Department of Regenerative Medicine, Kanagawa Children's Medical Center, Yokohama, Kanagawa, Japan
Advanced Medical Research Center, Yokohama City University, Yokohama, Kanagawa, Japan
Correspondence: Hideki Taniguchi, M.D., Ph.D., Department of Regenerative Medicine, Yokohama City University Graduate School of Medical Science, 3–9 Fuku-ura, Kanazawa-ku, Yokohama 236-0004, Japan. Telephone: +81-45-787–2621; Fax: +81-45-787–8963, e-mail: email@example.com
In healthy joints, hyaline cartilage covering the joint surfaces of bones provides cushioning due to its unique mechanical properties. However, because of its limited regenerative capacity, age- and sports-related injuries to this tissue may lead to degenerative arthropathies, prompting researchers to investigate a variety of cell sources. We recently succeeded in isolating human cartilage progenitor cells from ear elastic cartilage. Human cartilage progenitor cells have high chondrogenic and proliferative potential to form elastic cartilage with long-term tissue maintenance. However, it is unknown whether ear-derived cartilage progenitor cells can be used to reconstruct hyaline cartilage, which has different mechanical and histological properties from elastic cartilage. In our efforts to develop foundational technologies for joint hyaline cartilage repair and reconstruction, we conducted this study to obtain an answer to this question. We created an experimental canine model of knee joint cartilage damage, transplanted ear-derived autologous cartilage progenitor cells. The reconstructed cartilage was rich in proteoglycans and showed unique histological characteristics similar to joint hyaline cartilage. In addition, mechanical properties of the reconstructed tissues were higher than those of ear cartilage and equal to those of joint hyaline cartilage. This study suggested that joint hyaline cartilage was reconstructed from ear-derived cartilage progenitor cells. It also demonstrated that ear-derived cartilage progenitor cells, which can be harvested by a minimally invasive method, would be useful for reconstructing joint hyaline cartilage in patients with degenerative arthropathies. Stem Cells2014;32:816–821
Since the regenerative capacity of joint hyaline cartilage is limited, age- and sports-related injuries to the knee may lead to degenerative arthropathies [1, 2]. Surgical treatments of this defect include mosaicplasty, a technique for transplanting small cylindrical osteochondral autografts, and bone marrow stimulation. These techniques are characterized by major drawbacks such as low engraftment rates of transplanted cartilage and the formation of fibrocartilage with inferior biomechanical stiffness [3, 4]. Hence, cartilage defects are a primary target of regenerative medicine .
Recently, we have succeeded in isolating cartilage progenitor cells from ear elastic cartilage. Human cartilage progenitor cells have high chondrogenic and proliferative potential to form elastic cartilage with long-term tissue maintenance . However, it is unknown whether ear-derived cartilage progenitor cells (eCPCs) can be used to reconstruct joint hyaline cartilage, whose mechanical and histological properties differ from those of elastic cartilage. Here, we showed the use of eCPCs for joint hyaline cartilage reconstruction.
Materials and Methods
Detailed Materials and Methods were provided as Supporting Information.
Results and Discussion
First, we performed histological analysis of canine ear elastic cartilage. Similar to human ear elastic cartilage, the perichondrium layer was alcian blue negative, elastica van gieson negative, collagen type I positive, and collagen type II negative. Imunohistochemical analysis of CD44 and CD90, markers for human eCPCs, revealed a rare population of double-positive cells 0.95 (± 0.12)% specifically residing in the perichondrium layer (Supporting Information Fig. S1). We separated canine ear elastic cartilage into the perichondrium layer and the chondrium layer. The separation was confirmed by alcian blue (Fig. 1A, 1B). After digesting each layer, the cells were cultured to yield eCPCs and chondrocytes. Single canine eCPCs formed colonies that contained over 100 cells each after 2 weeks. Canine eCPCs and chondrocytes formed 23.9 (± 4.5) and 9.9 (± 6.8) colonies, respectively (Fig. 1D). These results showed that canine eCPCs were highly clonogenic.
