Department of Orthopaedic Surgery, Toyama Medical and Pharmaceutical University, Toyama, 1 Department of Orthopaedic Surgery, Shinsyu University, Nagano and 2 Department of Orthopaedic Surgery, Faculty of Medicine, Osaka University, Osaka, Japan.
Correspondence to: T. Kimura or (reprints) R. Katayama, Department of Orthopaedic Surgery, Toyama Medical and Pharmaceutical University, Toyama, Japan. E-mail (Kimura): tkimura{at}ms.toyama-mpu.ac.jp; (Katayama) riek{at}ms.toyama-mpu.ac.jp
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Abstract |
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Methods. BMMCs, which had a fibroblastic morphology and pluripotency for differentiation, were isolated from bone marrow of the tibia of rabbits, grown in monolayer culture, and transfected with the CDMP1 gene or a control gene (GFP) by the lipofection method. The autologous cells were then implanted into full-thickness articular cartilage defects in the knee joints of each rabbit.
Results. During in vivo repair of full-thickness articular cartilage defects, cartilage regeneration was enhanced by the implantation of CDMP1-transfected autologous BMMCs. The defects were filled by hyaline cartilage and the deeper zone showed remodelling to subchondral bone over time. The repair and reconstitution of zones of hyaline articular cartilage was superior to simple BMMC implantation. The histological score of the CDMP1-transfected BMMC group was significantly better than those of the control BMMC group and the empty control group.
Conclusion. Modulation of BMMCs by factors such as CDMP1 allows enhanced repair and remodelling compatible with hyaline articular cartilage.
KEY WORDS: Cartilage repair, Mesenchymal cell, Chondrogenic differentiation, CDMP1
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Introduction |
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We and others have investigated the use of mesenchymal cells derived from bone marrow as a biological method for the repair of articular cartilage defects [1417]. It is already established that bone marrow-derived mesenchymal cells (BMMCs) contain pluripotent cells that are capable of differentiating into various types of cells, including chondrocytes, osteoblasts and adipocytes [1824]. Since BMMCs are easily isolated from the bone marrow and can be rapidly amplified, they are likely to be the most suitable cell type for the repair [17]. However, there are still arguments about the efficiency of chondrogenic differentiation, reconstitution of hyaline articular cartilage zone, the integration of the regenerated and surrounding tissues, and the long-term integrity of the repaired tissues. Although, culture-expanded and implanted BMMCs form cartilaginous tissue in vivo, the regeneration is sometimes limited to certain portion of the defect and the repair does not always result in reconstitution of the sustainable zones of articular cartilage [14]. Clearly, there is a need to further develop methods for the reliable repair of damaged cartilage using BMMCs.
Cartilage-derived morphogenetic protein 1 (CDMP1) is a member of the transforming growth factor ß (TGF-ß) superfamily and has been shown to be involved in chondrogenesis [2529]. CDMP1 has been shown to promote aggregation of mesenchymal cells and enhance chondrocyte differentiation [30, 31]. These roles of CDMP1 during chondrogenesis from undifferentiated mesenchymal cells led us to hypothesize that the modulation of BMMCs with biologically active factor(s), such as CDMP1, could assist in the maintenance of cell viability and chondrogenic differentiation in vivo, and improve the repair of damaged cartilage. In the present study, we transfected autologous BMMCs with CDMP1, implanted them into full-thickness articular cartilage defects in rabbits.
