The purpose of this study was to optimize the 3D printed PCL meniscus scaffolds seeded with BMSCs and cultured in vitro, and to investigate the effect of PTH (1–34) on the repairing of the tissue-engineered meniscus in vivo after implantation in total meniscectomy canine model. The results showed that the BMSCs–scaffold construct synthesized and accumulated more cartilage ECM in vitro when cultured for 21 days as compared to other culture time points. Furthermore, intra-articular injection of PTH (1–34) decreased the cell hypertrophy of the tissue-engineered meniscus during regeneration and increased the chondroprotective effects of the tissue-engineered meniscus for knee cartilage in vivo. This demonstrated the satisfactory efficacy of PTH (1–34) and tissue-engineered meniscus combination on meniscal replacement.
There are various methods to prepare tissue-engineered meniscus scaffolds. However, 3D printing provides a high controllability of the internal structure and geometric shape and personalization of printed entities. Due to the complicated anatomical structure and stress environment of meniscus, 3D printing has greater advantages in the preparation of tissue-engineered meniscus scaffold as compared to other fabrication technologies. In this study, a PCL scaffold was 3D printed to prepare tissue-engineered meniscus scaffolds, which showed reproduction of the native meniscus anatomical shape, low degradation rate, and good cell compatibility, and could anatomically respond to the knee joint pressure as well as support the growth of cells and tissues in vivo.
Currently, there is no uniform standard for in vitro culture of cell-seeded scaffolds in tissue-engineered meniscus studies. In many studies, cell-free scaffold or cell-seeded scaffold were transplanted without in vitro culture into animals; although certain regeneration effects were achieved, joint degeneration often occurred after transplantation[27, 28]. In this study, 3D printed scaffolds were seeded with canine BMSCs and cultured under chondrogenic culture medium in vitro, and the BMSCs–scaffold showed an increasing trend of cell proliferation and ECM, GAG, and Col2 synthesis, which reached the highest levels on day 21 of culture, and declined on day 28; the decline may be related to hypertrophy of BMSCs during long-term chondrogenic induction in vitro. Therefore, the BMSCs–scaffold construct was cultured in vitro for 21 days before implantation, which makes it more conducive to the synthesis and accumulation of cartilage ECM, resulting in more mature structure and function, so that it responds more quickly to knee joint pressure after implantation.
BMSCs are frequently used as seed cells for tissue-engineered meniscus[10, 29]; however, chondrogenesis leads to undesired terminal differentiation of the generated chondrocyte, which reduces the quality of regenerative tissue and decreases its repair efficacy . In our study, the immunohistochemistry of meniscus implants in BMSCs–scaffold group showed high expression of the chondrocyte terminal differentiation markers Col10 and MMP13, indicating the terminal differentiation of BMSCs.PTH (1–34) has demonstrated its potential in inhibiting the terminal differentiation of BMSC chondrogenesis, and it is often used in research concerning cartilage tissue engineering. Mueller et al.  found that intermittent PTHrP stimulation can promote cartilage formation and inhibit hypertrophy of BMSC chondrogenesis in vitro. Among the four PTHrP isoforms (1–34, 1–86, 7–34, and 107–139), Lee et al. found that PTHrP (1–34) most significantly enhanced chondrogenesis and inhibited hypertrophic differentiation of human BMSCs. In this study, at 12 weeks postoperatively, the cells composition and ECM deposition of the neo-tissue of meniscus implants in PTH treated group were similar to those of native meniscus. Furthermore, this tissue also showed lower expression of terminal differentiation markers Col10 and MMP13. These observations suggested that intra-articular injection of PTH (1–34) enhanced tissue regeneration and ECM deposition as well as inhibited the terminal differentiation of tissue-engineered meniscus with BMSCs as seed cells in vivo.
At the same time, we also observed the chondroprotective effect of PTH (1–34) on the tissue-engineered meniscus implant. Although recent studies have attempted to make tissue-engineered meniscus that simulate the anatomy and mechanical properties of native meniscus in order to alleviate the stress environment of knee joint and better protect the knee cartilage, their protective effect after transplantation needs to be strengthened[3, 33, 34]. Hannink et al. implanted PCL-PU meniscus scaffolds in dogs and found articular cartilage degeneration and chondrocyte hypertrophy. Similar to their findings we also found that the articular cartilage in the tissue of BMSCs–scaffold group showed significant damage, although it showed a certain reduction in cartilage degeneration and chondrocyte loss when compared with the Meniscectomy group. This may be due to the variation of cartilage friction coefficient in the knee joint after the transplantation, resulting in wear on the cartilage surface, meanwhile, the biomechanical properties of knee joint may have changed, which would disrupt the normal homeostasis of the joint, leading to cartilage degeneration[34–36]. The degeneration of cartilage may also have an impact on the meniscus function. Adamts5 and MMP13 are considered important catabolic enzymes that degrade aggrecan(AGG) and Col2, key ECM components of functional cartilage, and their expression is related to the cartilage degeneration[19, 38, 39]. The potential of PTH (1–34) in protecting against cartilage degeneration and inducing matrix regeneration after articular cartilage injury has been demonstrated in in vivo studies[19, 32, 40]. Dai et al. subcutaneously injected PTH (1–34) into guinea pigs of meniscectomy model and found the inhibition of cartilage degeneration by PTH (1–34), which may be related to the inhibition of Adamts4 and MMP13 expression. In this study, the injection of PTH (1–34) after transplantation of tissue-engineered meniscus reduced the degree of lesions in the knee cartilage showed a higher expression of Col2 and lower expression of Adamts5 and MMP13. This suggested that PTH (1–34) inhibited the degeneration of cartilage caused by the total substitution of tissue-engineered meniscus, protected the integrity of the knee joint cartilage, and thus enhanced the repairing effects of tissue-engineered meniscus.
There are several limitations of this study. The recent studies report different application dosage, frequency, and duration of PTH (1–34)[18, 42, 43]. The regeneration of tissue-engineered meniscus in vivo is a multi-step dynamic process, and the variations in PTH (1–34) application to a certain factor may cause different effects[21, 44]. In the present study, although the PTH (1–34) promoted the effects of BMSCs-3D printed scaffold for total meniscal substitution, cartilage degradation was not completely prevented. Therefore, further optimization of the administration time, dosage, and mode is needed, and further follow-up studies should be undertaken to enhance the adjuvant effect of PTH (1–34) on the regeneration of tissue-engineered meniscus with BMSCs as seed cells in vivo.