Tissue Engineering and Regenerative Medicine, Vol. 9, No. 1, pp 43-50 (2012) DOI : 10.1007/s13770-012-0043-3
｜Original Article｜
Cartilage Tissue Engineering using Chondrocyte-Derived Extracellular Matrix Scaffold Suppressed Vessel Invasion During Chondrogenesis of Mesenchymal Stem Cells In Vivo Kyoung-Hwan Choi1,2, Bo Ram Song1, Byung Hyun Choi3, Minhyoung Lee4, So Ra Park5, and Byoung-Hyun Min1,2,6* 1
Department of Molecular science and Technology, Ajou University, Suwon, Korea 2 Cell Therapy Center, Ajou Medical Center, Suwon, Korea 3 Division of Biomedical and Bioengineering Sciences, College of Medicine, Inha University, Incheon, Korea 4 Department of Biochemistry, McGill University, Montreal, Canada 5 Departments of Physiology, College of Medicine Inha University, Incheon, Korea 6 Departments of Orthopedic Surgery, School of Medicine, Ajou University, Suwon, Korea (Received: November 11th, 2011; Revision: November 17th, 2011; Accepted: December 7th, 2011)
Abstract : Loss of chondrogenic phenotypes of tissue engineered cartilage using mesenchymal stem cells (MSCs) in vivo are thought to be influenced by environmental factors like vessel invasion in particular. This study investigated effect of a chondrocyte-derived extracellular matrix (CD-ECM) scaffold on the hypertrophic changes and vessel invasion into tissue engineered cartilage using rabbit MSCs in comparison with a synthetic polyglycolic acid (PGA) scaffold. Rabbit MSCs in CD-ECM or PGA scaffold were differentiated for 1 week in vitro and implanted in the back of nude mice for 6 weeks in vivo. Gross observation showed red stains, indicative of vessel invasion, increased along with the loss of chondrogenic phenotype in safranin-O stains, which was more prominent in the PGA constructs. The area showing loss of chondrogenic phenotypes in safranin-O stain was correlated well with the mineralized area in the von kossa stain and the area with vessel-like structures in the gomori aldehyde fuchsin stain at 6 weeks in terms of their size and distribution. Also, vessel invasion took place more deeply and intensively into the constructs, in accordance with the expression of angiogenic markers (CD31, VEGF-A and HIF-1α) and a macrophage marker (CD68). This phenomenon progressed much more rapidly in the PGA constructs than in the CDECM constructs, and correlated well with the loss of chondrogenic phenotypes. In conclusion, this study showed that tissue engineered cartilage using the CD-ECM scaffold maintained better chondrogenic phenotypes in vivo and showed lower levels of hypertrophic changes and vessel invasion. Key words: cartilage tissue engineering, mesenchymal stem cells (MSCs), hypertrophic change, vessel invasion, extracellular matrix (ECM) scaffolds
culture of MSCs in vivo display signs similar to those of chondrocyte hypertrophy, including the expression of type x collagen and matrix mineralization.4,5 It is not clear, but the hypertrophic changes of MSCs in vivo appears to involve infiltration of blood vessels into the implanted constructs. The vessel formation is normally a critical event in the early stage of reparative processes and tissue regeneration. However, persistence of a vascular network can interfere with later transformation or maturation in natural, avascular tissue such as articular cartilage.6,7 Other studies also demonstrated that bone formation initiates at the periphery of the hypertophic cartilage core which is invaded by blood vessels.8-10 For this reason, the influence of the contact blood vessels on the chondrogenic phenotype of implanted MSCs constructs represents an
1. Introduction Mesenchymal stem cells (MSCs) are widely used in cartilage tissue engineering because of their pluripotency and selfrenewing capability. However, they still have a critical limitation that they do not fully differentiate into chondrocytes and eventually suffer from degenerative changes involving loss of chondrogenic phenotypes and matrix mineralization.1-3 This phenomenon is more marked when MSCs are implanted and differentiated in in vivo environment such as the subcutaneous injection in mice.3,4 Some studies also showed that long term *Tel: +82-31-219-5225; Fax: +82-31-219-5229 e-mail:
[email protected] (Byoung-Hyun Min)
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and vessel invasion. The MSCs-derived cartilage constructs using the CD-ECM and PGA scaffolds were implanted in the back of nude mice, and the extent of hypertrophic changes and vessel invasion-related events were examined.
