Repair of articular cartilage defects in rabbits using CDMP1 gene-transfected autologous mesenchymal cells derived from bone marrow

R. Katayama, S. Wakitani1, N. Tsumaki2, Y. Morita, I. Matsushita, R. Gejo and T. Kimura

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objective. Cartilage-derived morphogenetic protein 1 (CDMP1), which is a member of the transforming growth factor-ß superfamily, is an essential molecule for the aggregation of mesenchymal cells and acceleration of chondrocyte differentiation. In this study, we investigated whether CDMP1-transfected autologous bone marrow-derived mesenchymal cells (BMMCs) enhance in vivo cartilage repair in a rabbit model.

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


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Articular cartilage is a highly differentiated avascular tissue with abundant extracellular matrix. Once damaged by various causes, such as trauma, osteoarthritis, articular cartilage often shows progressive deterioration without healing [1–3]. A number of methods have been developed to treat such damaged articular cartilage. These attempts can be categorized principally into restoration, replacement, relief, and resection of cartilage [4, 5; for review see 6]. Among them, biological resurfacing of cartilage is one of the methods that could restore joint function. In addition to tissue-based methods, such as osteochondral grafts [7, 8], development of cell therapy has aroused considerable interest. Human and experimental studies on the transplantation of cultured cells into areas of damage have shown promise in the repair of cartilage defects [9–13].

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 [14–17]. 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 [18–24]. 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 [25–29]. 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Isolation and expansion of autologous BMMCs
Forty-six mature New Zealand White rabbits weighing 3.5 to ~4 kg were used. The rabbits were anaesthetized by intramuscular injection of ketamine hydrochloride (60–70 mg/kg) and xylazine (6 mg/kg). The BMMCs were obtained from the tibia as described previously [14]. Briefly, the aspirate from the tibia was washed, centrifuged and resuspended in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal calf serum (FCS) and antibiotics (100 U/ml of penicillin G and 100 µg/ml of streptomycin). Then the cells from the bone marrow were cultured in 100-mm plastic dishes containing the same medium at 37°C under 5% CO2/95% air. One day after seeding, each culture dish was washed three times by mild agitation with new medium to remove non-adherent cells. When the adherent cells reached subconfluence, they were freed from the dish with 0.05% trypsin/0.02% EDTA and subcultured (passage 2). The cells were further subcultured at subconfluency (passage 3).

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 guanidine–phenol–chloroform 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 4–0 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 Kruskal–Wallis test, followed by the Scheffe method for multiple comparisons. Differences of the scores between two groups were analysed by the Mann–Whitney 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 avidin–biotin reaction (Vectastain ABC kit; Vector Laboratories, Burlingame, CA, USA).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Rabbit BMMCs and CDMP1 gene transfer
Quantitative PCR analysis indicated that CDMP1-transfected BMMCs started expression of CDMP1 by day 5 (Fig. 1A). By immunostaining, approximately 20% of the cells reproducibly expressed the transgene (Fig. 1B) and the expression was maintained for at least 3 weeks in monolayer culture (not shown). CDMP1-transfected BMMCs showed enhanced expression of aggrecan and Col2a1 with decreased expression of Col1a2 during culture (Fig. 1C).



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FIG. 1. Expression of CDMP1 and matrix genes. (A) CDMP1 expression in BMMCs was detected by real-time PCR analysis. There was a marked increase in the mRNA level for CDMP1 during CDMP1-transfected BMMC culture for 5 days. Results are the mean of three independent experiments. The value of each CDMP1 mRNA was normalized to the amount of HPRT mRNA. The standardized value of CDMP1-transfected BMMCs was arbitrarily set to 100. White bar indicates control BMMCs; black bar indicates CDMP1-transfected BMMCs. (B) Immunohistochemical staining for CDMP1. Arrows indicate CDMP1 expressing BMMCs in culture after transfection (upper panel). There were no CDMP1-positive cells in control BMMCs (lower panel). Scale bar is 100 µm. (C) RT-PCR analysis of matrix genes. The expression of aggrecan and Col2a1 was more prominent in the CDMP1-tranfected BMMCs after 5 days in culture. a, control BMMCs. b, CDMP1-transfected BMMCs. HPRT was used as internal control.

 
To analyse the possible adverse effect of CDMP1 gene transfer on BMMCs, the MTT assay was performed (see supplementary data at Rheumatology Online). There was an initial decrease in cell growth activity during culture in the defined medium. This was in accordance with the report of Sekiya et al. [35] which indicated loss of a portion of marrow stromal cell population during culture in defined medium, apparently through apoptosis. However, by comparing with control GFP gene transfer, CDMP1 alleviated the initial decline of cell growth and helped to maintain a higher level of activity thereafter.

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. 2A–C). In the control BMMC-implanted rabbits (Fig. 2D–F), 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 2G–I shows autologous CDMP1-transfected BMMC-implanted right knees of the same animals shown in Fig. 2D–F 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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FIG. 2. Representative histological appearance of the defects after 4 weeks. (A, B, C) Empty control group. (D, E, F) Left knees of GFP-transfected BMMC group. (G, H, I) Right knees of CDMP1-transfected BMMC group. D and G, E and H, F and I show bilateral knee specimens from the same rabbit, respectively. J is a higher magnification of I. Safranin O/Fast Green staining. (K, L) Immunohistochemical staining specific for type II collagen and type I collagen, respectively. Scale bar is 500 µm.

 
Immunohistochemical staining indicated that regenerated cartilage after the implantation of CDMP1-transfected BMMCs showed intense staining for type II collagen (Fig. 2K), again supporting the differentiated hyaline cartilage nature of the repair tissue. Staining for type I collagen was mostly limited to the reconstituted subchondral bone (Fig. 2L).

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 (A–C in Table 1) and deeper zone filling/remodelling (D–E 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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TABLE 1. Results of histological grading scale1

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The current investigation demonstrated that full-thickness articular cartilage defects were repaired with hyaline cartilage after implantation of autologous CDMP1-transfected BMMCs. The repair was superior to previously reported simple BMMC implantation, seemingly because of better surface zone repair and reconstitution.

Transplantation of cultured allogeneic or autologous chondrocytes into areas of cartilage damage has been shown to faithfully produce hyaline cartilage [10–12]. 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 [20–22], 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 [25–31]. 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.


    Acknowledgments
 
We would like to thank Dr Frank P. Luyten for providing us with CDMP1. This work was supported by Grants in Aid for Scientific Research (14370458 and 11470305) from the Japan Society for the Promotion of Science and a Research grant from the Ministry of Health and Welfare, Japan.

The authors have declared no conflicts of interest.

Supplementary data

    Supplementary data are available

    at Rheumatology Online.


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Submitted 4 November 2003; revised version accepted 19 April 2004.



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