Anterior Cruciate Ligament Tissue Engineering Research Articles

Distinctive collagen maturation process in fibroblasts derived from rabbit anterior cruciate ligament, medial collateral ligament, and patellar tendon in vitro.

PurposeDifferences in the tissue-specific collagen maturation process between tendon and ligament are still unknown. Collagen cross-link formation is crucial for the collagen maturation process. The aim of this study is to examine collagen maturation processes of anterior cruciate ligament (ACL), medial collateral ligament (MCL), and patellar tendon (PT) in vitro, in order to determine the optimal cell source for tissue engineering of ligament.MethodsCells derived from the ACL, MCL, and PT of New Zealand white rabbits were isolated. Each cell type was cultured for up to 4 weeks after reaching confluence. Cell–matrix layers were evaluated and compared for their morphology, collagen cross-links, and gene expression levels of lysine hydroxylase 1 and 2, lysyl oxidase (LOX), tenomodulin, collagen1A1 (Col1A1), and collagen3A1 (Col3A1).ResultsTransmission electron microscopy photomicrographs verified that collagen fibrils were secreted from all three types of fibroblasts. A higher ratio of dihydroxylysinonorleucine/hydroxylysinonorleucine was evident in the ligament compared to the tendon, which was consistent with lysine hydroxylase 2/lysine hydroxylase 1 gene expression. The gene expression of LOX, which regulates the total amount of enzymatic cross-linking, and the gene expression levels of Col1A1 and Col3A1 were higher in the ACL matrix than in the MCL and PT matrices.ConclusionACL, MCL, and PT cells have distinct collagen maturation processes at the cellular level. In addition, the collagen maturation of ACL cells is not necessarily inferior to that of MCL and PT cells in that all three cell types have a good ability to synthesize collagen and induce collagen maturation. This bioactivity of ACL cells in terms of ligament-specific mature collagen induction can be applied to tissue-engineered ACL reconstruction or remnant preserving procedure with ACL reconstruction.

Current tissue engineering strategies in anterior cruciate ligament reconstruction

Rupture of the anterior cruciate ligament (ACL) is one of the most common ligamentous injuries of the knee. Limitations of allografts and autografts in ACL reconstruction as well as recent advancements in biology and materials science have spurred interest in developing tissue-engineered ACL replacements that have the potential to mimic the native ACL in terms of both biological and mechanical properties. This article reviews the current literature regarding contemporary tissue engineering strategies. The four basic components of tissue engineering, biomaterial scaffolds, cell sources, growth factors, and mechanical stimuli, as applied to the development of tissue-engineered ACL replacement grafts, will be systematically addressed. In addition, animal models that have been used to test these tissue-engineered ACL replacements will also be reviewed. To date, there is no tissue-engineered ACL construct that has been successfully implanted in humans. We expect that continued progress in designing a viable tissue-engineered ACL replacement will accompany rapidly advancing techniques in materials science and biology.

Wired silk architectures provide a biomimetic ACL tissue engineering scaffold

Silk has been increasingly investigated as a scaffold for tissue-engineered anterior cruciate ligament (ACL) grafts, primarily due to a uniquely advantageous combination of biocompatibility and robust biomechanical strength in the short and middle terms. While previous studies have explored the biomechanical and biological effects of graft geometry, these studies have largely ignored the effects of repeated loading on long term biomechanical performance—an important consideration considering the relatively slow rate with which the silk scaffold is remodeled. In the present study, we utilized a tensile bioreactor to carry out cyclic loading tests on various silk ACL scaffold designs. Silk scaffolds were fabricated with three different architectures (wired, braided, and straight fibered). These were tested in static loading, low cyclic loading to 250 cycles, and high cyclic loading to 100,000 cycles. Different scaffold conditions including dry, wet, with cells, without seeded cells were tested and compared. The ultimate tensile strength (UTS), linear stiffness and construct elongation rate were used to compare the structural behavior of each graft architecture. Based upon this analysis, silk scaffolds with a wired structure exhibited biomechanical behavior most similar to the native human ACL. We thus conclude that the wired silk scaffold design we present provides a biofidelic mechanical basis for tissue engineering strategies for ACL reconstruction.