important issue to understand the cause of the phenomenon and find the way of inhibiting it. The hypertrophic changes of MSCs-derived cartilage constructs in vivo may depend on the differentiation state of the constructs before implantation. Some studies showed that more differentiated MSCs in vitro maintain better the chondrogenic phenotypes and form more stable cartilage-like tissue in vivo.1113 These findings suggest that the stability of chondrogenic phenotype of a MSCs construct in vivo is controlled by the balance between environmental cues and the inherent phenotypic stability of the construct. Importantly, production of highly differentiated constructs from MSCs in vitro could be an important prerequisite to guarantee stable cartilage formation preventing hypertrophic changes and the undesired tissue formation in vivo. Various approaches have been investigated for chondrogenic differentiation of MSCs, such as using mechanical stimulations, soluble factors, different cell sources and scaffolds.2,14-16 For example, Cui et al. used low intensity ultrasound (LIUS), a mechanical stimulation and showed that the chondrogenic phenotypes of MSCs construct were better maintained in the back of nude mice by pre-treatment of LIUS before implantation.2 Xiao Yang et al. used TGF-β1, a soluble factor, and showed it inhibited terminal hypertrophic differentiation of chondrocyte and helped maintaining articular cartilage.17 Likewise, three dimensional (3-D) scaffolds can be also used as a method to maintain the chondrogenic phenotype of a differentiated construct from MSCs. Scaffolds play a critical role in chondrocyte function and chondrogenic differentiation of MSCs during cartilage regeneration.14,17,18 Some studies also deal with a question of whether vessel invasion in situ is dependent on chemical or physical cues particularly on scaffold type.18,19 These results have demonstrated difference of blood vessel formation in other ECM, including type I collagen, fibrin and puramatrix. Therefore, the choice of scaffold materials and types could be important in the chondrogenic differentiation of MSCs and maintaining chondrogenic phenotypes of differentiated constructs in vivo as well. We have previously shown that an chondrocytes-derived extracellular matrix (CD-ECM) scaffold provided a good environment for cartilage tissue formation using chondrocytes.20, 21 The CD-ECM scaffold also enhanced chondrogenic differentiation of rabbit MSCs better than the polyglycolic acid (PGA) scaffold in vitro and helped the construct maintain chondrogenic phenotypes for longer time in vivo.22 In this study, we investigated if the beneficial effect of the CD-ECM scaffold on maintaining chondrogenic phenotypes in vivo is correlated with the inhibition of hypertrophic changes
2. Materials and Methods 2.1 Cell Isolation and Culture Rabbit bone marrow-derived mesenchymal stem cells (rMSCs) were obtained from 2-week-old female New Zealand white rabbits (n=1; Joong Ang Experimental Animal Center, Seoul, Korea). The bone marrow aspirates were obtained aseptically from the tibia and femur, and mononuclear cells (MNCs) were obtained by ficoll-gradient centrifugation at 1,500 g for 5 min. The MNCs were resuspended in Minimum essential medium eagle-alpha modification (α-MEM; Sigma, MO, USA) supplemented with 1% antibiotics-antimycotic (Gibco, CA, USA) and 10% fetal bovine serum (FBS; Hyclone, MA, USA). MNCs were seeded at 1.5×10 7 cells/plate (150 mm) and cultured at 37oC in a 5% CO2 incubator. After 2 days, non-adherent cells were removed and adherent cells were further cultured. At day 14 after cell seeding, the adherent MSCs were retrieved by trypsin treatment and replated at 1×106 cells/plate (150 mm) for expansion. Rabbit chondrocytes were freshly isolated from rabbit articular cartilage of 2-week-old female New Zealand white rabbits (n=1). For this, cartilage pieces were digested with 1 mg/ml collagenase (Worthington, NJ, USA) in Dulbecco’s modified Eagle’s medium (DMEM). The obtained cell suspension was passed through a nylon mesh cell strainer (BD, MA, USA) and centrifuged at 1700 g for 10 min. The cell pellet was resuspended in DMEM with 10% FBS and 1% antibiotics-antimycotic. The chondrocyte were seeded at 1.5×106 cells/plate (150 mm) and cultured at 37oC in a 5% CO2 incubator. The culture medium was changed every three days. 2.2 Preparation of CD-ECM and PGA Scaffolds The CD-ECM scaffold was prepared from porcine chondrocytes by our proprietary technique as previously described.20,21 In brief, primary chondrocytes from porcine knee joints were first expanded in monolayer culture for 3 weeks and further cultured in a 3 dimensional (3-D) pellet for another 3 weeks. The 3-D cartilage-like tissue was freeze-dried for 48 h at -56oC under 5 m Torr to remove cellular components and produce a porous, sponge type scaffold. The scaffold produced was treated with 200 U/ml DNase I and washed thoroughly 3 times with phosphate-buffered saline (PBS). Then, the scaffold was dried and trimmed off of its surface area using a biopsy punch (5 mm in diameter). We used the PGA scaffold in a sheet
