**4. Discussion**

The development of Kartigen® was started from our previous study using atelocollagenembedded chondrogenic BMSCs to repair the full thickness of cartilage defect in miniature pigs [51]. The promising results encouraged us to initiate a case series study to test the safety and efficacy of Kartigen® for treating cartilage defects. In the 9 years of followup study, improvement of knee functions was satisfactory and sustainable [34]. Most importantly, infection, inflammation, joint adhesion, loose body, and tumor formation were not found in the Kartigen®-implanted knees. However, in the case series study, the contralateral knees of the same patients were used as a control group. To further validate the therapeutic efficacy of Kartigen® in comparison with standard surgical treatments, such as microfracture, a new controlled and randomized phase I clinical trial was conducted. As expected, no severe adverse events were found, and all the TEAEs were not treatment-related. Although clinical outcome was similar for Kartigen implantation and microfracture in 1-year follow-up, a sustained improvement of knee functions was found in the Kartigen® group in the 2 years follow-up.

Microfracture was introduced for focal articular cartilage repair in the 1980s and soon became a standard treatment for cartilage defects [9,46,52]. However, its results are usually associated with fibrocartilage production [12,13], and its long-term efficacy remains poor [16,53]. Mosaicplasty provides a better improvement than microfracture, but there are still several disadvantages, such as the donor-site morbidity for autografts [54], limited sources of grafts, a technical challenge in leveling the graft during operation [55], and poor integration of the implant into host tissue [56,57]. On the contrary, the procedure of Kartigen® implantation is technically simple and can be carried out with a small arthrotomy or using arthroscope. Furthermore, there is no donor site morbidity, and sources of cells are not limited.

The ability of engrafted cells to integrate into the recipient site and participate in the repair process is crucial for successful clinical outcomes. It has been reported that intra-articular injection of autologous MSCs reduced the degeneration of cartilage defects and provided pain relief [58], but integration of injected MSCs into damaged cartilage defects is unclear and doubtful. On the contrary, using atelocollagen as a cell carrier, Kartigen® prevents cell loss, is easy for implantation for cartilage defects of any shape, and achieves uniform cell distribution at the recipient site. These advantages contribute to the efficacy and durability of Kartigen® implantation. In addition, atelocollagen has been proven to enable a gradual proliferation and matrix synthesis of chondrocytes, which allow chondrocytes to maintain their phenotype for up to 4 weeks in vitro [40]. Atelocollagen gel can also support cell proliferation, matrix synthesis, and chondrogenic differentiation of MSCs [42]. Recently, the Adachi group reported the efficacy of repairing osteochondral defects with minced cartilage embedded in atelocollagen gel [41]. Our previous clinical trial with 9 years of follow-up also demonstrated the safety and efficacy of atelocollagen in repairing cartilage defects together with CPs [34].

Unlike previous studies using mature chondrocytes for cartilage repair in ACI [59], our study showed CPs in Kartigen® exhibiting sufficient integration capacity. Under arthroscopic examination, the integration between the graft and the recipient site was complete (Figure 3). The histological analyses of the biopsy specimens also demonstrated the integration of the implanted tissue into the surrounding articular cartilage (Figure 4). The accumulation of GAG and collagen type II was confirmed in the biopsy specimens (Figure 4). However, there are several limitations to this study. Because this is a phase I study, the sample size number is small. The 2 years of follow-up are not long enough to reach a final conclusion for cartilage defect repair.

Recent advances in 3D bioprinting [60–63] have enabled reconstructions of functional living cartilage to recapitulate the complexity and architecture [64,65] of an articular surface. We are looking into partnerships to integrate that technology and potentially further improve the outcome with Kartigen® implantation. This may result in more favorable biomechanical properties at the recipient sites and allow earlier weight-bearing and range

of motion without concern of graft detachment. Since MSCs are multipotent, additional biochemical and biomechanical stimulations can delicately manipulate the chondrogenic differentiation and maturation of seeded cells [66,67] to improve the functional properties of the derived neo-cartilage tissues [29]. The integration of 3D bioprinting and biomimetic in vitro chondrogenesis will drive advanced therapeutic innovations for cartilage repairs.
