OBJECTIVE: To evaluate clinical, radiographic, and magnetic resonance (MR) results of costal chondrocyte-derived pellet-type scaffold-free autologous chondrocyte implantation (CCP-ACI) in osteochondral defects (ODs) up to 10-mm depth during 5 years of follow-up.
METHODS: Ten patients with CCP-ACI performed in ODs with depth up to 10 mm were retrospectively analyzed. The minimum follow-up period was 5 years. The median age was 36.5 (range 20-55) years. The median size and the depth of the OD lesion were 4.25 cm2 (range 2-6) and 7.0 mm (6-9), respectively. Clinically, the International Knee Documentation Committee, Lysholm, and visual analog scale pain scores were evaluated. Radiographically, the hip‒knee‒ankle (HKA) angle and the Kellgren‒Lawrence (K‒L) grade were assessed. On MR imaging, the magnetic resonance observation of cartilage repair tissue (MOCART) 2.0 score and the defect depth were evaluated.
RESULTS: All average clinical scores improved significantly by 1, 2, and 5 years postoperatively. The average HKA angle and the proportion of K‒L grade did not change significantly within 5 years. The median total MOCART scores were 50 (range 45-65), 50 (35-90), 57.5 (40-90), and 65 (50-85) at 6 months, 1 year, 2 years, and 5 years postoperatively, respectively (p = 0.001), with significant improvement at 2 years compared to that at 6 months postoperatively. The signal intensity of the repair tissue and subchondral change significantly improved from 10 (range 10-10) to 12.5 (10-15) (p = 0.036), and from 10 (10-10) to 17.5 (0-20) (p = 0.017), respectively. Significant improvements were seen at 5 years postoperatively for the former and at 2 years postoperatively for the latter. The average depths on MR imaging were 6.7, 6.7, 6.8, 6.6, and 6.6 mm preoperatively and at 6 months, 1 year, 2 years, and 5 years postoperatively with no significant changes (n.s).
CONCLUSIONS: CCP-ACI provided acceptable mid-term outcomes in ODs up to 10-mm in depth without bone grafting despite of no scaffold. The procedure can be one of minimally invasive treatment options for ODs without scaffold-related problems.
METHODS: IV.

Seeding cells are key factors in cell-based cartilage tissue regeneration. Monoculture of either chondrocyte or mesenchymal stem cells has several limitations. In recent years, co-culture strategies have provided potential solutions. In this study, directly co-cultured rat costal chondrocytes (CCs) and human Wharton\'s jelly mesenchymal stem (hWJMSCs) cells were evaluated as a candidate to regenerate articular cartilage.
Rat CCs are directly co-cultured with hWJMSCs in a pellet model at different ratios (3:1, 1:1, 1:3) for 21 days. The monoculture pellets were used as controls. RT-qPCR, biochemical assays, histological staining and evaluations were performed to analyze the chondrogenic differentiation of each group. The 1:1 ratio co-culture pellet group together with monoculture controls were implanted into the osteochondral defects made on the femoral grooves of the rats for 4, 8, 12 weeks. Then, macroscopic and histological evaluations were performed.
Compared to rat CCs pellet group, 3:1 and 1:1 ratio group demonstrated similar extracellular matrix production but less hypertrophy intendency. Immunochemistry staining found the consistent results. RT-PCR analysis indicated that chondrogenesis was promoted in co-cultured rat CCs, while expressions of hypertrophic genes were inhibited. However, hWJMSCs showed only slightly improved in chondrogenesis but not significantly different in hypertrophic expressions. In vivo experiments showed that all the pellets filled the defects but co-culture pellets demonstrated reduced hypertrophy, better surrounding cartilage integration and appropriate subchondral bone remodeling.
Co-culture of rat CCs and hWJMSCs demonstrated stable chondrogenic phenotype and decreased hypertrophic intendency in both vitro and vivo. These results suggest this co-culture combination as a promising candidate in articular cartilage regeneration.

BACKGROUND: Costal chondrocytes (CCs), as a promising donor cell source for cell-based therapy for cartilage repair, have strong tendency of hypertrophy and calcification, which limited CCs from further application in cartilage regenerative medicine. Synovium-derived stromal cells (SDSCs), have shown their beneficial effect for chondrocytes to maintain phenotype. This study aims to investigate whether SDSCs could help CCs to maintain chondrogenic phenotype and suppress hypertrophic differentiation in cartilage repairs.
METHODS: CCs were directly cocultured with SDSCs in pellet or indirectly cocultured using a conditioned medium in vitro for 3 weeks. Cartilage matrix formation and hypertrophic differentiation of CCs were analyzed by RT-PCR, biochemical assays, and histological staining. Cocultured pellets were implanted into the osteochondral defects made on the femoral groove of the rats. Then, macroscopic and histological evaluations were performed.
RESULTS: Pellets formed by CCs alone and CCs cocultured with SDSCs reveal equal cartilage matrix deposition. However, the gene expression of type X collagen was significantly downregulated in cocultured pellets. Immunohistochemistry analysis revealed suppressed expression of type X collagen in cocultured pellets, indicating SDSCs may suppress hypertrophic differentiation of chondrocytes. Further in indirect coculture experiment, SDSCs suppressed type X collagen expression as well and promoted the proliferation of CCs, indicating SDSCs may influence CCs by paracrine mechanism. The pellets implanted in the osteochondral defects showed good restoration effects, whereas the grafts constructed with CCs and SDSCs showed lower type X expression levels.
CONCLUSIONS: These results suggest that SDSCs may maintain the phenotype of CCs and prevent the hypertrophic differentiation of CCs in cartilage repair.The Translational Potential of this Article: CCs is a promising donor cell source for cell-based therapy for cartilage repair. Based on our study, cocultured with SDSCs weakened the tendency of hypertrophy and calcification of CCs, which provide a potential usage of SDSCs in CCs-based cartilage repair therapy to suppress newly formed cartilage calcification and improve clinical outcomes.