Modulation of cell attachment and collagen production of anterior cruciate ligament cells via submicron grooves/ridges structures with different cell affinity

This study aimed to investigate the effects of submicron-grooved topography and surface cell affinity on the attachment, proliferation and collagen synthesis of anterior cruciate ligament (ACL) cells. Two grooved polystyrene (PS) surfaces (equal groove/ridge width of 800 nm) with a groove depth of 100 or 700 nm were fabricated and modified by oxygen plasma treatment, dopamine deposition and conjugation of RGD-containing peptides to enhance cell affinity. The elongation and alignment of ACL cells was enhanced by grooved structures with increasing groove depths regardless of surface chemistry. On the other hand, cell spreading and proliferation mainly depended on surface chemistry, in accordance with surface cell affinity: O(2) plasma < dopamine deposition < RGD conjugation. The synthesis of type I collagen was the highest by the ACL cells cultured on the 700 nm grooved surface conjugated with RGD peptides, indicating that both surface grooved topography and chemistry play a role in modulating collagen production of ACL cells. Furthermore, the type I collagen deposited on the 700 nm PS surface was aligned with grooves/ridges. Our results indicated that both ligand presentation and cell alignment are important in the physiological activities of ACL fibroblasts. Such information is critical for design of biomaterials for ACL tissue engineering.

Most British Surgeons Would Consider Using a Tissue-Engineered Anterior Cruciate Ligament: A Questionnaire Study

Donor site morbidity, poor graft site integration, and incorrect mechanical performance are all common problems associated with autografts for anterior cruciate ligament (ACL) reconstructions. A tissue-engineered (TE) ligament has the potential to overcome these problems. We produced an online questionnaire relating to tissue engineering of the ACL to obtain input from practising clinicians who currently manage these injuries. 300 British orthopaedic surgeons specialising in knee surgery and soft tissue injury were invited to participate. 86% of surgeons would consider using a TE ACL if it were an option, provided that it showed biological and mechanical success, if it significantly improved the patient satisfaction (63%) or shortened surgical time (62%). 76% felt that using a TE ACL would be more appropriate than a patellar tendon, hamstring, or quadriceps autograft. Overall, most surgeons would be prepared to use a TE ACL if it were an improvement over the current techniques.

Stem Cell and Tissue Engineering Applications in Orthopaedics and Musculoskeletal Medicine

Stem Cell and Tissue Engineering Applications in Orthopaedics and Musculoskeletal Medicine

Prediction of the morphological and mechanical properties of a novel scaffold for anterior cruciate ligament tissue engineering

"Prediction of the morphological and mechanical properties of a novel scaffold for anterior cruciate ligament tissue engineering." Computer Methods in Biomechanics and Biomedical Engineering, 14(sup1), pp. 45–46Keywords: tissue engineeringanterior cruciate ligamentscaffold designfinite element simulationmorphological characterisation

Morphological Characterization of a Novel Scaffold for Anterior Cruciate Ligament Tissue Engineering

Tissue engineering offers an interesting alternative to current anterior cruciate ligament (ACL) surgeries. Indeed, a tissue-engineered solution could ideally overcome the long-term complications due to actual ACL reconstruction by being gradually replaced by biological tissue. Key requirements concerning the ideal scaffold for ligament tissue engineering are numerous and concern its mechanical properties, biochemical nature, and morphology. This study is aimed at predicting the morphology of a novel scaffold for ligament tissue engineering, based on multilayer braided biodegradable copoly(lactic acid-co-(e-caprolactone)) (PLCL) fibers The process used to create the scaffold is briefly presented, and the degradations of the material before and after the scaffold processing are compared. The process offers varying parameters, such as the number of layers in the scaffold, the pitch length of the braid, and the fibers' diameter. The prediction of the morphology in terms of pore size distribution and pores interconnectivity as a function of these parameters is performed numerically using an original method based on a virtual scaffold. The virtual scaffold geometry and the prediction of pore size distribution are evaluated by comparison with experimental results. The presented process permits creation of a tailorable scaffold for ligament tissue engineering using basic equipment and from minimum amounts of raw material. The virtual scaffold geometry closely mimics the geometry of real scaffolds, and the prediction of the pore size distribution is found to be in good accordance with measurements on real scaffolds. The scaffold offers an interconnected network of pores the sizes of which are adjustable by playing on the process parameters and are able to match the ideal pore size reported for tissue ingrowth. The adjustability of the presented scaffold could permit its application in both classical ACL reconstructions and anatomical double-bundle reconstructions. The precise knowledge of the scaffold morphology using the virtual scaffold will be useful to interpret the activity of cells once it will be seeded into the scaffold. An interesting perspective of the present work is to perform a similar study aiming at predicting the mechanical response of the scaffold according to the same process parameters, by implanting the virtual scaffold into a finite element algorithm.