Prior to cell transplantation, it is crucial to obtain a highly chondrogenic population with a large amount of cartilage extracellular matrix (ECM) following in vitro cultivation. We used a layered culture system combined with several cytokines, which we previously showed enhanced cartilage regenerative capacity in vitro . After chondrogenic induction, canine eCPCs differentiated into chondrocytes that produced proteoglycans (Fig. 2A). To quantify and compare the chondrogenic potential of canine eCPCs with that of chondrocytes, we performed dimethylmethylen blue method to evaluate the secretion of sulfated glycosaminoglycans (sGAGs), a major component of the cartilage ECM. The concentrations of both extracellular and intracellular sGAGs of canine eCPC under differentiation conditions were 43.60 (± 14.10) ng per 1.0 × 103 cells. The comparable concentration of total sGAGs in chondrocytes was 52.64 (± 3.13) ng/1.0 × 103 cells, highlighting the high chondrogenic potential of canine eCPCs (Fig. 2B).
Moreover, to examine the elastic cartilage reconstruction capacity of canine eCPCs, cultured cells were expanded, subjected to cartilage differentiation conditions, and transplanted into autologous subcutaneous tissue. After 60 days, reconstructed tissue was harvested from autologous subcutaneous tissue. Histological analysis of this tissue demonstrated that canine eCPCs differentiated into mature chondrocytes and formed an elastic cartilage rich with proteoglycans and elastic fibers (Fig. 2C). In addition, immunohistochemical analysis revealed that reconstructed elastic cartilage contained a collagen type I+ capsule enveloping a collagen type II+ chondrium layer, which was observed to reconstruct a perichondrium layer (Fig. 2C). Reconstruction of perichondrium layer containing a number of CD44+ CD90+ cartilage progenitor cells suggested that canine eCPCs would have the capacity of forming elastic cartilage with long-term structural maintenance. Furthermore, these data showed that eCPCs transplantation into recipient with normal immune system succeeded to reconstruct elastic cartilage without scaffold. This transplantation method will be cardinal technology for elastic cartilage reconstruction in human.
To investigate whether eCPCs could be used to reconstruct joint hyaline cartilage, canine eCPCs were transplanted onto full-thickness cartilage defects after chondrogenic induction (Fig. 3A). After 60 days, we performed histological analysis. Reconstructed tissues contained Safranin O-positive proteoglycans, demonstrating that these were cartilaginous tissues (Fig. 3C). Immunohistochemical staining showed that reconstructed tissues were collagen type II positive, collagen type X positive, collagen type I negative, collagen type VI positive, and elastin negative, indicating the formation of mature cartilage (Fig. 3B; Supporting Information Fig. S2). To evaluate the healing effect, we determined the International Cartilage Repair Society (ICRS) histological score and the Wakitani score (these criteria are listed in Supporting Information Fig. S3). The transplanted group demonstrated an ICRS score of 10.25 (± 4.15) and a Wakitani score of 4.75 (± 2.95) (Fig. 3C). The scores of this group were higher than those of the nontransplanted group, suggesting that the transplanted group exerted a healing effect.
To confirm that reconstructed cartilage consisted of hyaline cartilage, we then performed further analysis. Generally, joint hyaline cartilage contains few elastic fibers, in contrast to ear elastic cartilage [7, 8]. To quantify elastic fibers, pixel intensities no greater than 75 points were measured with a color extraction method. The elastic fiber of ear elastic cartilage was 52.79 (± 9.23)%, that of joint hyaline cartilage was 0.81(± 1.15)%, and that of reconstructed cartilage was 1.34 (± 1.66)% (Supporting Information Fig. S4). The reconstructed cartilage contained few elastic fibers, suggesting that the cartilage reconstructed by autologous transplantation of eCPCs had histological properties similar to normal joint hyaline cartilage.
