Introduction

Anterior cruciate ligament (ACL) rupture is a common injury 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 chapter reviews current strategies that surround the development of a tissue-engineered ACL substitute and explores the limitations as well as future direction of these technologies. The four basic components of tissue engineering, biomaterial scaffolds, cell sources, growth factors, and mechanical stimuli, will be addressed. In addition, animal models that have been used to evaluate these tissue-engineered ACL replacements will also be reviewed.

ACL reconstruction is a common surgical procedure that is necessary because of the inherent inability of a ruptured ACL to heal. Current treatment consists of reconstruction with autograft or allograft rather than primary repair. While autografts have been successfully employed, the chief drawback is donor site morbidity, which can result in pain, infrapatellar contracture, tendonitis, weakness, and patella fracture. Additionally, in multiligamentous knee injury, recurrent injury, or revision surgery, autograft may be unavailable. The use of allograft can circumvent most of the these issues, but can be associated with limited supply, delayed biological incorporation, and the risk of disease transmission. Also, allografts are associated with a higher re-rupture rate, particularly in younger, more active patients.

Due to these limitations, there has been interest in developing synthetic ACL substitutes since the early 1970s. Nonbiodegradable synthetic grafts such as Proplast, Dacron, and GORE-TEX have all been previously evaluated for ACL reconstruction. While these grafts had sufficient initial strength, all had complications with particulate wear debris, the associated synovitis, and ultimately graft failure. Currently, there are no synthetic replacements for primary ACL reconstruction approved for clinical use in the United States.

Recent advances in biology and materials science have prompted researchers to develop a tissue-engineered ligament substitute. Tissue engineering is a multidisciplinary field that incorporates principles of engineering, biology, and materials science to replace or regenerate tissues in order to restore normal function. This field was popularized by Langer and Vacanti in the late 1980s, and operates under the paradigm of four basic components: a structural scaffold, a cell source, biologic modulators, and mechanical modulators. In orthopaedics, tissue engineering techniques have been applied to developing therapies for cartilage loss and bony nonunion, as well as tendon and ligament insufficiency. The ACL’s unique mechanical properties and inability to heal are the major challenges that have limited the development of tissue-engineered solutions to ACL rupture.

Biomaterial Scaffolds

The ideal scaffold for a tissue-engineered ACL graft should be biocompatible and possess mechanical strength comparable to the native ACL. Additionally, it should be biodegradable to allow for tissue ingrowth and facilitate cellular processes required for the regeneration of the new ACL. Many biomaterials have been evaluated, including biologic materials, biodegradable polymers, and composite materials.

Since type I collagen constitutes roughly 90% of the native ACL, collagen-based scaffolds have been extensively studied. In the 1990s, Dunn et al. assembled extruded collagen fibrils into scaffolds and were able to demonstrate rabbit ACL fibroblast adherence and viability in vitro and in vivo that were completely resorbed in 6 weeks. Goulet et al. showed a decrease in mechanical strength over time in collagen scaffolds seeded with caprine ACL fibroblasts. Collagen-glycosaminoglycan and collagen-elastin composite scaffolds have demonstrated the ability to support cell growth and elaboration of fibroblast markers, but require further characterization of mechanical properties. Cross-linking of collagen with ultraviolet light or chemical reagents and a braid-twist scaffold design have been shown to result in improved but still less than desired mechanical properties of the scaffolds. The inadequate mechanical properties, the immunogenicity associated with bovine collagen, and concerns regarding leaching of chemical cross-linking agents have spurred investigation of scaffold materials with more favorable properties than collagen.

Silk is a biocompatible biologic material that has long been used as suture material and has been studied as a scaffold material for ligament tissue engineering, as its tensile strength is similar to native ACL. Additionally, silk is biodegradable, loses its tensile strength in 1 year, and undergoes complete proteolytic degradation within 2 years in vivo. Altman et al. developed a silk scaffold composed of processed fibers woven into a wire-rope geometry, which has demonstrated promise for a ligament replacement graft. This silk scaffold has a hierarchical structure with silk fibers wound into strands and then twisted into cords and arranged into a three-dimensional matrix. This construct has mechanical properties similar to native ACL and also demonstrates appropriate viscoelastic properties. Silk scaffolds have also been shown to support human bone marrow stromal cell attachment as well as proliferation in a three-dimensional environment and synthesis of fibroblastic markers with the application of dynamic mechanical loading. A silk scaffold with tricalcium phosphate–based anchors for bony integration has shown comparable mechanical properties to allograft and autograft at 3 and 6 months postoperatively in a porcine model.

Other biologic materials such as hyaluronic acid, chitosan, and alginate have also been investigated. To address some of the inherent weaknesses of these biological materials, investigators have developed different modification methods as well as formed various composites. While these biologic materials and their composites present interesting possibilities, further studies are necessary to define their potential use in ligament tissue engineering.