Potential of Skin Fibroblasts for Application to Anterior Cruciate Ligament Tissue Engineering

Fibroblasts isolated from skin and from anterior cruciate ligament (ACL) secrete type I and type III collagens in vivo and in vitro. However, it is much easier and practical to obtain a small skin biopsy than an ACL sample to isolate fibroblasts for tissue engineering applications. Various tissue engineering strategies have been proposed for torn ACL replacement. We report here the results of the implantation of bioengineered ACLs (bACLs), reconstructed in vitro using a type I collagen scaffold, anchored with two porous bone plugs to allow bone-ligament-bone surgical engraftment. The bACLs were seeded with autologous living dermal fibroblasts, and grafted for 6 months in goat knee joints. Histological and ultrastructural observations ex vivo demonstrated a highly organized ligamentous structure, rich in type I collagen fibers and cells. Grafts' vascularization and innervation were observed in all bACLs that were entirely reconstructed in vitro. Organized Sharpey's fibers and fibrocartilage, including chondrocytes, were present at the osseous insertion sites of the grafts. They showed remodeling and matrix synthesis postimplantation. Our tissue engineering approach may eventually provide a new solution to replace torn ACL in humans.

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) hydrogelto 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 100μm 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.

Comparison of Potentials Between Stem Cells Isolated from Human Anterior Cruciate Ligament and Bone Marrow for Ligament Tissue Engineering

We have previously isolated and identified stem cells from human anterior cruciate ligament (ACL). The purpose of this study was to evaluate the differences in proliferation, differentiation, and extracellular matrix (ECM) formation abilities between bone marrow stem cells (BMSCs) and ACL-derived stem cells (LSCs) from the same donors when cultured with different growth factors, including basic fibroblast growth factor (bFGF), epidermal growth factor, and transforming growth factor-beta 1 (TGF-beta1). Ligament tissues and bone marrow aspirate were obtained from patients undergoing total knee arthroplasty and ACL reconstruction surgeries. Proliferation, colony formation, and population doubling capacity as well as multilineage differentiation potentials of LSCs and BMSCs were compared. Gene expression and ECM production for ligament engineering were also evaluated. It was found that BMSCs possessed better osteogenic differentiation potential than LSCs, while similar adipogenic and chondrogenic differentiation abilities were observed. Proliferation rates of both LSCs and BMSCs were enhanced by bFGF and TGF-beta1. TGF-beta1 treatment significantly increased the expression of type I collagen, type III collagen, fibronectin, and alpha-smooth muscle actin in LSCs, but TGF-beta1 only upregulated type I collagen and tenascin-c in BMSCs. Protein quantification further confirmed the results of differential gene expression and suggested that LSCs and BMSCs increase ECM production upon TGF-beta1 treatment. In summary, in comparison with BMSCs, LSCs proliferate faster and maintain an undifferentiated state with bFGF treatment, whereas under TGF-beta1 treatment, LSCs upregulate major tendinous gene expression and produce a robust amount of ligament ECM protein, making LSCs a potential cell source in future applications of ACL tissue engineering.

The Future: Optimizing the Healing Environment in Anterior Cruciate Ligament Reconstruction

The therapeutic use of autologous platelet-rich plasma constitutes a breakthrough in the stimulation and acceleration of soft-tissue healing and bone regeneration. Platelet-rich technologies seek to facilitate anterior cruciate ligament replacement by mimicking the native tissue and improving the adequacy of tissue function with appropriate cues, ultimately leading to better patient care. There are different technical protocols for preparing platelet-rich plasma, and the resultant products are typically heterogeneous; moreover, protocols for administrating the products in patients vary extensively. Poor standardization in the field makes full evaluation of different plasma products and establishing standards for the most beneficial applications of this technology difficult. This article presents the current data on the use of platelet-rich plasma in the reconstruction of the anterior cruciate ligament. Although the findings are not conclusive, the use of autologous platelet-rich plasma is shown to be safe, reproducible, and effective in mimicking the natural processes of soft tissue and bone healing. Platelet-rich technologies offer new opportunities for research and the application of anterior cruciate ligament tissue engineering.

The phenotypic responses of human anterior cruciate ligament cells cultured on poly(ϵ‐caprolactone) and chitosan

The purpose of this study is to evaluate the phenotypic responses of human anterior cruciate ligament (ACL) cells on two biodegradable materials: poly(epsilon-caprolactone) (PCL) and chitosan. ACL cells cultured on PCL displayed phenotypes that were well spread with a developed cytoskeleton. In comparison, chitosan was not an appropriate substrate to support the attachment and spreading of ACL cells, which was attributed to the low fibronectin (FN) adsorption of chitosan. However, ACL cells cultured on chitosan exhibited a dramatic effect on increasing transcripts of transforming growth factor beta1 (TGF beta1) and collagen III. After coating FN on chitosan surface, cell morphology and the mRNA levels of all tested genes had the similar levels on PCL and FN-coated chitosan. This indicates the expression of TGF beta1 and collagen III mRNA of human ACL cells was seem to correlate closely with the adhesion behavior of human ACL cells and was influenced by the underlying substrate properties. Since an ideal scaffold used in ACL tissue engineering is not only for cell attachment but also for extracellular matrix deposition during ligament regeneration, chitosan may be considered as a scaffold for ACL tissue engineering, which can upregulate the expression of specific genes of matrix production and wound healing in human ACL cells to synthesize more quantity of FN and TGF beta1 proteins.

