Meniscal Cells Mediated Bone Marrow Mesenchymal Stem Cells Differentiation

Xin Duan¹, Xingxing Dong², Qi Li¹,Xiang Zhou¹ Jian Li^1#

1. Department of Department of Orthopedics, West China Hospital of Sichuan University, Chengdu, China

2. Department of Pathology, No.9 Peoples Hospital, Chengdu, China

Address for Correspondence:
Li Jian
No 37 Guo Xue Xiang,
Department of Orthopaedics,
West China Hospital, Chengdu,
China.

Abstract:

Objective: Seed cell was the most important factor restricting the clinical application of meniscal tissue engineering. Bone mesenchymal stem cells（MSCs）which has its own advantages become the potential cell source of tissue engineering. The arms of the study is to investigate the induction effect of MSCs by meniscal cells under co-colutured without contact.

Methods: MSCs and meniscal cells were isolated from bone marrow and meniscus MSCs and meniscal cells were cultured in the either side of membrane of Millopore’s hanging cell culture insert speratedly and lasted for 7 days without different kinds of cells contact.(non-contact co-culture and cells ratio of the co-culture is 1:1). Immunohistochemistry and RQ-PCR was to applied.

Result:  Immunohistochemistry of type Ⅰ collagen of induced MSCs was positive and that of Ⅲ collagen was negative. mRNA expression of aggrecan type I and III collagen were increased and that of type II collagen was not increased in MSCs after induced by meniscal cell

Conclusion: MSCs which induced by meniscal cells with non-contact co-culture method have differentiation potential to meniscal cells. MSCs which induced by meniscal cells with non-contact co-culture method may be the seed cells of tissue engineering of the meniscus.

J.Orthopaedics 2010;7(4)e9

Keywords:

tissue engineering; meniscus; seed cell; bone marrow mesenchymal stem cells

Introduction:

Background

Meniscal function is essential to the normal function of the knee joint, but meniscal injuries are very common, especially in athletes. ⁽¹⁾ Due to the lack of vasculature in the fibrocartilage, such as the inner 1/3 portion of meniscus, injuries to them usually result in formation of fibrous tissue which greatly alters joint function and predisposes the joint to degenerative changes. Surgical interventions like partial and total meniscectomy, allogous meniscal transplantation, meniscal repairing and so on, from some studies, have their relative limitations, such as osteoarthritis, immunogenicity, disease transmission, limited availability, limited indications.^(2-8)  Tissue engineering may offer new treatment modalities for the regeneration of meniscus lesions or for the complete replacement of a degenerated or injured (part of the) meniscus by a tissue-engineered meniscus. Tissue engineering is based on a smart and unique combination of cells, growth factors and scaffolds. The seed cell is a very important part of tissue-engineered meniscus. The meniscal cells from the patients which are excellent cell source for a tissue-engineered meniscus seem to be less than ideal for expansion or production sufficient matrix.^（8,9）Mesenchymal stem cells (MSCs) are pluripotent cells present in many adult mesenchymal tissues, such as synovium, muscle, adipose tissue and bone marrow and usually isolated from bone marrow.⁽¹⁰⁾ Bone marrow mesenchymal stem cells(MSCs) will maintain cell morphology and potent of multi-differentiation when culture in vitro.⁽¹¹⁾  In exchange for a more painful harvesting technique, human bone marrow mesenchymal stem cells (MSCs) may offer significant advantages in terms of great proliferative capacity and potential for differentiation toward fibrocartilaginous lineages. ^{(8, 9)} Hence, in this study, the induction effect of MSCs by meniscal cells was investigated under co-colutured without contact.

Materials and Methods:

The research protocol of this experiment was reviewed and approved by the ethical committee of Sichuan University.

