Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

Summary

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.

Abstract

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.

Introduction

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% ^....

Protocol

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.

1. Administer general anesthesia and pre-operative antibiotics as per institution's protocol to the patient.
2. Apply tourniquet and inflate 100 mg above systolic blood pressure.
3. Situate the patient in the supine position. Make a horizontal anterolateral incision with knee in 30-45° flexion for a 5 mm portal and insert the arthroscopic trocar and camera into t....

Results

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.

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Petri dish	Fisher Scientific	08-757-103C
Phosphate Buffered Saline (PBS)	Fisher Scientific	BP3994
Collagenase	Gibco	17018-029	Store at 4 °C
DMEM/F-12	Cellgro	10-092-CV	Store at 4 °C. Warm in 37 °C water bath before use.
Fetal Bovine Serum (FBS)	Gibco	10082	Store at -80 °C
Penicillin/Streptomycin	Lonza	17-602E	Store at 4 °C
Centrifuge Tubes- 15 ml	Corning	430790
T-25 flasks	BD Falcon	3013
Trypsin-Versene mixture	Lonza	17-161E	Store at 4 °C. Warm in 37 °C 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 -80 °C
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 4 °C
Tween	Fisher Scientific	BP337-500
mouse Anti-β-actin antibody (1° Ab)	Sigma Aldrich	A5441	Store at -20 °C
goat-anti-mouse antibody (2° Ab)	Cell Signaling	4408	Store at -20 °C
Hoechst dye	Sigma Aldrich	14530	Store at -20 °C
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 12 mm 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 1 g i.v.
Arthroscopy trocar	Smith and Nephew
Arthroscopy Camera	Smith and Nephew
Arthroscopic grasper and bitter	Arthrex
4.5 mm shaver	Arthrex
Interference Screws	Arthrex		stainless steel screws
Sputter Coater	Polaron	E5400