Unlike ear elastic cartilage that has high flexibility due to elastic fibers, healthy joint hyaline cartilage has high biomechanical strength and stiffness to assist in distributing the high forces (approximately 2.5–10 times the body weight), which are generated during walking and running [8-11]. The amounts of proteoglycans and other extracellular matrix components in cartilage are the major determinants of its mechanical properties [12, 13]. In the result of measurement of the total sGAGs, the amount of sGAGs in reconstructed cartilage (8.86 (± 1.91) µg/mg wet tissue) was equivalent to that in joint hyaline cartilage (9.30 (± 2.17) µg/mg wet tissue) (Fig. 3D). The elastic modulus, which is a biomechanical parameter, was determined on the basis of compression-deformation and viscoelastic shock-absorbing responses . To measure the biomechanical stiffness of reconstructed cartilage, elastic modulus was determined using atomic force microscopy (Fig. 3E). Reconstructed cartilage exhibited a trend toward a higher elastic modulus compared with ear elastic cartilage and was comparable to that of joint hyaline cartilage (Fig. 3F). This data demonstrated that the biomechanical stiffness of reconstructed cartilage (197.5 (± 102.5) kPa) differed from that of ear elastic cartilage (48.4 (± 16.7) kPa) and was similar to that of joint hyaline cartilage (162.8 (± 68.2) kPa) (Fig. 3G). These data suggested that eCPCs reconstructed cartilage by producing hyaline cartilage. Overall, these results demonstrated that autologous transplantation of eCPCs into the canine knee joint resulted in the formation of mature hyaline cartilage, suggesting that eCPCs may be able to differentiate into hyaline cartilage in the environment of joint.
eCPCs have several advantages over the other cell sources for the purposes of joint cartilage repair and reconstruction, such as bone marrow mesenchymal stem cells and synovial stem cells. Bone marrow mesenchymal stem cells possess multipotent differentiation capacities. However, high levels of osteocalcin and alkaline phosphatase expression suggest that these stem cells are prone to differentiate into osteocytes [15, 16], and their low chondrogenic potential is compounded by their propensity for ectopic mineralization. Although synovial stem cells are considered to be the most promising cell source [17, 18], proliferative and chondrogenic capacities are often impaired when cells were harvested from osteoarthritis patients [19, 20]. Thus, joint hyaline cartilage reconstruction capacity of eCPCs had come close to synovial stem cells. The use of eCPCs are expected especially for severe arthropathies, such as osteoarthritis or rheumatoid arthritis, because, these cells can be collected from sites independent of any knee pathologies. However, direct functional comparison with various stem cell sources remains to be solved.
The number of patients with osteoarthritis is increasing worldwide. In US, it is estimated that 27 million individuals currently have this disease [21, 22]. Our study demonstrates that autologous cells, harvested by a minimally invasive procedure with local anesthesia [23-25], can be used to restore damaged cartilage to gain mechanical and histological properties similar to hyaline cartilage. Thus, this study offers hope for a new and effective therapy to regenerate the joint hyaline cartilage of patients with osteoarthritis or degenerative arthropathies.
We thank N. Sasaki, N. Kobayashi, K. Yasumura, H. Suzuki, and S. Murata for technical support. This work was supported by the Grants-in-Aid of the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan to T.T. (No. 24106510, 24689052), to S.K. (No. 20592101), and to H.Y.Y. (No. 24680050, 24106505) and by Health and Labor Sciences Research Grants Research to T.T. (No. 12103252) and to S.K. (Research on intractable disease No. 2011-164), and Japan-Korea Basic Scientific Cooperation Program by Japan Society for the Promotion of Science (JSPS) to T.T. This work was also supported by a grant to T.T. from Yokohama Foundation for Advanced Medical Science (Research and Development Project III No. 22).
M.M.: collection of data, data analysis and interpretation, and manuscript writing; T.T. and S.K.: conception and design, data analysis and interpretation, and manuscript writing.; H.K., Y.Y., T.M., H.Y.Y., S.N., and L.J.I.: collection of data; H.T.: conception and design, and final approval of manuscript.
Disclosure of Potential Conflicts of Interest
The authors indicate no potential conflicts of interest.