1. Meniscal cells source and culture

The New Zealand white rabbit were purchased from West China experimental animal station of Sichuan University. For 4-week-old rabbits were anesthetized by an intraperitoneal injection of sodium pentobarbital. Menisci were taken from the knee joint and the meniscal cells were isolated and harvested, according to previous studies.  ^(12,13)  After removing soft tissue and slicing meniscus(1mm³), the isolation starts with a short digestion in 0.05% hyaluronidase for 5 min and a subsequent digestion in 0.2% trypsin for 30 min, followed by the regularovernight incubation in 0.2% collagenase type I . The digested tissue/cell suspension was passed through 200μm cell strainer to remove tissue debris and cells centrifuged. Cells were cultured in 1800rpm for 5 min. The cells were suspended in culture media consisting of Dulbecco’s modified Eagle medium and Ham's F12 (DMEM/F12 1:1) with 10% fetal bovine serum (Hyclone, USA), 100U/ml penicillin and 100μg/ml streptomycin. The cells were put in a humid atmosphere containing 5% CO2, with medium changed first 6 days and every 3 days thereafter.

2. Bone marrow mesenchymal stem cells source and culture

2ml of MSCs was aspirated from each tibia of rabbits mentioned above with a 5ml syringe containing 0.2 ml heparin(1000 Unit per ml). Mononucleated cells were isolated using a Histopaque-1090 (Haoyang,China) density gradient method. These cells were cultured in a 75 cm2 flask with Dulbecco’s modified Eagle medium- low glucose (L-DMEM)( Gibco, USA )supplemented with 10% fetal bovine serum (Hyclone US), 100U/ml penicillin and 100μg/ml streptomycin. The cells were put in a humid atmosphere containing 5% CO2, with medium changed first 5 days and every 3 days thereafter.

3. Co-culture of meniscal cells and bMSC

After 10–14 days of primary culture, when the proliferating colonies had nearly reached confluence, the adherent cells were harvested with 0.25% trypsin-ethylenediaminetetraacetic acid. The cells were prepared to co-culture without contact. All co-cultures were conducted in 6-well plates and using 1.0 um PET transparent hanging cell culture inserts (Millipore,USA). MSCs were seeded on 6-wells plates, while meniscal cells were seeded on the upper surface of the membrane of the cell culture inserts. 1x10⁵ cells were seeded into each well or membrane of the tissue insert. Co-cultured cells were maintained for 7 days in DMEM/F12 with 10% fetal bovine serum at 37°C and 5% CO2 in a humidified atmosphere with medium being exchanged every 3 days. As controlling, MSCs and meniscal cells were seed into 6-well plates separately 1x10⁵ cells per well in 6-wells plates.

4. Immunohistochemistry of type I Collagen and type III expression

Expression of type I collagen and type III collagen was assessed by immunohistochemistry(IHC), using monoclonal anti- collagen type I(Sigma, USA) and collagen III( Calbiochem USA). All incubations were performed in a humified chamber. All adherent cells were fixed by acetone.the samples of cells were incubated overnight at 4°C with anti- collagen type I and collagen III at a dilution of 1:200. The Collagen I reaction was visualized by using EnVision+ Dual Link DAB (zhongshanjinqiao,Beijing, China) according to manufacturers’ list with hematoxylin counterstaining.

5. Real-time PCR analysis of gene expression

Cells were rinsed in PBS and lysed in TRIzol (Invitrogen). Total RNA was then extracted following manufacturers constructions. Briefly, chloroform was added to each sample and sample tubes centrifuged to enable phase separation. RNA was precipitated by addition of isopropanol to the aqueous phase, followed by centrifugation. Precipitated RNA pellets were washed in 75% ethanol and then resuspended in distilled RNAse-free water. cDNA was prepared from RNA using Revert Aid™ Frist Strand cDNA Synthesis Kit (MBI). RNA (1<μg) was mixed with random prime hexamers (200ng) then incubated at 70°C for 5 minutes. Tubes were cooled on ice, then 5x first strand buffer, 10mM dNTP mix 2ul, 20U Ribonuclease Inhibitor (recombinant) and 200U RevertAid™ M-MuLV Reverse Transcriptase were added, giving a final volume of 20μl. Samples were then incubated at 20°C for 10 min, 42°C for 60 minutes and finally heated to 70°C for 10 minutes.

