, Mueller I, Slaper Cortenbach I, Marini FC, et al. Clarification of the nomenclature for MSC: the International Society for Cellular Therapy position statement. Cytotherapy 2005;7:393–5. [3] Murphy JM, Fink DJ, Hunziker EB, Barry FP. Stem cell therapy in a caprine model of osteoarthritis. Arthritis Rheum 2003;48:3464–74. [4] Tuan R. Stemming cartilage degeneration: adult mesenchymal stem cells as a cell source for articular cartilage tissue engineering. Arthritis Rheum 2006;54:3075–88. [5] De Bari C, Dell'Accio F, Vanlauwe J, Pitzalis C, Luyten FP. Distinct biological properties of human mesenchymal stem cells from different sources. Arthritis Res Ther 2005;7: 40–6. [6] Sakaguchi Y, Sekiya I, Yagishita K, Muneta T. Comparison of human stem cells derived from various mesenchymal tissues. Superiority of synovium as a cell source. Arthritis Rheum 2005;52:2521–9. [7] McGonagle D, De Bari C, Arnold P, Jones E. Lessons from musculoskeletal stem cell research: the key to successful regenerative medicine development. Arthritis Rheum 2007;56:714–21. [8] Chen FH, Tuan RS. Mesenchymal stem cells in arthritic diseases. Arthritis Res Ther 2008;10:223–7. [9] English A, Jones EA, Corscadden D, Henshaw K. A comparative assessment of cartilage and joint fat pad as a potential source of cells for autologous therapy development in knee osteoarthritis. Rheumatology 2007;46:1676–83. [10] Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284: 143–7. [11] Gimble JM, Katz AJ, Bunnell BA. Adipose-derived stem cells for regenerative medicine. Circ Res 2007;100:1249–60. [12] Bouffi C, Djouad F, Mathieu M, Noël D, Jorgensen C. Multipotent mesenchymal stromal cells and rheumatoid arthritis: risk or benefit? Rheumatology 2009;48: 1185–9. [13] Chen FH, Rousche KT, Tuan RS. Technology insight: adult stem cells in cartilage regeneration and tissue engineering. Nat Clin Pract Rheumatol 2006;2:373–82.

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