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Cartilage Tissue Engineering using Chondrocyte-derived Extracellular Matrix Scaffold Suppressed
4 µm in thickness were prepared and stained with safranin-O/ fast green (accumulation of sulfated proteoglycans), von kossa (calcified tissues), and Gomori aldehyde fuchsin (elastin fiber). Sections from all 5 samples were obtained and analyzed for each staining. The ratio analysis of stain area was carried out using an image J program.23
form purchased from Albany International Inc. (Mansfield, MA, USA). The PGA scaffold was also cut by the biopsy punch to make its dimension same as that of the CD-ECM scaffold. The dimensions of the CD-ECM and PGA scaffolds were 5 mm diameter and 2 mm in thickness. Both the CD-ECM and PGA scaffolds were submerged in 99% alcohol for 10 h for sterilization and kept at 4oC before use. After washing 3 times with PBS containing antibiotics, the scaffolds were preincubated in chondrogenic defined medium for 12 h at 37oC.
2.6 Immunohistochemical Analysis The sections of samples prepared as described above were rinsed with PBS and treated sequentially with 3% H2O2 for 10 min, 0.15% Triton X-100 for 10 min, and 1% BSA in PBS for 10 min. The sections were then incubated with primary antibodies (1:200) for 1 h at room temperature. The primary antibodies used were mouse anti-rabbit antibodies for type X collagen, CD31, CD68, HIF-1α, and VEGF (all from Abcam, Cambridge, UK) after washing 3 times in PBS, the sections were incubated with a biotinylated secondary antibody against mouse IgG (1:200) for 1 h and peroxidase-conjugated streptavidin solution for 30 min at room temperature (both from Dakocytomation, CA, USA). The sections were finally counterstained with Mayer’s hematoxylin (Sigma, MO, USA) and then mounted for microscopic observation (Nikon E600, Japan). Sections from all 5 samples/ time point/group were obtained and analyzed for each staining. The ratio analysis of stain area is using an image J program.23
2.3 Induction of Chondrogenic Differentiation of Rabbit MSCs and Chondrocytes In Vitro The MSCs and chondrocytes at passage 1 were suspended in a chondrogenic defined medium (DMEM supplemented with ITS m ixture, 50 µg/ml ascorbate 2-phosphate, 100 nM dexamethasone, 40 mg/ml proline, and 1.25 mg/ml BSA) without TGF-β, a typical chondrogenic inducer. For cell seeding, 5×106 cells in 1 ml medium were loaded dynamically into an ECM or PGA scaffold for 90 min. The cell-seeded scaffolds were placed in 24-well plates and incubated until 1 week at 37oC under 5% CO2 to induce chondrogenic differentiation. 2.4 Chondrogenic Differentiation of Rabbit MSCs and Chondrocytes In Vivo The constructs containing rabbit MSCs and chondrocytes in the CD-ECM or PGA scaffold (n=80/scaffold) were prepared and subjected to chondrogenic differentiation for 1 week in vitro as described above. Six-week old male nude mice (n=20 for each scaffold group) were anthesized with a mixture of ketamine hydrochloride and rumpun. The back skin of nude mice was incised and 4 differentiated constructs were implanted in the subcutaneous tissue of each mouse, where 2 constructs were implanted longitudinally in each side of the spine. Total of 80 constructs were implanted in 20 mice for each scaffold group. Five mice were sacrificed for a scaffold group at each time point of 1, 2, 4, and 6 weeks postimplantation (20 samples/time point/group). Among the samples retrieved, 5 samples each were used for gross image, histological analysis, respectively. The experimental protocol was approved by the Animal Care and Use Committee of Ajou University.
2.7 Statistical Analysis Data from the stain ratio was analyzed for statistical significance between two comparative samples by one-tailed p value using GraphPad software. The experiments were repeated at least 3 times (n=3). The statistical significance was assigned by *** p