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Materials and methods |
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CDMP1 gene transfer into BMMCs
CDMP1 cDNA insert from the p742CDMP1Int vector [31] was used under the control of CMV-IE promoter (Clontech, Palo Alto, CA, USA). A green fluorescent protein (GFP) expression vector, pEGFP-C1 (Clontech), was used as the control vector. The passage-3 BMMCs from each rabbit were transfected with the CDMP1 or the control GFP gene by the lipofection method using FuGENETM6 (Roche, Indianapolis, IN, USA). Approximately 1 x 106 cells in a 100-mm culture dish were washed twice with Hanks solution and covered with 6 ml of serum-free DMEM. Then the DNA-FuGENETM6 mixture (3 µg of the DNA mixed with 9 µl of FuGENETM6) was added to each dish, and the cells were incubated at 37°C for 6 h. Next, the medium was removed and replaced with a defined medium [22], consisting of DMEM with ITS+Premix; insulin 6.25 µg/ml, transferrin 6.25 µg/ml, selenous acid 6.25 µg/ml, linoleic acid 5.33 µg/ml, bovine serum albumin 1.25 mg/ml, pyruvate 1 mM, ascorbate 2-phosphate 0.17 mM, proline 0.35 mM, dexamethasone 0.1 µM, and recombinant human TGF-ß3 10 ng/ml (No. 531-82501; Wako, Osaka, Japan). To confirm cell viability after gene transfer, the MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide] assay was performed during culture as described previously [32].
Expression of CDMP1 and matrix genes in BMMCs
Total RNA from the transfected BMMCs after a 5-day culture was prepared using the modified acid guanidinephenolchloroform method [33]. Five micrograms of the RNA was converted to cDNA using the Super ScriptTM First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA, USA). Quantitative PCR was performed using an ABI prism 7000 (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's recommendations. The primers were as follows: CDMP1 forward primer, 5'-TCCAGACCCTGATGAACTCC-3', CDMP1 reverse primer, 5'-TCCACGACCATGTCCTCATA-3', CDMP1 TaqMan probe, 5'-CATTGACTCTGCCAACAACGTGGTGTATAA-3'; HPRT forward primer, 5'-GACCTTGCTTTCCTTGGTCA-3', HPRT reverse primer, 5'-TCCAACAAAGTCTGGCCTGT-3', HPRT TaqMan probe, 5'-CAGTATAATCCAAAGATGGTCAAGGTCGCA-3'. PCR was performed at 50° for 2 min, 95° for 10 min, and 50 cycles of 95° for 30 s and 60° for 1 min. Standardization was performed using RNA extracted from rabbit chondrocytes and quantitation was normalized to an endogenous control (HPRT). RT-PCR for matrix genes was performed with initial denaturation at 94° for 5 min, 30 cycles of 94° for 1 min, 57° for 1 min, 72° for 2 min, and final extension at 72° for 10 min. The primers were as follows: rabbit type II collagen (Col2a1) forward primer, 5'-CAACAACCAGATCGAGAGCA-3', reverse primer, 5'-CCAGTAGTCACCGCTCTTCC-3'; rabbit aggrecan forward primer, 5'-TCTCCAAGGACAAGGAGGTG-3', reverse primer, 5'-AGGCTCTGGATCTCCAAGGT-3'; rabbit type I collagen (Col1a2) forward primer, 5'-CAATCACGCCTCTCAGAACA-3', reverse primer, 5'-TCGGCAACAAGTTCAACATC-3'.
Implantation of CDMP1-transfected autologous BMMCs for in vivo cartilage repair into full-thickness articular cartilage defect
Three days after CDMP1 and GFP gene transfer, BMMCs were freed from the culture dishes with trypsin/EDTA. Then 1 x 106 autologous cells were embedded in 200 µl of type-I collagen gel (at a final concentration of 0.15%; Nitta Gelatin, Osaka, Japan) and implanted into a large full-thickness articular cartilage defect. The defect (4 mm in diameter and 4 mm in depth) was created through the articular cartilage and into the subchondral bone of the patellar groove in 46 rabbits using an electric drill equipped with a 4-mm diameter drill bit. In 30 rabbits, the defects were implanted with individual autologous BMMCs; the defect in the right knee was filled with CDMP1-transfected BMMCs and the defect in the left knee was filled with control GFP-transfected BMMCs. In the remaining 16 rabbits, defects made in the right knees were not filled, as an empty control. The incision was closed using 40 Vicryl and all rabbits were allowed to move freely after surgery.
