Strain-Controlled Organ Culture of Intact Human Anterior Cruciate Ligaments – An Ex-vivo Model to Investigate Degenerative and Regenerative Approaches

Krismer, Anna; Geissberger, Christian Andreas; Thomi, Gierin Florence; Cabra, Romina Silvia; Kohl, Sandro; Ahmad, Sufian (2 March 2016). Strain-Controlled Organ Culture of Intact Human Anterior Cruciate Ligaments – An Ex-vivo Model to Investigate Degenerative and Regenerative Approaches (Unpublished). In: ORS Annual Meeting. Orlando. 02.03.2016.

ABSTRACT INTRODUCTION: Injuries of the anterior cruciate ligament (ACL) are among the most common ligament injuries.1 Current strategies used for ACL reconstruction are the local autograft, allograft and ligament prostheses. Graft implantation evaluated by long-term studies showed that the restoration of knee stability can be achieved in more than in 90% of cases. Nevertheless these grafts had some associated complications such as ligament laxity, donor site morbidity, and pathogen transfer. These unresolved challenges in treatment of ACL ruptures demanded the establishment of an in-vitro culture model for ACLs as a base for further research. The present study aims to establish an appropriate in-vitro culture model for human ACLs by investigating the general ability of ACL cells to maintain their phenotype in culture. Moreover, this study evaluates whether culturing ACLs in a strain- controlled dynamic loading bioreactor system is superior to a free-swelling static loading culture model. Now, there is increased interest in tissue-engineered solutions like mechanobiological models of ACL culture that allow testing of different regenerative approaches.
METHODS: A novel bioreactor to harbor and mechanically stimulate full human cruciate ligaments was designed and manufactured (Fig. 1A-D). Fresh and full ACLs with attached femoral and tibial bone were obtained from full-knee prosthesis surgery with written consent of the patients (ethically approved). Initial height was measured as shown in Fig. 1C. The ability of ACL cells to maintain their phenotype was analyzed by DNA content (Hoechst), by collagen content (hydroxy-proline = HYP assay), by cellular activity (resazurin red assay) and by reverse transcriptase polymerase chain reaction (RT-qPCR) to check for relative gene expression of ligament specific markers. Human ACLs were either cultured under static loading conditions in a free-swelling culture or under dynamic loading conditions using a custom-made bioreactor applying 7% dynamic strain with 0.2Hz and 7% strain (~±2mm amplitude) to the ligaments for 24h for 7 and 21 days (Fig. 1). ACL cell activity and viability was determined close to the tibial region (T), the mid region (M) and close to the femoral region (F) by performing a resazurin salt cell activity assay and investigating 3D stacks of confocal laser scanning microscopy (cLSM) on cells treated with a LIVE/DEAD staining kit.
RESULTS SECTION: Among the 3 different ACL zones of dynamically loaded ACLs, DNA content was somewhat higher in tissue close to the F zone than in the tissue close to the M and T zone (P = 0.59). There was no difference in cellular mitochondrial activity in ACL samples cultured under dynamic conditions compared to static conditions on day 1 (1481±415.4 vs. 844.7±155.8 RFU, respectively, P = 0.10, data not shown) as well as on day 7 (973.8±229.6 vs. 790.8±39.6 RFU, respectively, Fig. 1E, P = 0.70). There was a trend for higher mitochondrial activity after 21 days in the T Zone (Fig. 1E, P = 0.082). Cell viability in ACL cells cultured under dynamic loading compared to cells cultured under static loading was similar (78.8±19.5% vs. 76.6±3.4% respectively, t-test P = 0.4467, measured after 7 days, Fig. 1F,G). After 7 days of dynamic culturing, ACL-fibroblasts still expressed their ligament-specific genes (data not shown). Large differences in relative gene expression between each ACL sample were observed, indicating an inter- individual variability.
DISCUSSION: ACL cells are able to survive for 7 and 21 days and to maintain their phenotype in organ culture as shown by qPCR, although further studies are required. DNA and cellular activity revealed that the ACL is an inhomogenous tissue in terms of cell density and cell phenotypes.1 Culturing ACLs in a dynamic bioreactor system seems to be beneficial for cell viability and activity.
SIGNIFICANCE: A strength of this research is the use of intact primary biological samples of ACL with written consent of patients and has to be considered top relevant for clinical research. Future research will focus on in vitro degenerative rupture models and to test regenerative approaches.
REFERENCES: 1. Kiapour AM, Murray MM. 2014. Basic science of anterior cruciate ligament injury and repair. Bone Joint Res 3:20-31.
ACKNOWLEDGEMENTS: We thank Eva Roth for assistance with the biochemical assays. The machine shop of the ISTB manufactured the bioreactor. This project was partially funded by the Swiss Orthopedic Society grant No. S99083814080618560 to SS Ahmad.

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