Materials and methods
Animals and reagents
A total of 10 one-year-old male canines were provided by the College of Agriculture at the Shanghai Transportation University (Shanghai, China; permit no. 2007-0004). Study approval was obtained from the Shanghai Animal Care and Use Committee (Shanghai, China) prior to performing the study. The following reagents were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA): pcDNA™6.2-GW/EmGFP, plenti6/v5, one shot Stbl3 chemically competent Escherichia coli, BLOCK-iT polymerase II (Pol II) expression vector kit with emerald green fluorescent protein (EmGFP) and BLOCK-iT™ lentiviral Pol II expression system. The following reagents were also used: AxyPrep plasmid purification kit (Axygen, Union City, CA, USA), polylactic acid (PLA) and polyglycolic acid (PGA) (Sigma, St. Louis, MO, USA), recombinant human CDMP-2 (PeproTech, London, UK), 0.25% F-10 basic culture media and Safranin-O dye (Shanghai Reagent Company, Shanghai, China), toluidine blue dye (Shanghai Reagent Company), anti-collagen-I antibodies (mouse and canine; Abcam, Cambridge, UK), anti-collagen-II antibodies (mouse anti-canine; Neomarkers, Fremont, CA, USA), anti-S-100 antibodies (rabbit anti-canine; Neomarkers) and Alcian Blue staining buffer (Pierce Biotechnology, Inc., Rockford, IL, USA).
Lentiviral infection of canine myoblasts
Canine myoblasts were purified and cultured, as previously described (7). The cells were infected with lentiviruses containing the empty vector or the hCDMP-2 gene for two days. GFP+ clones were selected for further studies. The expression of GFP and hCDMP-2 genes in different clones were quantified by quantitative polymerase chain reaction (qPCR) using total RNA. The sequences of the primers were: GAPDH forward, 5′-GACAACTTTGGTATCGTGGAAGG-3′ and reverse, 5′-CCAGTAGAGGCAGGGATGATGT-3′; GAPDH probe, fam-5′-CTCATGACCACAGTCCATGCCATCACT-3′-tamra; GFP forward, 5′-AAGCAGCACGACTTCTTCAAGTC-3′ and reverse, 5′-TCGCCCTCGAACTTCACCTC-3′; GFP probe, 5′-CATGCCCGAAGGCTACGTCCAGGAG-3′; hCDMP-2 forward, 5′-CAGAAGTATTTGTTTGATGTGTCCA-3′ and reverse: 5′-AAAGGCAAGGGAAGAGCTGCA-3′; and hCDMP-2 probe, 5′-CAGACAAAGAAGAGCTGGTGG GCGC-3′.
Western blot analysis
Western blot analyses were performed with total protein extracts prepared using T-PER® tissue protein extraction reagent (Pierce). Filters were probed with anti-CDMP-2 (mouse anti-human; Sigma).
In vivo repair of meniscal fibrocartilage injury in canines
The PLA/PGA (Sigma) scaffold was made using a specific mold. The canine model of meniscal injury was established by cutting across the entire meniscus (0.5 cm from the front edge, with a width of 2 mm, and a thickness of 2 mm at the meniscocapsular junction and 1.5 mm at the other side) (Fig. 1A). The injury encompassed all three zones (the red-red zone, red-white zone and white-white zone). The animals were divided into four groups: Group A, suture only; group B, suture with the addition of the recombinant hCDMP-2 on the PLA/PGA scaffold; group C, PLA/PGA scaffold with canine myoblasts carrying the empty vector; D, PLA/PGA scaffold with canine myoblasts expressing hCDMP-2 (six menisci for each group) (Fig. 1B and C). Samples of repaired tissue extracted at weeks 3, 8 and 12 post-repair were subjected to gross morphological studies and hematoxylin and eosin (H&E) and Safranin-O staining. Immunohistochemistry (IHC) studies were performed to assess the expression of collagen I and II and S-100 protein. Quantitative assays were also performed to assess the levels of collagen I and II (by ELISA), and the glycosaminoglycans (GAGs) (by Alcian Blue staining) in the red-red and white-white zones. The normal value was obtained by assessing the meniscal tissue at the uninjured part using the same methods as those used for the repaired tissue.