Unlike nondegradable synthetic ACL replacements, the use of synthetic biodegradable polymers has shown great promise in ACL tissue engineering. Many of these polymers are currently approved by the US Food and Drug Administration for other surgical applications. Polymer selection and scaffold fabrication techniques can allow customization of characteristics such as structure, mechanical properties, degradation rate, and cellular response.

Various methods of manufacturing, processing, and assembling porous scaffolds for use in tissue engineering have been proposed, including braiding, drawing, phase separation, freeze drying, molecular self-assembly, and electrospinning. Sung and colleagues demonstrated fibroblast attachment and proliferation on scaffolds composed of braided poly glycolic acid (PGA) sutures coated with polycaprolactone (PCL). Later, a braided polydioxanone construct was evaluated in a caprine model but was found to be unsuitable due to the rapid loss of mechanical properties. Lu et al. compared braided scaffolds composed of poly l -lactic acid (PLLA), PGA, and, polylactic-co-glycolic acid (PLGA). They concluded that PLLA scaffolds supported the highest rates of ACL fibroblast proliferation and had the most favorable mechanical properties for an ACL substitute. PLLA also has a relatively slow degradation rate, while exhibiting greater strength than PLGA. Laurencin and colleagues developed a three-dimensional, braided fibronectin-coated PLLA scaffold, with distinct regions for the bony insertions and the intra-articular region. A comparison of PLLA fiber scaffolds with different geometries (aligned fibers, twist, braid, and braid-twist) demonstrated improved viscoelastic properties in the braid-twist scaffolds as compared with the other configurations. In order to improve the viscoelastic properties of this scaffold, 10% polyethylene glycol diacrylate hydrogel was added. However, this decreased pore size in the scaffold, and further investigation regarding its effects on cell proliferation and metabolism are needed. Another method is electrospinning, which produces ultrafine fibers by drawing a polymer solution onto a collecting plate using an electrical charge. Electrospinning can be used for fabricating scaffolds with fiber diameters in the nanometer to micron range, and has been shown to yield favorable mechanical properties compared with bulk polymer, while also allowing for better cell adhesion and diffusion of nutrients. Parameters such as porosity, fiber diameter, and stiffness can be controlled by altering polymer solution, while fiber alignment can be controlled by modifying the collecting apparatus. James et al. recently demonstrated that electrospun PLGA fibers in the nanometer range supported increased human tendon fibroblast proliferation and extracellular matrix deposition in vitro. Similarly, Cardwell et al. demonstrated that electrospun fiber diameter less than 1 mm supported improved mesenchymal stem cell proliferation. Peach et al. demonstrated that electrospun PCL nanofiber scaffolds functionalized with polyphosphazene were more hydrophilic and supported greater cell protein synthesis but demonstrated no difference in cell proliferation or mechanical properties as compared with controls.

Novel synthetic and composite biomaterials, along with different fabrication techniques, are under constant investigation. A bioactivated form of polyethylene terephthalate has undergone preliminary investigation in vitro. Bourke et al. investigated braided poly(desaminotyrosyl-tyrosine ethyl carbonate) scaffold and demonstrated the ability to support fibroblast growth and display the necessary strength for use as an ACL graft. Hayami et al. reported on electrospun poly(epsilon-caprolactone-co- d , l -lactide) fibers embedded in a non-cell-adherent photo-cross-linked N-methacrylated glycol chitosan hydrogel seeded with primary ligament fibroblasts, with cell viability and extracellular matrix deposition demonstrated in vitro. Blends of hydrophobic PCL and hydrophilic PGA-PCL-PGA triblock copolymer were electrospun into scaffolds. Properties of these scaffolds, such as hydrophilicity, mechanical strength, and degradation rate, were controlled by varying the ratios of PCL and PGA-PCL-PGA copolymer, the latter being a commonly used suture material. Finite element analysis has been utilized to model the optimal configuration for a braided copoly(lactic acid-co-[ε-caprolactone]) scaffold. While these data are interesting, characterization of the biological response to these scaffolds is needed.

Overall, there have been great advances in the study of biomaterials for use in ACL tissue engineering. Because of the complexity of native ligament, it is likely that composite materials will continue to be investigated, as they allow the combination of advantageous traits of multiple materials and the offsetting of their weaknesses.

Cell Sources

Another aspect of tissue engineering an ACL replacement is cell source. An ideal cell source for ligament tissue engineering should be readily available, have the capacity to proliferate, and possess the potential to elaborate an extracellular matrix (ECM) similar to that seen in native ligaments. While ligament fibroblasts are the logical candidate for ACL regeneration, the limited quantity and modest proliferative potential limit their use. Additionally, there is an age-dependent decrease in proliferation and migration potential. With the advancement of stem cell technology, more and more investigators have started using pluripotent and multipotent stem cells for ligament tissue engineering.