Histological examination of repair tissue
The animals were killed 2, 4 or 8 weeks after the operation. The distal part of each femur was removed, fixed in 4% paraformaldehyde, decalcified in 10% EDTA and embedded in paraffin. Then sections were cut through the centre of each defect, stained with safranin O/Fast Green, examined in a blinded manner by two evaluators, and were graded with use of a histological scale (see supplementary data at Rheumatology Online), which was a modification of those described by Wakitani et al. [14] and Pineda et al. [34]. The scale is composed of two categories. The first category evaluates surface layers (hyaline articular cartilage zone) repair and contains three parameters: cell morphology and matrix staining graded from 0 to 8 points, surface regularity graded from 0 to 3, integration of donor with host adjacent cartilage graded from 0 to 2. The second category evaluates filling and remodelling of the defect of the deeper zone, and contains two parameters: filling of defect graded from 0 to 4, reconstitution of subchondral bone and osseous connection graded from 0 to 3. Differences of the histological scores between three groups were analysed with the KruskalWallis test, followed by the Scheffe method for multiple comparisons. Differences of the scores between two groups were analysed by the MannWhitney U test. A P value <0.05 was considered significant.
Immunohistochemistry
To investigate expression of the transgene in vitro, immunohistochemical staining for CDMP1 was performed using a goat polyclonal antibody specific for CDMP1 (N-17; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and standard procedures. Immunohistochemical analysis of the repaired tissue in vivo was also performed using antibodies specific for types I or II collagen (F-56, F57, Fuji Chemical, Takaoka, Japan). Immunoreactivity was detected using a biotinylated horse anti-mouse antibody and avidinbiotin reaction (Vectastain ABC kit; Vector Laboratories, Burlingame, CA, USA).
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Results |
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Repair of cartilage defects with autologous BMMCs
In the empty control group 2 weeks after the operation, the defect was incompletely filled and contained newly formed fibrous tissue as expected. On the other hand, the defects implanted with BMMCs were filled with repair tissue that contained hyaline cartilage-like elements. This hyaline repair was more obvious in the CDMP1-transfected BMMC group. Figure 2 shows the representative histological appearance of the defects at 4 weeks. In the empty control group, the defects were almost filled with fibrous tissue and cancellous bone at this stage. Although there was spotted safranin O staining in the deeper zone of the defects, cells in the surface zone of each defect were entirely non-chondrogenic (Fig. 2AC). In the control BMMC-implanted rabbits (Fig. 2DF), the defects were filled with repair tissue that contained hyaline cartilage. In most of the rabbits, the base of the defect was replaced by new bone. Although some knees showed repair by differentiated cartilage, safranin O staining tended to be more distinct in the deep zone of the regenerated tissue. The surface zone often showed a fibrous structure or had only moderate safranin O staining. Figure 2GI shows autologous CDMP1-transfected BMMC-implanted right knees of the same animals shown in Fig. 2DF respectively. In the CDMP1-transfected BMMC group, the defects were mostly filled with hyaline cartilage at 4 weeks. It was noteworthy that hyaline cartilage was formed up to the level of original articular surface and safranin O staining was intense throughout most of the regenerated articular surface zone.
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Eight weeks after the autologous CDMP1-transfected BMMC implantation, the appearance of the repaired cartilage was comparable to differentiated hyaline cartilage, and the subchondral tissue was completely replaced by new bone of a thickness close to that of the host subchondral bone (see supplementary data at Rheumatology Online).
Histological score of the repair tissue
In comparison with the empty control group, the scores of the control autologous BMMC implantation were better (i.e. lower) at 2, 4 and 8 weeks (Table 1). However, not all joints behaved uniformly and the scores tended to become worse at 8 weeks, which was compatible with our previous observation after BMMC implantation [14]. On the other hand, the scores of the CDMP1-transfected autologous BMMC implantations were significantly better than those for the empty control. The scores were maintained at 8 weeks and were significantly better than those for control BMMC implantation and the empty control. The comparison of two categories, surface zone repair (AC in Table 1) and deeper zone filling/remodelling (DE in Table 1), indicates that CDMP1-transfected autologous BMMC implantation results in significantly better repair, especially in the surface layer (hyaline cartilage zone).
