Abstract
Background
A cell-based tissue-engineered construct can be employed for treating meniscal lesions occurring in the non-vascularized inner two-thirds. The objective of this study was to test the hypothesis that both pre-differentiation of human bone marrow derived stromal cells (hBMSCs) into chondrogenic lineage before cell seeding and platelet-rich plasma (PRP) pretreatment on a PLGA mesh scaffold enhances the healing capacity of the meniscus with hBMSCs-seeded scaffolds in vivo.
Methods
PRP of 5 donors was mixed and used for the experiments. The woven PLGA mesh scaffold (VicrylTM, Ethicon) measuring 20×8 mm (thickness, 0.2 mm) was prepared. The scaffolds were immersed into 1,000 μl of PRP and were centrifuged at 150g for 10 min. Then, the scaffold was flipped 180° and the same procedure was done for the other side. After washing, the scaffolds were soaked into 1,000 μl of DMEM media. hBMSCs from an iliac crest of 10 patients after informed consent and approval of our IRB were induced into chondrogenic differentiation with chondrogenic media containing 10 ng/ml rhTGF-ß3 in 1.2% alginate bead culture system for 7 days. Then, 2×105 hBMSCs were recovered, seeded onto the scaffold, and cultured under dynamic condition. Based on the presence of pre-differentiation into chondrogenic lineage and the PRP pretreatment, 4 study groups were prepared. (no differentiation without PRP, no differentiation with PRP, chondrogenic differentiation without PRP, chondrogenic differentiation with PRP) Cell number for each cell-seeded scaffold was determined at 24 hours after seeding. Then, scaffolds were placed between human meniscal discs and were implanted subcutaneously in nude mice for 6 weeks (n=10 per group).
Results
Cell attachment analysis revealed no significant difference among groups (p>0.05). The average cell number attached on the scaffold was ranged 1.1×105 to 1.2×105 among groups after 24 hours, so the initial cell seeding efficiency was ranged 55 to 60%.
Histologic results from the 10 constructs containing hBMSCs undifferentiated and seeded onto non-PRP treated scaffolds revealed none had healed at all. Of the constructs containing hBMSCs undifferentiated and seeded onto PRP-pretreated scaffolds, three menisci healed and seven did not heal. Of the constructs containing hBMSCs pre-differentiated into chondrogenic lineage and seeded onto non-PRP treated scaffolds, six menisci healed and four did not heal. Of the constructs containing hBMSCs pre-differentiated into chondrogenic lineage and seeded onto PRP-pretreated scaffolds, seven menisci healed and three did not heal. Histological evaluation demonstrated a continuous hypercellular new fibrous tissue integrating into the native devitalized meniscus disc tissue in healed samples. The histological outcome between the groups was significant (p<0.05) (Table 1) (Figure 1).
Conclusion
hBMSCs, which were differentiated into chondrogenic lineage before cell seeding and attached PRP-pretreated PLGA mesh scaffolds, demonstrated enhanced healing capacity of human meniscus in a meniscal repair mouse model. These findings demonstrate that both pre-differentiation of hBMSCs into chondogenesis and the PLGA scaffold modified by PRP pretreatment provides more biomimetic and biocompatible strategy for cell-mediated meniscal repair.
Acknowledgements
This study was supported by Basic Science Research Program through the National Research Foundation of Korea (#2015-01004099).
