The Repair of Critically Sized Osteochondral Defects with Re-differentiated Chondrocytes Results in the Formation of Collagen Type 2-Containing Granulation Tissue

Vanessa J Bianchi 1, 2 ; Adrienne Lee 3 ; Jesse Anderson 3 ; Justin Parreno 4 ; John Theodoropoulos 3 ; David Backstein 3 ; Rita Kandel 1, 2, 5

 1. Lunenfeld-Tanenbaum Research Institute; 2. Institute of Biomaterials and Biomedical Engineering; 3. Division of Orthopaedics, Mt. Sinai Hospital; 4. The Scripps Research Institute;  5. Pathology and Laboratory Medicine, Mt Sinai Hospital

Introduction: The integrity of articular cartilage (AC) tissue is essential for smooth, painless joint movement. When damaged, AC is has limited ability to repair. Autologous chondrocyte implantation (ACI), which utilizes passaged chondrocytes, is one therapeutic approach to repair damaged AC. However, this treatment commonly leads to the formation of fibrocartilage which is mechanically inferior to hyaline cartilage. This tissue is the result of the cells that are being used. When chondrocytes are passaged, they lose their phenotype (termed “de-differentiate”) and the ability to form hyaline cartilage. The use of transforming growth factor β (TGFβ) to re-differentiate passaged chondrocytes has been validated in vitro, however it has not been established if re-differentiated chondrocytes will enhance defect repair when implanted in vivo. Furthermore, fibrin gel is used clinically as a glue and in in vitro studies as a cell scaffold, and thus could be an appropriate carrier to enhance cell retention in the repair site. We hypothesize that re-differentiated chondrocytes in fibrin gel will form cartilage tissue in vitro and will form hyaline cartilage when implanted into critical size osteochondral defects in rabbits. 

Methods: Rabbit or human chondrocytes were serially passaged twice (P2) in monolayer culture. P2 cells were used directly (de-differentiated control) or re-differentiated in high density culture with TGFβ3. Cells were mixed with fibrin gel (1.4 x106/ 20uL) to form fibrin clots which were cultured in vitro to assess the use of fibrin gel as a scaffold. Additionally, fibrin clots were formed with autologous rabbit chondrocytes (de-differentiated or re-differentiated) and implanted in critically sized osteochondral defects in NZW rabbit knee joints. Rabbits were sacrificed 6 weeks post-implantation. Tissues were assessed histologically and by immunohistochemistry. Tissue was graded using ICRS-II scale. 

Results: TGFβ3 enhanced the formation of cartilaginous tissues by both human and rabbit passaged chondrocytes in vitro. The cartilage tissue was rich in aggrecan and collagen type 2 (Col2) and the re-differentiated phenotype was retained when cells were cultured in fibrin gel. For human chondrocytes, re-differentiation reduced the accumulation of the fibrocartilage marker collagen type 1 (Col1), and was necessary to promote accumulation of the hyaline marker Col2 in the in vitro formed tissues. Analysis at six weeks post-implantation showed that implants containing de-differentiated rabbit chondrocytes resulted in fibrocartilaginous repair, the standard outcome of ACI. Implantation of re-differentiated chondrocytes resulted in granulation tissues. Immunohistochemistry demonstrated that these tissues contained Col1, Col2, and aggrecan. Tissues had similarly low scores by ICRS-II grading indicating poor repair in both groups. 

Conclusions: In vitro experiments showed that re-differentiated human and rabbit chondrocytes maintain their chondrogenic differentiation in fibrin gel. This confirms that fibrin is a suitable scaffold for cell delivery. Unexpectedly, when implanted re-differentiated chondrocytes show a different reparative response than de-differentiated chondrocytes and they do not appear to enhance repair at early time point. The reason for this is not clear but given the presence of Col2 in the repair tissue another study of longer duration is required to determine if tissue maturation to hyaline cartilage will occur.