Bone marrow–derived mesenchymal stem cells (BMSCs) are the most studied adult stem cells for skeletal tissue regeneration and have demonstrated broad potential. They have a potential for self-renewing and for directed differentiation into cells, such as fibroblasts, chondrocytes, osteoblasts, and adipocytes. Mesenchymal stem cells (MSCs) are known to have robust synthetic and proliferative systems, and the ability to adapt readily to their local niche. BMSCs are easily harvested, can differentiate into a fibroblast-like phenotype, and avoid the ethical issues surrounding embryonic stem cells. In a head-to-head comparison of caprine BMSCs, ACL fibroblasts, and skin fibroblasts, BMSCs showed the highest cell proliferation and extracellular matrix collagen production. However, this source demonstrates significant cell heterogeneity, including high numbers of nonstem cells, nonviable cells, and the presence of differentiation-inhibiting endothelial cells, thus limiting bone marrow as a source of MSCs. Novel cell sorting techniques may improve the identification and yield of MSC harvest, obviating the need for prolonged culture and the co-culture of nonstem cell lines for this application.

Embryonic stem cells (ESCs) are a pluripotent cell source that has been investigated in many tissue engineering applications. However, the ethical controversies surrounding these cells can be avoided by selecting a different cell source. Induced pluripotent cells (iPSCs) are pluripotent cells that are derived from nonpluripotent adult cells. However, the methods of reprogramming utilized may be tumorigenic and immunogenic in humans. Due to the relative ease and efficacy of BMSCs, ESCs and iPSCs are not widely utilized in ACL tissue engineering.

More recently, MSCs have been isolated from different organs of the body. One of the most popular is adipose derived stem cells (ASCs). Similar to BMSCs, ASCs are a heterogeneous population with varying proliferation and differentiation potentials, even when isolated by density-gradient fractionation. Although the use of the noncultured total stromal vascular fraction (SVF) from adipose tissues may remove the need for culture, available studies using SVF show lower regenerative efficacy relative to cultured ASCs. Also, Eagan et al. found that ASCs did not consistently upregulate production of ligament markers when treated with growth factors in vivo. Therefore multiple drawbacks make ASCs a less than ideal source for ligament tissue engineering. In recent years there has been a prevailing theory that a subset of MSCs is associated with the vasculature, which explains why MSCs can be found in almost all of the organs in the body. These cells have been identified as perivascular stem cells, are multipotent, and have similar morphology as traditional MSCs. They have been shown to express markers of stem cells and tendon cells simultaneously, and thus may hold potential for use in ligament tissue engineering as well.

Human foreskin fibroblasts (HFFs) have gained increasing popularity for ACL tissue engineering due to their availability and homogeneity, and are already used in Dermagraft skin substitute. Currently there are commercial HFF cell banks that have already tested these cells for viruses, retroviruses, tumorigenicity, endotoxins, and mycoplasma, making them suitable for clinical use. HFFs possess proliferative potential and immunomodulatory properties similar to those of adult stem cells. This is an important characteristic, as the unabated inflammatory response to cell-scaffold constructs may promote disorganized scar and diminish the synthesis of functional collagen. However, HFFs were not found to be beneficial when seeded onto PCL scaffolds in a rat model of ACL reconstruction.

Cell source is an important consideration in ligament tissue engineering. The appropriate cells not only produce an extracellular matrix that is similar to that of native tissue, but they can also secrete trophic factors that will recruit host cells to assist in the regenerative process. During in vivo testing of tissue-engineered constructs, the exact contribution of implanted cells remains largely unknown and requires further study. Additionally, the necessity of implanting cells rather than simply allowing the ingrowth of native cells should be further studied.

Growth Factors

It is well accepted that cell proliferation, ECM elaboration, neovascularization, as well as mechanical properties can be dramatically influenced by the presence of growth factors. While the exact mechanisms involved in ligament repair are still unclear, many growth factors known to have mitogenic effects on musculoskeletal tissues have been empirically investigated. Specifically, epidermal growth factor, fibroblast growth factor, growth and differentiation factor, insulin-like growth factor (IGF), platelet-derived growth factor, and transforming growth factor beta-1 have all been shown to increase cell proliferation, fibroblastic differentiation, or matrix production in ACL tissue engineering ( Table 142.1 ).

However, not all studies have consistently demonstrated these effects, which could be attributed to the suggestion that ACL fibroblast response to growth factors is age-dependent. Some investigators have taken the approach of modeling extra-articular ligament healing by using autologous conditioned serum (ACS) or platelet rich plasma (PRP) to simulate the process of hemostasis. Although these methods have been met with some success, the exact composition of growth factors in ACS or PRP is unknown and can vary drastically between batches. This variability can render the use of ACS or PRP impractical for tissue engineering applications. With the recent advances in biomaterials and molecular biology, more investigators are incorporating growth factors into biomaterials for controlled release or using gene therapy techniques to upregulate cellular production of growth factors. Future investigation is likely to continue to focus on the controlled delivery or production of relevant growth factors from biodegradable polymer constructs to enhance ligament tissue engineering.