TRITON-X is most effective among three decellularization agents for ACL tissue engineering.

Ruptures of the anterior cruciate ligament (ACL) are still associated with high rates of long-term complications, even in patients undergoing modern, state-of-the-art replacement. Tissue-engineering approaches have been shown to be of value in improving treatment of ACL ruptures. However, the success of tissue-engineering procedures depends on the choice of an appropriate biomaterial. Decellularized ACL tissue potentially combines the structural composition of the targeted tissue with a reduced risk of graft rejection or disease transmission. In this study, we tested the effectiveness of currently available decellularization methods based on TRITON-X, sodium dodecyl sulfate (SDS), and trypsin. After identifying the most effective decellularization method, the capacity for reseeding with ACL fibroblasts was studied. All decellularization protocols reduced DNA content, with TRITON-X treatment having the greatest effect. Concurrently, decellularization did not affect tissue collagen or total protein content, but did decrease glycosaminoglycan content. TRITON-X also resulted the least glycosaminoglycan depletion. Porcine ACL tissue after decellularization with TRITON-X could be successfully reseeded with human ACL fibroblasts as demonstrated by steady DNA content and increasing pro-collagen expression.

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.

The differentiation of mesenchymal stem cells by mechanical stress or/and co-culture system

Differentiation of mesenchymal stem cells (MSCs) into anterior cruciate ligament (ACL) cells is regulated by many factors. Mechanical stress affects the healing and remodeling process of ACL after surgery in important ways. Besides, co-culture system had also showed the promise to induce MSCs toward different kinds of cells on current research. The purpose of this study was to investigate the gene expression of ACL cells’ major extracellular matrix (ECM) component molecules of MSCs under three induction groups. In addition, to follow our previous study, cell electrophoresis technique and mRNA level gene expression of MSC protein were also used to analyze the differentiation of MSCs. The results reveal that specific regulatory signals which released from ACL cells appear to be responsible for supporting the selective differentiation toward ligament cells in co-culture system and mechanical stress promotes the secretion of key ligament ECM components. Therefore, the combined regulation could assist the development of healing and remolding of ACL tissue engineering. Furthermore, this study also verifies that cell electrophoresis could be used in investigation of cell differentiation. Importantly, analysis of the data suggests the feasibility of utilizing MSCs in clinical applications for repairing or regenerating ACL tissue.

Evaluation of the anterior cruciate ligament, medial collateral ligament, achilles tendon and patellar tendon as cell sources for tissue-engineered ligament

This study investigated four different connective tissue cell types to determine which cell type should be the source for seeding a tissue-engineered anterior cruciate ligament (ACL) replacement. Cells derived from the ACL, medial collateral ligament (MCL), achilles tendon (AT), and patellar tendon (PT) of New Zealand White rabbits were isolated and cultured. Each cell type was cultured in vitro after seeding on three-dimensional (3-D) braided polymer scaffolds and on tissue culture polystyrene that served as a control. Samples were evaluated and compared for their morphology, proliferation, and gene expression of fibronectin, type I and type III collagen. Scanning electron microscopy (SEM) photomicrographs verified cell attachment of all four types of connective tissue fibroblasts to the scaffolds. Preliminary results comparing proliferation indicate that cells obtained from the PT and AT have the fastest proliferation. Whereas gene expression of the phenotypic markers measured using real-time reverse transcription polymerase chain reaction (RT-PCR) indicates ACL cells have the highest gene expression for the matrix markers. This leads to the question of which cell type should be the cell source for tissue-engineering of ligament, the highly proliferating cells or the differentiated matrix producing cells. This study would suggest that ACL differentiated matrix producing cells are the most suitable cells for further study and development of a tissue-engineered ligament.

