that ensures dissipation of stress and load on a joint. Injuries to tendons and ligaments are fairly common in sports or coupled with other orthopedic injuries, but restoration to original condition is almost never achieved and reinjuries are expected even after healing. It has been observed that natural healing after injuries to joints occurs through formation of fibrovascular scar tissue and not by regeneration of the original graded tissues, making the site susceptible to reinjury. Cartilage, on the other hand, is a firm and tough connective tissue that acts as a cushion between bones. Injury or gradual erosion of the cartilage can lead to painful joints, affecting the quality of life as it has limited capacity for healing.
Tissue engineering has emerged as a viable technique for repair and regeneration of damaged connective tissues. It utilizes a scaffold of a suitable material and shape, seeded with precursor cells and loaded with appropriate factors promoting growth and differentiation. The outer ear, a small piece of trachea as well as a patella, of cartilaginous and osteochondral origin respectively, have been successfully grown in vitro using this technique.
Interface tissue engineering (ITE) goes a step further to synthesize tissue for complex interfaces such as ligament to bone, tendon to bone, and cartilage to bone. The interface is actually only 100 μm to 1 mm in length but ITE necessitates thorough understanding of the cell composition, maturation of various cell types, as well as the structure–function relationships at the interface site. It is expected that the unique nature of the site would need the presence of specific transcription factors/proteins and its function would also be regulated by the extent of load bearing by the associated muscles.
The scaffolds constitute the most crucial component in the regeneration of connective tissues. Ideally, they should be biodegradable, porous, nontoxic and nonantigenic, flexible, and support nutrient and waste transport. They provide the framework for adhesion, growth, and differentiation of cells and facilitate cellular interactions. Scaffolds were made of a single component, calcium phosphate, 30 years ago but a number of novel materials have been tested for composite scaffolds over the last decade. These include synthetic microfibers such as porous polyethylene (PPE), poly-L lactic acid (PLLA), polylactide-co-glycolide (PLGA), and polyurethane, as well as biological materials such as collagen and silk. There are several studies reporting both the advantages and disadvantages of collagen-based scaffolds. Nanofibers form excellent scaffolds owing to their biocompatibility and possible variations in fiber diameter and alignment. The interaction of clay with biomolecules is also being studied to explore its suitability in tissue engineering.
Located in the knee and performed arthroscopically, an anterior cruciate ligament reconstruction surgery removes the torn ligament and replaces it with a tissue graft of either autografts or allografts. (Wikimedia Commons/Phalinn Ooi)
Scaffolds that mimic in vivo conditions at the interfaces should compulsorily be stratified and should attempt to replicate the gradation in structure and function found in native tissues. A triphasic scaffold that has been used for the regeneration of the anterior cruciate ligament (ACL) interface with bone involves three distinct, yet continuous phases: Phase A comprising PLGA 10:90 mesh was designed for fibroblast culture, Phase B made up of PLGA microspheres is for fibrochondrocyte culture and fibrocartilage formation, and Phase C composed of PLGA and bioactive glass (BG) composite microspheres was designed for bone formation. Each of the phases was seeded with the relevant cell types—fibroblasts, articular chondrocytes, and osteoblasts—and the matrix heterogeneity and density were evaluated over a period of eight weeks. Spatially separated but extensive tissue infiltration and matrix deposition with continuity was observed in Phases A and C. Migration of cells to Phase B and its vascularization were observed too. The PLGA-BG phase is instrumental in promoting mineralization and calcification of the cartilage and bone-like matrices in Phase B and C, respectively. The mechanical properties were also observed to improve with higher cell density. This graft has also been successfully grown in an athymic rat model. Nanofibers of PLGA and hydroxyapatite (HA) nanoparticles have also been used to assemble the triphasic scaffold with similar results.
Further work in the field needs to focus on optimization of the growth conditions of cells on the scaffold and its in vivo evaluation. The effect of biological, chemical, and mechanical stimulation on regeneration has to be studied under both in vitro and in vivo conditions. Incorporation of biologically active molecules into the nanofiber scaffold is a biomimetic approach to promoting cell growth and differentiation. Scaffolds coated with type 1 collagen and laminin showed enhanced proliferation of cells. Plasma treatment of the scaffold also promoted cell division reproducibly. Another successful and innovative approach has involved the fabrication of a scaffold by electrospinning basic fibroblast growth factor (bFGF), releasing PLGA nanofibers onto knitted silk microfibers. Two transcription factors, SOX-9 and scleraxis (Scx), have been identified to promote chondrogenesis and tenogenesis at the tendon/ligament to bone interface, and their incorporation into the scaffold structure is being planned.
After a multi-tissue graft has been successfully generated, its biological fixation or functional integration with each other as well as the host environment should ideally follow. Two essential prerequisites to aid this process are angiogenesis (the development of a viable blood circulation) and the establishment of cellular communication among the three resident cell populations: the fibroblasts, fibrochondrocytes, and osteoblasts. Angiogenesis and cellular communication are absolutely vital to achieving homeostasis under in vivo conditions. It is proposed that multiphasic scaffolds for ACL-to-bone integration may be fabricated as a cylinder that can be inserted directly into the bone tunnels for smooth interface formation. For rotator cuff repair of the shoulder, scaffold patches could be surgically sutured to the tendon to facilitate repair and bridge the gap between the tendon and bone.
The structure of an interface is naturally organized to reduce stress and optimize load bearing at the joint. The angle of attachment and the gross shape of the insertion are critical factors in preventing stress at the interface, and reducing the angle has been shown to aid the process. Interlocking of the tissues by interdigitation increases the strength and toughness of the graft. The orientation/alignment of the collagen fibers and mineral deposition has also been shown to be critical for functional repair of the interface. The transition between the unmineralized and mineralized tissues at the graded interface is particularly challenging and is key to attaining the mechanical properties of the interface.
Another requirement for a functionally graded transition between tendon and bone is physiologic muscle loading. Experiments have shown that reduction in muscle loading impaired mineralization and fibrocartilage formation, leading to disorganized fiber distribution and inferior mechanical properties at the interface. It becomes imperative to achieve a fine balance between excessive loads, which can cause microdamage, and insufficient loads so that healing is faster.
Tissue engineering brings with it several ethical, medical, and regulatory issues that will need to be addressed for clinical progress to continue. Considerable success has been achieved in animal models and the results have to be replicated in human subjects. A clinical trial enrolled six subjects for mesenchymal stem cell injections in an attempt at resurfacing of the articular cartilage in osteoarthritis. The results are not known, even though the study is over. Similar clinical trials for evaluation of a resorbable PLLA implant for regeneration of the ACL interface, bone marrow stem cells on protein scaffolds to heal the articular cartilage of the knee, and the impact of platelet-rich plasma on healing of rotator cuffs are in various stages of study.
Research inputs ranging from the ideal scaffold material (material science), growth factors (biochemistry), in vivo transition for structural (histology) and functional integration (biophysics) will have to be effectively incorporated for successful tissue regeneration. It follows that techniques for sterilization and long-term storage of stratified scaffolds will also need to be developed. The strategy for soft tissue and integrative orthopedic repair through tissue regeneration and implantation thus has wide-ranging implications.
Ruby