Gene expression was analyzed by real-time PCR using an FTC2000 (Funglyn Canada). Housekeeping genes was GAPDH. Primers for collagen type I, II, III and aggrecan were designed using the Primer Premier 5.0 software(Table 1). Reactions were carried out in duplicate in 96-well plates in a final volume of 30μl. For GAPDH , the reaction mix contained 10×buffer(Mg²⁺ free), 3 µl MgCl₂(25mM), 0.36 µl dNTP(25mM), 1 µl, upstream primer (10 µM), 1 µl downstream primer(10 µM), primer(10 µM),3U Taq, ,ddH₂O 18.74 µl, 5 µl cDNA. The PCR reaction consisted of an initial enzyme activation step at 94°C for 2 minutes, followed by 40 cycles of 94°C for 20 seconds, 55°C for 30 seconds and 60°C for 40 seconds. A cycle threshold value (Ct) value was obtained for each sample and duplicate sample values were averaged. The 2-ΔΔCt method was then used to calculate relative expression of each target gene.

Table 1. Real-time PCR primer details. Accession number given for primers designed using Primer Premier 5.0 software.

Fig 1.type I collagen protein was found in MSCs cells which had co-cultured with meniscal cells without contact(A 400X) and meniscal cell(B 400X) and type I collagen protein wasn’t found in controlled MSCs.(C 400X)

Fig 2: collagen type III protein was not found in MSCs cocultured meniscal cell(A 200X), meniscal cell(B 200X) and controlled MCSs(C 100X)

Fig 3 expression of aggrecan mRNA by controlled MSCs , MSCs following co-culture without contact and meniscal cell following 7 days in culture.

. * = statistical significance (P<0.05) from MSCs following co-culture without contact

Fig 4 expression of type I collagen mRNA by controlled MSCs , MSCs following co-culture without contact and meniscal cell following 7 days in culture.

. * = statistical significance (P<0.05) from MSCs following co-culture without contact

Fig 5 expression of type II collagen mRNA by controlled MSCs , MSCs following co-culture without contact and meniscal cell following 7 days in culture.

. * = statistical significance (P<0.05) from MSCs following co-culture without contact

Statistical analysis

Statistical significance was determined using Pair Wise Fixed Reallocation Randomization Test with calculation SPSS 13.00 software, where P <0.05 was considered significant.

Results :

Immunohistochemistry

Collagen type I protein was found in MSCs cells which had co-cultured with meniscal cells without contact (fig 1 A). While in controlled samples, the expression of collagen type I protein was found in meniscal cells (Fig 1B).In the other hand, the expression of collagen type I protein wasn’t found in MSCs solo cultured as control.(Fig 1C).

The collagen type III protein wasn’t found in MSCs cells which had co-cultured with meniscal cells without contact(fig 2 A), meniscal cells(fig 2 B) and MSCs solo cultured as control(fig 2 C) .

Gene expression following meniscal cells and MSC co-culture without contact

The mRNA expression of aggrecan, type I collagen, type II collage and type III collagen were assessed by real-time quantitative RT-PCR (Fig. 3). Group I was the MSCs solo cultured, Group II was the MSCs co-cultured with meniscal without contact, Group III was meniscal cells as control.

The resulting data were expressed as a ratio of group I. As shown in (Fig. 3), the expression of Aggrecan mRNA of Group II was significantly (P<0.05) increased than Group I and also significantly(P< 0.05) lower express than that in Group III after 7 days culture. In addition, the ratios of Aggrecan for group II and group III were 4.15 ± 3.19 and 54.14 ± 19.8, respectively. The expression of type I collagen in three group was show in (Fig. 4). the type I collagen mRNA of group II was significantly (P< 0.05)increased than Group I，and then slightly lower than that of the Group III(P>0.05) after 7 days culture. The ratios of type I collagen of group one for group II and group III were 2.91 ± 0.80 and 1.78 ± 0.69. In (Fig.5), the type II collagen mRNA of group II was higher but not significantly (P>0.05) and significantly lower than that of Group III (P < 0.05) after 7 days culture. The ratios of type II collagen for group II and group III were 0.99 ± 0.70 and 10.27 ± 5.44.In addition, the type III collagen mRNA of group II was increased than that of group I but not significantly (P<0.05) and then as the same as the group III(P<0.05) after 7 days culture. The ratios of type III collagen for group II and group III were 1.72 ±0.84 and 1.52 ± 0.25. (Fig.6)