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Discussion |
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Transplantation of cultured allogeneic or autologous chondrocytes into areas of cartilage damage has been shown to faithfully produce hyaline cartilage [1012]. However, there remain questions about the fate of the transplanted cells, limits on the number of available cells and poor integration of the newly formed cartilage plug with host cartilage, and doubts about the ability of dedifferentiated cells to form hyaline cartilage. To overcome these potential drawbacks of chondrocyte-based cell therapy, we attempted to employ BMMCs for cartilage repair [14, 17]. In these experiments, however, we also noticed that the repair of articular cartilage after BMMC implantation was not yet satisfactory. Although the regeneration of cartilage after BMMC implantation was impressive, the articular surface was not always repaired by a layer of hyaline cartilage in the case of larger defects. Such insufficient hyaline repair often fails to reconstitute well-remodelled cartilage surface zone and tends to become deteriorated with time [14]. The problem of insufficient hyaline repair by BMMCs can be explained in two ways. First, the number of BMMCs used to repair the cartilage defect may be too low relative to the defect size. This is partly supported by the fact that small defects show spontaneous repair by regenerating cartilage through the migration of relatively sufficient mesenchymal progenitor cells from the bone marrow [36, 37].
Secondly, not all of the BMMCs may differentiate into chondrocytes within the cartilage defect after implantation. For in vitro chondrogenesis from mesenchymal stem cells, TGF-ß and dexamethasone are reported to be essential [2022], and addition of other factors, such as bone morphogenetic proteins (BMCs), could improve differentiation. During in vivo repair after BMMC or mesenchymal stem cell implantation, these bioactive factors may be supplied at the site of the chondro-osseous defect from the host tissues and initiate cells into the chondrogenic lineage. However, the availability of such bioactive factor(s) may not be always sufficient to achieve chondrogenesis. In order to overcome these obstacles to BMMC-based repair, it seems likely that engineered BMMCs expressing soluble factor(s), such as BMP2, recently reported by Gelse et al. [38], should be useful. Use of cells that have already been engineered to enter chondrogenic lineage may also have therapeutic potential.
The CDMP1 (GDF5) gene, which we used in the present study, has been shown to be involved in commitment of mesenchymal cells to the chondrogenic lineage and acceleration of chondrocyte differentiation [2531]. Taking advantage of such an in vivo role of CDMP1 during chondrogenesis from mesenchymal cells, we used engineered CDMP1-transfected BMMCs for cartilage repair in the present study. Although the repair was not perfect, implantation of CDMP1-transfected BMMCs resulted in better surface zone repair as well as deeper zone remodelling. There is no doubt that reconstitution of hyaline articular cartilage zone and its superficial layers is a prerequisite for the prolonged integrity of the repaired tissue. Why, then, did the CDMP1 transfection result in better surface zone repair with hyaline cartilage? It is possible that CDMP1-transfection helped to maintain cell growth activity, as indicated in the in vitro study (see supplementary data at Rheumatology Online). Knowledge from previous studies [30, 31] and the present in vitro study also suggests that CDMP1 helped the implanted BMMCs to enter chondrogenic lineage in the defect.
If cells with differentiated chondrogenic phenotype are desired for transplantation, use of further differentiated BMMCs or chondrocytes could be suitable. Such cells should enable immediate synthesis and formation of hyaline cartilage matrix in the defect. In our experience, transplantation of already differentiated cells or chondrocytes forms a good cartilage plug in the defect, but often fails to show the necessary remodelling and integration in the surface zone and is unable to reconstitute a good subchondral structure [10]. We speculate that use of BMMCs committed to the chondrogenic lineage, rather than already well-differentiated chondrocytes, should promote better remodelling and integration of the regenerated cartilage.
The use of engineered autologous BMMCs in future in vivo studies may enable us to regenerate extensive defects of articular tissues. However, therapeutic application in humans may pose several problems. The use of transient transfection by lipofection, as in the present study, should help to avoid possible toxicity, the provocation of an inflammatory response and technical complexity, although transfection efficiency is relatively low. Transfection of cells to express bioactive proteins, as well as other factors that are important for differentiation, cell viability or matrix synthesis, may eventually provide the basis for effective BMMC-based repair of damaged articular cartilage.
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Acknowledgments |
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The authors have declared no conflicts of interest.
Supplementary data
Supplementary data are available
at Rheumatology Online.
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References |
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