A subscription to JoVE is required to view this content. Sign in or start your free trial.
For future applications as a patch to repair partial tears of the Anterior Cruciate Ligament (ACL), human ACL derived cells were isolated from tissue obtained during reconstructive procedures, expanded in vitro and grown on tissue engineered scaffolds. Cellular adhesion and morphology was then performed to confirm biocompatibility on scaffold surface.
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.
The anterior cruciate ligament (ACL) is a commonly injured intra-articular ligament of the knee. Approximately 200,000 (ACL) injuries are reported annually in the United States. Over 75% of patients experiencing ACL injury opt for orthopedic reconstructive surgery 1,2,3,4. Surgical intervention is often indicated due to an inherently poor healing potential3. ACL reconstruction is typically accomplished by means of an autograft or allograft tendon. Autograft and allograft represent the gold standard for reconstruction as they boast high success rates, and primary suture repair, the other treatment option, has shown failure rates of up to 94% 5,6,7.
Partial tears of the ACL represent 10% to 28% of all ACL tears8. In a prospective study, Noyes et al. estimated that 50% of patients with partial tears affecting more than half of the ACL progressed to complete ACL insufficiency after non-operative treatment9. Other studies report persistent instability and decreased function with fewer than 30% of patients able to return to their pre-injury activity level 9,10,11,12,13. Treatment options are limited and include conservative modalities, thermal shrinkage of remaining ACL, or ACL reconstruction. Recently, there has been increased interest in augmented primary repair. These techniques use biologics to enhance primary suture repairs14. Recent research has attempted to harvest mesenchymal-like hACL derived stem cells to circumvent graft limitations,31,32 but the validity and efficacy of these cells is still unknown. The ideal cell source for tissue engineering applications seems to be non-mesechymal hACL derived fibroblast cells.
Current research is focused on identifying a suitable matrix material and cell source for the engineered scaffold. It is standard procedure for a torn ACL to be discarded as surgical waste during reconstruction surgery, however, this damaged ligament may be a quality source for the acquisition of cells needed to develop and enhance an ideal tissue engineered ACL replacement. Our lab has developed a protocol for the in vitro expansion of these harvested hACL derived cells. Using an engineered 2D matrix infused with hACL derived cells, we have designed a patch that could potentially augment partial ACL repair and strengthen torn ligaments.
1. Surgical Retrieval
Note: An IRB approval was obtained to collect the ACL stump during ACL reconstruction surgery. IRB exemption was given as the ACL stump used were generally discarded as surgical waste. 20 patients were used to collect the ACL stump.
2. Tissue Digestion and hACL Isolation
3. Cellular Expansion, Freezing and Thawing
4. Fabrication of Tissue Engineered 2-D Poly Lactic-co-glycolic Acid (PLAGA) Scaffold
5. Scanning Electron Microscopy (SEM)
6. Immunofluorescence Staining
The working model for surgical retrieval, tissue digestion and isolation of the human Anterior Cruciate Ligament (hACL) derived cells is shown in Figure 1. The cells migrated from the explants and adhered to the T-25 flasks. These cells were cultured for 3 days and then were visualized under a light microscope (Figure 2). A confluent monolayer was obtained by day 7. The presence of healthy, viable cells indicated the successful retrieval and culture of hACL derived cells.
The primary objective of this hACL/2D scaffold study was to use the obtained cells in a patch to augment primary repair of partial ACL tears. Nonoperative management of partial ACL tears may include a short period of immobilization, bracing, a progressive rehabilitation program, and regular follow-up evaluations 13,16,17. However, many studies show that conservative treatment in athletes has been associated with poor results and failure. Buckley et al. evaluated 25 patients with partial ACL tears at i...
The authors declare that they have no competing financial interests.
The authors would like to acknowledge the start-up fund and department of surgery research grant from Southern Illinois University, School of Medicine; and the Memorial Medical Foundation grant.
Name | Company | Catalog Number | Comments |
Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
Petri-dish | Fisher Scientific | 08-757-103C | |
Phosphate Buffered Saline (PBS) | Fisher Scientific | BP3994 | |
Collagenase | Gibco | 17018-029 | Store at 40C |
DMEM/F-12 | Cellgro | 10-092-CV | Store at 40C. Warm in 370C water bath before use. |
Fetal Bovine Serum (FBS) | Gibco | 10082 | Store at -800C |
Penicillin/Streptomycin | Lonza | 17-602E | Store at 40C |
Centrifuge Tubes- 15 ml | Corning | 430790 | |
T-25 flasks | BD Falcon | 3013 | |
Trypsin-Versene mixture | Lonza | 17-161E | Store at 40C. Warm in 370C water bath before use. |
DMSO | Fisher Scientific | BP231-100 | Combustible liquid. Can cause skin, eye and respiratory tract irritation. |
Cryogenic vials | Corning | 430489 | |
PLAGA | Purac Biomaterials | Purasorb PLG8523 | Store at -800C |
TCPS disks | Fisher Scientific | 12-545-82 | |
Dichloromethane | Fisher Scientific | AC36423-0010 | Possible cancer hazard. Store in a dry, cool place. |
Bytac paper | Saint gobin performance plastics | 1420652 | |
Scintillation vial | Fisher Scientific | 03-339-21G | |
Bovine Serum Albumin (BSA) | Sigma Aldrich | A7906 | Store at 40C |
Tween | Fisher Scientific | BP337-500 | |
mouse Anti-β-actin antibody (10 Ab) | Sigma Aldrich | A5441 | Store at -200C |
goat-anti-mouse antibody (20 Ab) | Cell Signaling | 4408 | Store at -200C |
Hoechst dye | Sigma Aldrich | 14530 | Store at -200C |
Glycerol | Fisher Scientific | BP229-1 | |
Glutaraldehyde | Fisher Scientific | BP2547-1 | Toxic by inhalation and if swallowed. Causes burns by all exposure routes. |
Hexamethyldisilazane | Fisher Scientific | AC43085-1000 | Flammable liquid and vapor. Causes burns by all exposure routes. |
Sterile Scissors | McKesson | 25-716 | |
Centrifuge | Eppendorf | 5804R | |
Light Microscope | Olympus | CK40 | |
Water Bath | Thermo scientific | 2845 | |
Vortex | Labnet | VX-200 | |
Glass Petri plates | fisher Scientific | S31473 | |
Acu-Punch | Acuderm Inc. | P1225 | Acu-Punch was used to cut 12mm disks |
Cacodylate buffer | Sigma Aldrich | 97068 | Flammable liquid, carcinogen and irritant. |
Osmium tetraoxide | Sigma Aldrich | 201030 | Highly toxic |
Triton X-100 | Sigma Aldrich | T8787 | Harmful if swallowed |
Ethanol | Decon Labs | 2705 | Keep away from heat, sparks, flame and other form of ignition. |
General/regional Anesthesia | Amphastar pharmaceuticals | 1% Lidocaine, 0.25% Bupivacaine,Anesthetic agents for induction and maintenance | |
Antibiotics | Hospira | 0409-0805-01 | Ancef 1g i.v. |
Arthroscopy trocar | Smith and Nephew | ||
Arthroscopy Camera | Smith and Nephew | ||
Arthroscopic grasper and bitter | Arthrex | ||
4.5mm shaver | Arthrex | ||
Interference Screws | Arthrex | stainless steel screws | |
Sputter Coater | Polaron | E5400 |
Request permission to reuse the text or figures of this JoVE article
Request PermissionThis article has been published
Video Coming Soon
Copyright © 2025 MyJoVE Corporation. All rights reserved