The Effects of Bone Marrow-Derived Mesenchymal Stem Cells and Fascia Wrap Application to Anterior Cruciate Ligament Tissue Engineering

After an anterior cruciate ligament (ACL) injury, surgical reconstructions are necessary in most cases, either with autografts, allografts, or artificial ligaments. Potential tissue-engineered ligaments would circumvent the disadvantages apparent in these methods. While seeding of mesenchymal stem cells (MSCs) and fascia wrap could potentially improve tissue regeneration and mechanical properties, their exact roles were evaluated in the current study. Knitted biodegradable scaffolds of poly-L-lactic acid (PLLA) and poly-glycolic-lactic acid (PGLA) yarns were used to reconstruct ACL in 48 rabbits. These were divided into four equal groups: only knitted scaffolds were used in group I; knitted scaffolds and mesenchymal stem cells were used in group II; knitted scaffolds, MSCs, and fascia lata were used in group III; knitted scaffolds and fascia lata were used in group IV. Carboxyfluorescein diacetate (CFDA)-labeled MSCs were used to trace the fate of seeded cells in groups II and III. Histology, Western blot analysis, and mechanical properties of reconstructed ACL were analyzed after 20 weeks. Fibroblast ingrowths were seen in all four groups while CFDA-labeled MSCs could be found after 8 weeks of implantation in groups II and III. Both the amount of collagen type I and collagen type III in groups III and IV were significantly higher than in group II, which was much higher than in group I. Both maximal tensile loads and stiffness of the reconstructed ACLs in groups I, II, III, and IV were significantly lower than normal controls after 20 weeks of implantation. It is concluded that MSCs could promote synthesis of collagen type I and collagen type III in tissue-engineered ligaments, while fascia wraps have stronger effects. Both MSC seeding and fascia wrap could not enhance ultimate tensile load and stiffness.

Selection of Cell Source for Ligament Tissue Engineering

Use of appropriate types of cells could potentially improve the functionality and structure of tissue engineered constructs, but little is known about the optimal cell source for ligament tissue engineering. The object of this study was to determine the optimal cell source for anterior cruciate ligament (ACL) tissue engineering. Fibroblasts isolated from anterior cruciate ligament, medial collateral ligament (MCL), as well as bone marrow mesenchymal stem cells (MSC) were compared using the following parameters: proliferation rate, collagen excretion, expression of collagen type I, II, and III, as well as alpha-smooth muscle actin. Green fluorescent protein (GFP) transfected MSCs were used to trace their fate in the knee joints. MSC, ACL, and MCL fibroblasts were all highly stained with antibodies for collagen types I and III and alpha-smooth muscle actin while negatively stained with collagen type II. Proliferation rate and collagen excretion of MSCs were higher than ACL and MCL fibroblasts (p < 0.05), and MSCs could survive for at least 6 weeks in knee joints. In summary, MSC is potentially a better cell source than ACL and MCL fibroblasts for anterior cruciate ligament tissue engineering.

Anterior cruciate ligament regeneration using braided biodegradable scaffolds: in vitro optimization studies

The anterior cruciate ligament (ACL) is the most commonly injured intra-articular ligament of the knee, and limitations in existing reconstruction grafts have prompted an interest in tissue engineered solutions. Previously, we reported on a tissue-engineered ACL scaffold fabricated using a novel, three-dimensional braiding technology. A critical factor in determining cellular response to such a graft is material selection. The objective of this in vitro study was to optimize the braided scaffold, focusing on material composition and the identification of an appropriate polymer. The selection criteria are based on cellular response, construct degradation, and the associated mechanical properties. Three compositions of poly- α-hydroxyester fibers, namely polyglycolic acid (PGA), poly- L-lactic acid (PLLA), and polylactic- co-glycolic acid 82:18 (PLAGA) were examined. The effects of polymer composition on scaffold mechanical properties and degradation were evaluated in physiologically relevant solutions. Prior to culturing with primary rabbit ACL cells, scaffolds were pre-coated with fibronectin (Fn, PGA-Fn, PLAGA-Fn, PLLA-Fn), an important protein which is upregulated during ligament healing. Cell attachment and growth were examined as a function of time and polymer composition. While PGA scaffolds measured the highest tensile strength followed by PLLA and PLAGA, its rapid degradation in vitro resulted in matrix disruption and cell death over time. PLLA-based scaffolds maintained their structural integrity and exhibited superior mechanical properties over time. The response of ACL cells was found to be dependent on polymer composition, with the highest cell number measured on PLLA-Fn scaffolds. Surface modification of polymer scaffolds with Fn improved cell attachment efficiency and effected the long-term matrix production by ACL cells on PLLA and PLAGA scaffolds. Therefore based on the overall cellular response and its temporal mechanical and degradation properties in vitro, the PLLA braided scaffold pre-coated with Fn was found to be the most suitable substrate for ACL tissue engineering.