Statistical analysis
The data are expressed as the mean ± standard deviation. Statistical significance was determined using one-way analysis of variance and the level of statistical significance was set at a probability value of P<0.05.
Results
Expression of hCDMP-2 in canine myoblasts following lentiviral infection
The infection efficiency was determined to be 71±0.088% by qPCR, and the expression level of hCDMP-2 was 0.0015±0.0005. Western blot analysis confirmed the expression of hCDMP-2 protein upon lentiviral infection of the canine myoblasts.
H&E and Safranin-O staining for the evaluation of in vivo meniscal fibrocartilage injury repair
H&E and Safranin-O staining showed partial staining of the repaired tissue in group D at week 3 post-repair. A few cartilage lacunae mixed with the scaffold tissue were observed in the red-red zone, while no staining was observed in the white-white zone (Fig. 2A and B). At week 8, H&E staining showed increased cartilage lacunae in the red-red zone of group D, and fibrous structures started to appear (Fig. 2C), while a small amount of cartilage lacunae without fibrous structures were observed in the white-white zone (Fig. 2D). The red-red zone showed an increased intensity and volume of cartilage lacunae (Fig. 2E), while the white-white zone showed partial staining with Safranin-O (Fig. 2F). At week 12 post-repair, H&E staining showed obvious cartilage lacunae in the red-red zone, with increased surrounding fibrous structures (Fig. 2G), while the presence of cartilage lacunae was also increased in the white-white zone, with no fibrous structures (Fig. 2H). Safranin-O staining showed the increased intensity and volume of the regenerated tissues in the red-red and the white-white zones at week 12 (Fig. 2I and J).
Examination of the protein expression of collagen I, collagen II and S-100
IHC studies revealed low expression levels of the cartilage-specific proteins, collagen II, collagen I and S-100, in the red-red zone in group D at week 3 post-repair, while no expression was observed in the white-white zone (Fig. 3A–C). At week 8 post-repair, positive expression of these proteins was detected in the repaired tissue in the red-red and white-white zones (Fig. 3D–I). At week 12, the expression of these proteins remained positive in the two zones (Fig. 3J–O).
Quantitative assessment of collagen I, collagen II and GAG proteins
The expression of collagen I and II was further quantified by ELISA, while the GAGs were assessed by Alcian blue staining. At week 3 post-repair, the expression of collagen I, collagen II and the GAGs was detected in the repaired tissue of the red-red zone, while no expression of these proteins was observed in the white-white zone (Tables I–III). At week 8, the expression of these proteins was detected in the red-red and white-white zones (Fig. 4). Similar results were obtained at week 12 (Fig. 4). Statistical analyses indicated that the expression of these three proteins increased with increasing time, and the differences between each pair of time-points were significant (P<0.01). In addition, significant differences were revealed between the red-red and white-white zones at the same time-point (P<0.01).
Discussion
To facilitate the repair of injury to meniscal fibrocartilage, which lacks a sufficient blood supply, numerous methods have been assessed in the clinic, including the establishment of a blood supply channel, blood clots and implantation of pedunculated synovial flaps, isolated synovia, porous polymers and fibrin clots (8,9). In recent years, certain studies have employed purified growth factors, including epidermal growth factor, TGF, platelet-derived growth factor, basic fibroblast growth factor, hepatocyte growth factor, bone morphogenetic protein 2 and interleukin 1 to facilitate the in vitro expansion of chondrocytes from meniscal fibrocartilage (10,11). Certain studies have used gene transfer technology to improve the in vitro growth of chondrocytes from meniscal fibrocartilage (12), while others have combined scaffold materials with growth factors to facilitate the healing of meniscal fibrocartilage (13). Further studies have explored stem cell technologies in combination with scaffold materials (13,15). These studies have indicated that it is crucial to select appropriate cell types and an effective route of treatment in order to achieve the most favorable outcome for meniscal fibrocartilage injury repair.
