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.

Single walled carbon nanotube composites for bone tissue engineering.

The purpose of this study was to develop single walled carbon nanotubes (SWCNT) and poly lactic-co-glycolic acid (PLAGA) composites for orthopedic applications and to evaluate the interaction of human stem cells (hBMSCs) and osteoblasts (MC3T3-E1 cells) via cell growth, proliferation, gene expression, extracellular matrix production and mineralization. PLAGA and SWCNT/PLAGA composites were fabricated with various amounts of SWCNT (5, 10, 20, 40, and 100 mg), characterized and degradation studies were performed. Cells were seeded and cell adhesion/morphology, growth/survival, proliferation and gene expression analysis were performed to evaluate biocompatibility. Imaging studies demonstrated uniform incorporation of SWCNT into the PLAGA matrix and addition of SWCNT did not affect the degradation rate. Imaging studies revealed that MC3T3-E1 and hBMSCs cells exhibited normal, non-stressed morphology on the composites and all were biocompatible. Composites with 10 mg SWCNT resulted in highest rate of cell proliferation (p < 0.05) among all composites. Gene expression of alkaline phosphatase, collagen I, osteocalcin, osteopontin, Runx-2, and Bone Sialoprotein was observed on all composites. In conclusion, SWCNT/PLAGA composites imparted beneficial cellular growth capabilities and gene expression, and mineralization abilities were well established. These results demonstrate the potential of SWCNT/PLAGA composites for musculoskeletal regeneration and bone tissue engineering (BTE) and are promising for orthopedic applications.

Evaluation of a hydrogel-fiber composite for ACL tissue engineering.

The anterior cruciate ligament (ACL) is necessary for normal knee stability and movement. Unfortunately the ACL is also the most frequently injured ligament of the knee with severe disruptions requiring surgical intervention. In response to this, tissue engineering has emerged as an option for ACL replacement and repair. In this study we present a novel hydrogel-fibrous scaffold as a potential option for ACL replacement. The scaffold was composed of PLLA fibers, in a previously evaluated braid-twist structure, combined with a polyethylene glycol diacrylate (PEGDA) hydrogel to improve viscoelastic properties. Both hydrogel concentration (10%, 15%, and 20%) and amount of hydrogel (soaking the fibrous scaffold in hydrogel solution or encasing the scaffold in a block of hydrogel) were evaluated. It was found that the braid-twist scaffold had a greater porosity and larger number of pores above 100 µm than braided scaffolds with the same braiding angle. After testing for their effects on swelling, fiber degradation, and protein release, as well as viscoelastic and tensile testing (when combined with fibrous scaffolds), it was found that the composite scaffold soaked in 10% hydrogel had the best chemical release and mechanical properties. The optimized structure behaved similarly to natural ligament in tension with the addition of the hydrogel decreasing the ultimate tensile stress (UTS), but the UTS was still comparable to natural ACL. In addition, cellular studies showed that the hydrogel-PLLA fiber composite supported fibroblast growth.

Tissue engineering of the anterior cruciate ligament: the viscoelastic behavior and cell viability of a novel braid-twist scaffold.

The anterior cruciate ligament (ACL) is the most commonly injured ligament of the knee; it also contributes to normal knee function and stability. Due to its poor healing potential severe ACL damage requires surgical intervention, ranging from suturing to complete replacement. Current ACL replacements have a host of limitations that prevent their extensive use. Investigators have begun to utilize tissue-engineering techniques to create new options for ACL repair, regeneration and replacement. In this study we tested novel braid-twist scaffolds, as well as braided scaffolds, twisted fiber scaffolds and aligned fiber scaffolds, for use as ACL replacements composed of poly(L-lactic acid) fibers. Scaffolds were examined using stress relaxation tests, cell viability assays and scanning electron microscopy. The behaviors of the braid-twist scaffolds were modeled with Maxwell and quasi-linear viscoelastic (QLV) models. In stress relaxation tests, the braid-twist scaffolds behaved similarly to native ACL tissue, with final normalized stresses of 87% and 83% after an 8 N load. There was agreement between the experimental data and the Maxwell model when the model included an element for each structural element in the scaffold. There was also agreement between the experimental data and QLV model, scaffolds with similar braiding angles shared constants. In cell proliferation studies no differences were found between fibroblast growth on the braided scaffolds and the braid-twist scaffolds. SEM images showed the presence of new extracellular matrix. Data from this and previous tensile studies demonstrate that the braid-twist scaffold design may be effective in scaffolds for ACL tissue regeneration.

Tissue engineering of the anterior cruciate ligament using a braid-twist scaffold design.

The anterior cruciate ligament (ACL) is the most commonly injured intra-articular ligament of the knee. The insufficient vascularization of this tissue prevents it from healing completely after extreme tearing or rupture, creating a need for ACL grafts for reconstruction. The limitations of existing grafts have motivated the investigation of tissue-engineered ACL grafts. A successful tissue-engineered graft must possess mechanical properties similar to the ACL; to date no commercially available synthetic graft has achieved this. To accomplish this goal we have combined the techniques of polymer fiber braiding and twisting to design a novel poly L-lactic acid (PLLA) braid-twist scaffold for ACL tissue engineering. The scaffold is designed to accurately mimic the biomechanical profile and mechanical properties of the ACL. In this study, braid-twist scaffolds were constructed and compared to braided scaffolds and twisted fiber scaffolds. The addition of fiber twisting to the braided scaffold resulted in a significant increase in the ultimate tensile strength, an increase in ultimate strain, and an increase in the length of the toe region in these constructs over scaffolds that were braided. Based on the findings of this study, the braid-twist scaffold studied was found to be a promising construct for tissue engineering of the ACL.