Surgical retrieval, isolation and in vitro expansion of human anterior cruciate ligament-derived cells for tissue engineering applications.
Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical deficiencies that impair function and stability. Current options for the treatment of partial ACL tears range from nonoperative, conservative management to multiple surgical options, such as: thermal modification, single-bundle repair, complete reconstruction, and reconstruction of the damaged portion of the native ligament. Few studies, if any, have demonstrated any single method for management to be consistently superior, and in many cases patients continue to demonstrate persistent instability and other comorbidities. The goal of this study is to identify a potential cell source for utilization in the development of a tissue engineered patch that could be implemented in the repair of a partially torn ACL. A novel protocol was developed for the expansion of cells derived from patients undergoing ACL reconstruction. To isolate the cells, minced hACL tissue obtained during ACL reconstruction was digested in a Collagenase solution. Expansion was performed using DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). The cells were then stored at -80 ºC or in liquid nitrogen in a freezing medium consisting of DMSO, FBS and the expansion medium. After thawing, the hACL derived cells were then seeded onto a tissue engineered scaffold, PLAGA (Poly lactic-co-glycolic acid) and control Tissue culture polystyrene (TCPS). After 7 days, SEM was performed to compare cellular adhesion to the PLAGA versus the control TCPS. Cellular morphology was evaluated using immunofluorescence staining. SEM (Scanning Electron Microscope) micrographs demonstrated that cells grew and adhered on both PLAGA and TCPS surfaces and were confluent over the entire surfaces by day 7. Immunofluorescence staining showed normal, non-stressed morphological patterns on both surfaces. This technique is promising for applications in ACL regeneration and reconstruction.
Department of Medical Microbiology, Immunology & Cell Biology, Southern Illinois University School of Medicine.
Ashim
A
Gupta
Division of Orthopaedics and Rehabilitation, Department of Surgery, Southern Illinois University School of Medicine.
Kevin
K
Sharif
Division of Orthopaedics and Rehabilitation, Department of Surgery, Southern Illinois University School of Medicine.
Megan
M
Walters
Department of Medical Microbiology, Immunology & Cell Biology, Southern Illinois University School of Medicine.
Mia D
MD
Woods
Division of Orthopaedics and Rehabilitation, Department of Surgery, Southern Illinois University School of Medicine.
Anish
A
Potty
University of Illinois at Springfield.
Benjamin J
BJ
Main
Department of Medical Microbiology, Immunology & Cell Biology, Southern Illinois University School of Medicine; Division of Orthopaedics and Rehabilitation, Department of Surgery, Southern Illinois University School of Medicine; Department of Electrical and Computer Engineering, Biomedical Engineering Program, Southern Illinois University Carbondale; sel-amin@siumed.edu.
Saadiq F
SF
El-Amin
D000818:Animals; D016118:Anterior Cruciate Ligament / Q000166:cytology* / Q000601:surgery*; D059549:Anterior Cruciate Ligament Reconstruction / Q000379:methods; D002417:Cattle; D002448:Cell Adhesion / Q000502:physiology; D006801:Humans; D019344:Lactic Acid / Q000737:chemistry; D008856:Microscopy, Fluorescence / Q000379:methods; D011100:Polyglycolic Acid / Q000737:chemistry; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D023822:Tissue Engineering / Q000379:methods*; D054457:Tissue Scaffolds
D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D011100:Polyglycolic Acid; D019344:Lactic Acid