Discussion :

Tissue engineering or cell-based therapies for repair of the require a large number of cells (e.g. 25x10⁶ -37 x10⁶ cells/ml ^(14,15,16)]) with an fibrocarilage-like phenotype that can be implanted into the meniscal scaffold to produce a new matrix. It can be expected that only cells from the torn inner parts of meniscus will be available. The number of these cells will be small and the quality may be compromised by the trauma. So far no culture experiments have been described using this cell source and it can be doubted if these cells will proliferate and differentiate into the desired phenotype. While autologous MSCs would be the ideal choice, studies have shown that MSCs lack HLA class II receptors ⁽¹⁷⁾ and that human recipients receiving MSCs from sibling donors do not exhibit an immunogenic response ⁽¹⁸⁾suggesting that allogeneic MSCs could be used. However, only using MSCs has some limitations when combine with some scaffolds. Port et al. reported on meniscal repair supplemented with exogenous fibrin clot and BM-MSCs in a goat model, but the addition of BM-MSCs in conjunction with the fibrin clot did not enhance meniscal healing. ⁽¹⁹⁾ MSCs with hyaluronan-ester and gelatin scaffold have been used to treat fibrocartilage defects in a rabbit model, but the ‘meniscus’ didn’t completely restore the surface area and the tissue quality of normal meniscus. ⁽²⁰⁾ The main problem for these strategies is the generation of a usable cell population.

Until to now, there are no methodology that can be employed to induce and maintain a differentiated phenotype in MSCs, involving addition of growth factors or culture in a three-dimensional environment. The current study aimed to elucidate the effects co-culture had on the differentiation state of both meniscal cells and MSCs.

Our study suggested that co-culture without contact show some significantly change in matrix gene expression and protein secretion. This was similar with other studies using different cell types co-cultured with MSCs where no cell-cell contact has been shown to have some effect. But some studies demonstrate the different result that there was no effect when using co-culture without contact. ⁽²¹⁾ Our results may therefore be due to the specific cell type i.e. the meniscal cell, use for co-culture.

Our study shows that after co-culture has more collagen I protein mRNA expression and have secretion of collagen I protein comparing to controlled MSCs. This is important as collagen I protein is a mainly component of meniscal tissue ⁽²²⁾ and has been shown to be expressed by meniscal cells in our study. Moreover, collagen I protein mRNA expression of MSCs was only slightly lower than that of controlled meniscal cell. The co-culture have been shown to stimulate aggrecan expression in MSCs and this could count for fibro-cartilaginous lineage differentiation. There was, however, no increase in type II collagen in MSCs following co-culture with meniscal cell and type II collagen was still less than that of controlled meniscal cell. the same result was reported in previous study ⁽²²⁾. While type III collagen was only minor ^{(22 -24 )}, yet still significant changes in expression, results suggest that type III collagen was expressed at same levels by both meniscal cells and MSCs following co-culture. Base our study, the MSCs has some potential to differentiate to fibro-cartilaginous lineage because of increasing expression of type I collagen, aggrecan and type III collagen.

This study had several limitations. First, only the co-culture method without contact was applied in our study. Second, there was only one cell ratio (1:1) when apply cell culture. In further research, cell-to-cell culture method and more cell ratio cultures should be used.

In conclusion we have shown that co-culture of meniscal cells and MSCs causes MSCs differentiation to a fibrocartilage-like phenotype following co-culture without contact. Whereas the cell was not the same as the fibrochondrocyte, the MSCs have the potential to differentiate to fibrocartilage phenotype. However, the current strategy could be an alternative approach for supplying the seed cell of tissue engineering of meniscus.

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This is a peer reviewed paper 

Please cite as: Xin Duan: Meniscal Cells Mediated Bone Marrow Mesenchymal Stem Cells Differentiation.

J.Orthopaedics 2010;7(4)e9

URL: http://www.jortho.org/2010/7/4/e9

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