Chondrogenic differentiation of bone marrow-derived mesenchymal stromal cells via biomimetic and bioactive poly-ε-caprolactone scaffolds.

The objective of this study was to develop a scaffold for mesenchymal stromal cell (MSC) recruitment, proliferation, and chondrogenic differentiation. The concept behind the design is to mimic the cartilage matrix and contain stimulatory agents that make continuous supply of inductive factors redundant. Nanofibrous (N: ~400 nm) and microfibrous (M: ~10 µm) poly-ε-caprolactone (PCL) scaffolds were combined with 1% high-molecular-weight sodium hyaluronate (NHA/MHA), 1% hyaluronan (HA) and 200 ng transforming growth factor-beta 1 (TGF-ß1; NTGF/MTGF), or 0.1% bovine serum albumin (N/M). Scaffolds were seeded with MSCs from bone marrow and cultured without growth factors in vitro. Cultures with chondrogenic medium supplemented with TGF-ß1 served as controls. Proliferation, migration, and release of TGF-ß1 were investigated. Cell differentiation was evaluated by polymerase chain reaction (PCR) and real-time PCR. NTGF and MTGF exhibited primarily an initial release of TGF-ß1. None of the factors released by the scaffolds recruited MSCs. The expression of aggrecan was dependent on the scaffold ultrastructure with nanofibers promoting increasing and microfibers decreasing expression levels. Composites containing HA demonstrated elevated seeding efficiency and lower type I collagen expression. Expression of type II collagen was dependent on continuous or late supply of TGF-ß1, which was not provided by our scaffold design. The initial release of TGF-ß1 induced an expression of type I collagen and osteogenic marker genes. In conclusion, nanofibrous PCL scaffolds with or without augmentation are suitable for chondrogenic initiation of MSCs. Initial release of HA is sufficient in terms of directing the implanted MSCs toward a chondrogenic end, whereas a late release of TGF-ß1 is preferred to foster type II and avoid type I collagen expression.

Pretreatment of periosteum with TGF-beta1 in situ enhances the quality of osteochondral tissue regenerated from transplanted periosteal grafts in adult rabbits.

OBJECTIVE: To compare the efficacy of in situ transforming growth factor-beta1 (TGF-beta1)-pretreated periosteum to untreated periosteum for regeneration of osteochondral tissue in rabbits. METHODS: In the pretreatment group, 12 month-old New Zealand white rabbits received subperiosteal injections of 200 ng of TGF-beta1 percutaneously in the medial side of the proximal tibia, 7 days prior to surgery. Control rabbits received no treatment prior surgery. Osteochondral transverse defects measuring 5mm proximal to distal and spanning the entire width of the patellar groove were created and repaired with untreated or TGF-beta1-pretreated periosteal grafts. Post-operatively the rabbits resumed normal cage activity for 6 weeks. RESULTS: Complete filling of the defects with regenerated tissue was observed in both the TGF-beta1-pretreated and control groups with reformation of the original contours of the patellar groove. The total histological score (modified O'Driscoll) in the TGF-beta1-pretreated group, 20 (95% Confidence Interval (CI), 19-21), was significantly higher (P=0.0001) than the control group, 18 (16-19). The most notable improvements were in structural integrity and subchondral bone regeneration. No significant differences in glycosaminoglycan or type II collagen content, or equilibrium modulus were found between the surgical groups. The cambium of the periosteum regenerated at the graft harvest site was significantly thicker (P=0.0065) in the TGF-beta1-pretreated rabbits, 121 microm (94-149), compared to controls, 74 microm (52-96), after 6 weeks. CONCLUSIONS: This study demonstrates that in situ pretreatment of periosteum with TGF-beta1 improves osteochondral tissue regeneration at 6-weeks post-op compared to untreated periosteum in 12 month-old rabbits.

Tissue engineering of cartilage using poly-epsilon-caprolactone nanofiber scaffolds seeded in vivo with periosteal cells.

OBJECTIVE: To determine the potential of periosteal cells to infiltrate poly-epsilon-caprolactone (PCL) nanofiber scaffolds in vivo and subsequently produce cartilage in vitro. DESIGN: PCL nanofiber scaffolds, with or without chitosan-coating were implanted under periosteum in 6-month-old rabbits. Transforming growth factor-beta1 (TGF-beta1) or vehicle was injected into each implant site. After 1, 3, 5 or 7 days, scaffolds were removed, separated from the periosteum, and the scaffolds and periosteum were cultured separately for 6 weeks under chondrogenic conditions. Sulfated glycosaminoglycan (GAG), type II collagen, DNA content, cartilage yield, and calcium deposition were then analyzed. RESULTS: Cell infiltration was observed in all scaffolds. Cartilage formation in the uncoated scaffolds increased with duration of implantation (maximum at 7 days). Cells in the uncoated scaffolds implanted for 7 days produced significantly higher levels of both GAG [560 (95% confidence interval (CI), 107-1013) vs 228 (95% CI, 177-278) microg GAG/microg DNA] and cartilage yield [9% (95% CI, 3-14%) vs 0.02% (95% CI, 0-0.22%)] compared to chitosan-coated scaffolds (P=0.006 or less). There was no significant difference in GAG content or cartilage yield between the TGF-beta1-injected and vehicle-injected scaffolds. However, significantly more mineral deposition was detected in TGF-beta1-injected scaffolds compared to vehicle-injected scaffolds (P<0.0001). Cartilage yield from the periosteum, moreover, was significantly increased by subperiosteal TGF-beta1 injections (P<0.001). However, this response was reduced when chitosan-coated scaffolds were implanted. CONCLUSIONS: This study demonstrates that it is possible to seed PCL nanofiber scaffolds with periosteal cells in vivo and subsequently produce engineered cartilage in vitro.

Rejuvenation of periosteal chondrogenesis using local growth factor injection.

OBJECTIVE: To examine the potential for rejuvenation of aged periosteum by local injection of transforming growth factor-beta1 (TGF-beta1) and insulin-like growth factor-1 (IGF-1) alone or in combination to induce cambium cell proliferation and enhance in vitro periosteal cartilage formation. METHODS: A total of 367 New Zealand white rabbits (6, 12, and 24+ month-old) received subperiosteal injections of TGF-beta1 and/or IGF-1 percutaneously. After 1, 3, 5, or 7 days, the rabbits were sacrificed and cambium cellularity or in vitro cartilage forming capacity was determined. RESULTS: A significant increase in cambium cellularity and thickness, and in vitro cartilage formation was observed after injection of TGF-beta1 alone or in combination with IGF-1. In 12 month-old rabbits, mean cambium cellularity increased 5-fold from 49 to 237 cells/mm and in vitro cartilage production increased 12-fold from 0.8 to 9.7 mg 7 days after TGF-beta1 (200 ng) injection compared to vehicle controls (P<0.0001). A correlation was observed between cambium cellularity and in vitro cartilage production (R2=0.98). An added benefit of IGF-1 plus TGF-beta1 on in vitro cartilage production compared to TGF-beta1 alone was observed in the 2 year-old rabbits. IGF-1 alone generally had no effect on either cambium cellularity or in vitro cartilage production in any of the age groups. CONCLUSIONS: These results clearly demonstrate that it is possible to increase cambium cellularity and in vitro cartilage production in aged rabbit periosteum, to levels comparable to younger rabbits, using local injection of TGF-beta1 alone or in combination with IGF-1, thereby rejuvenating aged periosteum.