Because articular chondrocyte-based autologous chondrocyte implantations (ACIs) have restrictively restored articular cartilage defects, alternative cell sources as a new therapeutic option for cartilage repair have been introduced.
To assess whether implantation of a costal chondrocyte-derived pellet-type (CCP) ACI allows safe, functional, and structural restoration of full-thickness cartilage defects in the knee.
Case series; Level of evidence, 4.
In this first-in-human study, 7 patients with symptomatic, full-thickness cartilage lesions were enrolled. The chondrocytes isolated from the patients\' costal cartilage were expanded, followed by 3-dimensional pellet culture to prepare the CCP-ACI. Implantation of the pellets was performed via minimal arthrotomy and secured with a fibrin sealant. Clinical scores, including the International Knee Documentation Committee (IKDC) subjective, Lysholm, and Tegner activity scores, were estimated preoperatively and at 1, 2, and 5 years postoperatively. High-resolution magnetic resonance imaging was also performed to evaluate cartilage repair as well as to calculate the MOCART (magnetic resonance observation of cartilage repair tissue) score.
The costal chondrocytes of all patients formed homogeneous-sized pellets, which showed the characteristics of the hyaline cartilaginous tissue with lacunae-occupied chondrocytes surrounded by glycosaminoglycan and type II collagen-rich extracellular matrix. There were no treatment-related serious adverse events during the 5-year follow-up period. Significant improvements were seen in all clinical scores from preoperative baseline to the 5-year follow-up (IKDC subjective score, 34.67 to 75.86; Lysholm score, 34.00 to 85.33; Tegner activity score, 1.17 to 4.67; and MOCART score, 28.33 to 83.33). Two patients had complete defect filling on magnetic resonance imaging evaluation at 1 year. Moreover, at 5 years postoperatively, complete defect filling was observed in 4 patients, and hypertrophy or incomplete defect filling (50%-100%) was observed in 2 patients.
The overall results of this clinical study suggest that CCP-ACI can emerge as a promising therapeutic option for articular cartilage repair with good clinical outcomes and structural regeneration and with stable results at midterm follow-up.
NCT03517046 ( ClinicalTrials.gov identifier).

In this study, we developed the disc-type bio-cartilage reconstruction strategies for transplantable hyaline cartilage for reconstructive surgery using 3D-cell sheet culture of human bone marrow stromal cells and human costal chondrocytes. We compared chondrogenesis efficiency between different chondrogenic-induction methods such as micromass culture, pellet culture, and 3D-cell sheet culture. Among them, the 3D-cell sheet culture resulted in the best chondrogenesis with the disc-type bio-cartilage (>12 mm diameter in size) in vitro, but sometimes spontaneous curling and contraction of 3D-cell sheet culture resulted in the formation of bead-type cartilage, which was prevented by type I collagen coating or by culturing on amniotic membrane. Previously, it was reported that tissue-engineered cartilage reconstructed in vitro does not maintain its cartilage phenotype after transplantation but tends to transform to other tissue type such as bone or connective tissue. However, the disc-type bio-cartilage of 3D-cell sheet culture maintained its hyaline cartilage phenotype even after exposure to the osteogenic-induction condition in vitro for 3 weeks or after the transplantation for 4 weeks in mouse subcutaneous. Collectively, the disc-type bio-cartilage with 12 mm diameter can be reproducibly reconstructed by the 3D-cell sheet culture, whose hyaline cartilage phenotype and shape can be maintained under the osteogenic-induction condition as well as after the transplantation. This disc-type bio-cartilage can be proposed for the application to reconstructive surgery and repair of disc-type cartilage such as mandibular cartilage and digits.
BACKGROUND: Supplementary material is available for this article at 10.1007/s13770-016-9065-6 and is accessible for authorized users.

Generating neocartilage with suitable mechanical integrity from a cell source that can circumvent chondrocyte scarcity is indispensable for articular cartilage regeneration strategies. Costal chondrocytes of the rib eliminate donor site morbidity in the articular joint, but it remains unclear how neocartilage formed from these cells responds to mechanical loading, especially if the intent is to use it in a load-bearing joint. In a series of three experiments, this study sought to determine efficacious parameters of passive axial compressive stimulation that would enable costal chondrocytes to synthesize mechanically robust cartilage. Experiment 1 determined a suitable time window for stimulation by its application during either the matrix synthesis phase, the maturation phase, or during both phases of the self-assembling process. The results showed that compressive stimulation at either time was effective in increasing instantaneous moduli by 92% and 87% in the synthesis and maturation phases, respectively. Compressive stimulation during both phases did not further improve properties beyond a one-time stimulation. The magnitude of passive axial compression was examined in Experiment 2 by applying 0, 3.3, 5.0, or 6.7 kPa stresses to the neocartilage. Unlike 6.7 kPa, both 3.3 and 5.0 kPa significantly increased neocartilage compressive properties by 42% and 48% over untreated controls, respectively. Experiment 3 examined how the passive axial compression regimen developed from the previous phases interacted with a bioactive regimen (transforming growth factor [TGF]-β1, chondroitinase ABC, and lysyl oxidase-like 2). Passive axial compression significantly improved the relaxation modulus compared with bioactive treatment alone. Furthermore, a combined treatment of compressive and bioactive stimulation improved the tensile properties of neocartilage 2.6-fold compared with untreated control. The ability to create robust articular cartilage from passaged costal chondrocytes through appropriate mechanical and bioactive stimuli will greatly extend the clinical applicability of tissue-engineered products to a wider patient population.