Introduction: Articular cartilage injuries are very common, and if untreated can become symptomatic and progressively lead to premature osteoarthritis. It is well known that damaged cartilage has very limited potential to heal itself, and repair and regeneration of hyaline cartilage remain a clinical and scientific challenge. There are no pharmacological methods that can regenerate cartilage, and currently clinical treatments of debridement, chondrocyte transplantation and marrow stimulation have not been shown to restore consistently a durable articular surface. Tissue engineering and gene therapy concepts may improve cartilage repair by introducing cells, scaffolds, growth factors and other potential modulators of cartilage healing process. When analyzing cartilage treatment outcomes, traditionally we use macro- and microscopic assessment, immunohistochemistry, biochemical characterization etc. Recently, it has been postulated that biomechanical properties of newly formed cartilage are just as important, and novel methods of measurements have been proposed.

Materials and methods: 38 defects were created on weight-bearing part of the medial femoral condyle in sheep. The sheep were randomly assigned to one of four groups. In the bone marrow clot (BMC) group, the sheep were implanted with untreated autologous bone marrow clot that was aspirated from iliac crest of respected animal. In the bone marrow transduced with Ad. GFP (GFP) group, the sheep were implanted with autologous bone marrow clots genetically modified to over express green fluorescent protein (GFP). In the bone marrow transduced with Ad. TGF-β1 (TGF) group, the sheep were implanted with autologous bone marrow clots genetically modified to over express transforming growth factor-β1. Untreated sheep served as a control (defect without implant), and native cartilage served as positive control. Specimens were collected after 6 months and analyzed by single-impact micro-indentation (SIMI), atomic force microscope (AFM) and scanning electron microscope (SEM).

Results: SIMI and AFM measurements showed that repair tissue has greater Young’s elastic modulus then native cartilage. There was a statistically significant difference between TGF-β1, GFP and BMC groups. SEM analysis showed presence of structurally organized collagen molecules in TGF-β1, GFP and BMC groups.

Conclusion: The results of this study showed that it is possible to enhance cartilage repair process by means of genetically modified bone marrow. Furthermore, biomechanical data obtained with SIMI, AFM and SEM provided more detailed insight into articular cartilage function and structure, and in future may be of practical importance for a better understanding of both cartilage mechanics and cartilage disease progression.

Aims: This study investigates the use of novel autologous bone marrow plugs as a biological ÒmatrixÒ to support transgene expression following genetic modiþcation in vitro, and to deliver gene vectors to cartilage defects in vivo. Methods: Adenoviral vectors encoding marker genes (luciferase, green ßuorescent protein (GFP)) and bioactive genes (TGF-?) as well as genetically modiþed mesenchymalstem cells were used to characterize an autologous delivery system using clots of bone marrow aspirates in vitro, and within rabbit osteochondral defects in vivo. Results: Bone marrow clots were able to support expression of luciferase and TGF-? transgenes for up to 21d. In addition incubation of bone marrow clots with rTGF-? demonstrated, that the clots have chondrogenic potential, as evidenced by type II collagen and proteogly-can staining. Bone marrow clots seeded with cells genetically modiþed to express luciferase were able to support transgene expression following implantation into rabbit osteochondral defects for up to 14 days. Implanted clots were able to remain within the defects without þxation, and considerable integration with surrounding tissue was observed after 3 days. The bone marrow clots were also able to effectively localize transgene expression within the defects without leakage to surrounding tissue. Conclusion: These results demonstrate that genetically modiþed bone marrow plugs can support persistent transgene expression in vitro and within osteochondral defects in vivo. They provide an effective delivery system with chondrogenic potential.

Aims: This in vitro study investigates the use of Collagen/PRP Hydrogels as a biological matrix for containing genetically modified human ACL cells, and supporting transgene expression. Methods: Adenoviral vectors encoding marker genes (green fluorescent protein (GFP)) and bioactive) where used to infect cultured human ACL cells?genes (TGF- ex vivo. The cells were seeded in Collagen/PRP Hydrogels and maintained in culture. To expression over time, ELISA was performed at days 4, 8, 15, 23,?measure TGFand 29. GFP positive cells within the gel were viewed by fluorescence microscopy at the same time points. After 29 days, the cultures were fixed, sectioned and various sections were stained with H& E, toluidine blue to detect proteoglycans and by immunhistocemistry for collagen type I and II. Results: Collagen/PRP Hydrogels were transgenes for up to 29 days.?able to support expression of GFP and TGF- expressing gel/cell constructs produced an abundant?Compared to controls, TGF- amount of type I collagen, consistent with the ligament phenotype and appeared more cellular. Little or no proteoglycan staining was observed in either group. Conclusion: These results demonstrate that genetically modified human ACL cells can support persistent transgene expression in vitro, sufficient to stimulate growth of ligamentlike tissue within a Collagen/PRP Hydrogel. The high levels of transgene expression suggest that the Collagen/PRP Hydrogel can function as an effective gene delivery system for tendon repair in vivo.