CDMP-2 is able to facilitate cartilage repair without local tissue ossification and also to promote the ectopic generation of new tendons and ligaments (containing mainly type I collagen similar to fibrocartilage), and improve the quality of fibrocartilage tissue repair (16).
A previous study reported that myoblasts acted as adult stem cells for skeletal muscle, and that they maintained similar functions to multipotent mesenchymal cells that exhibit myogenic, osteogenic and adipogenic differentiation (17). Myoblast gene therapy has been successful in the treatment of muscle diseases and bone and joint problems.
The present study showed that canine myoblasts are able to produce hCDMP-2 RNA and protein in vitro following lentiviral infection with the hCDMP-2 gene. The infected canine myoblasts were transplanted into the meniscal injury site via a PLA/PGA scaffold in order to observe their effect on meniscal injury repair. H&E histology studies and Safranin-O staining, which assess cartilage GAGs in cells or tissue, showed that the treatments in groups A, B and C did not lead to the regeneration of new tissue in the canine meniscal injury region. By contrast, group D showed newly repaired tissue in different regions (red-red and white-white zones) with the characteristics of fibrocartilage tissue, demonstrating that the treatment of group D was able to promote the meniscal repair by fibrocartilage-like tissue regeneration. Furthermore, quantitative assessment of the fibrocartilage tissue-specific components, including collagen I, collagen II and GAGs, indicated that the red-red zone was regenerated more rapidly than the white-white zone during the fibrocartilage-like tissue repair of meniscal injury. This is consistent with the previous hypothesis, and may be due to the differences in the blood supply of these two regions.
In all four groups, the PLA/PGA scaffold completely dissolved at 8 weeks post-transplantation, indicating that the PLA/PGA scaffold used in this study did not affect meniscal injury repair. The scaffold material can be degraded in vivo, and the time for the complete degradation is approximately eight weeks.
Meniscal fibrocartilage has the characteristics of cartilage tissue as well as fibrous tissue. The white-white zone is more similar to the cartilage tissue (18), while the red-red zone resembles the fibrous tissue (19). Collagen fibers in the red-red zone are generally distributed longitudinally in a horizontal plain and aligned in a ‘C’ shape, while collagen fibers in the white-white zone exhibit a radial distribution (20). The IHC results of the present study revealed that the regenerated collagen fibers in the red-red zone exhibited a ‘C’-shaped distribution. Collagen I was aligned along the fibers, while collagen II was scattered irregularly among the mesenchyma of the tissue irrelevant to the fiber alignment. These observations are consistent with the previous description of the histology and structure of the meniscal fibrocartilage, indicating that the treatment adopted in the present study resulted in the meniscal regeneration by tissue that was similar to normal meniscal fibrocartilage tissue. Furthermore, a previous study indicated that the structure and components of the red-red and white-white regions are different from each other, as the former contains collagen I, which forms large beam fibers, while the latter contains high levels of collagen II and proteoglycans (21). These components have distinct functions to confer meniscal biomechanical properties. The content proportion of collagen I and II and the GAGs in the normal meniscal tissue obtained in the present study was similar to that of previous studies. However, the amounts and distribution of each component of the repaired tissue from group D showed differences from the normal tissue. For example, the red-red zone showed higher levels of the assessed proteins as compared to the white-white zone at all the examined time-points. Further evaluation is, however, required to assess whether these differences are capable of affecting the function of meniscal fibrocartilage.
The present study indicated that myoblasts infected with the hCDMP-2 gene are able to facilitate the regeneration of meniscal fibrocartilage-like tissue in the canine model of local meniscal injury, and that the repair of the red-red zone is more rapid than that of the white-white zone. Further studies are required to address the mechanism of the hCDMP-2-mediated effect on meniscal fibrocartilage injury repair. In addition, due to the limitation in the clinical use of lentiviral infections, further studies are required to explore the best way to combine the hCDMP-2 growth factor with myoblasts to be used in the clinic.