Introduction
In search of the ideal meniscal substitute, natural and synthetic materials for meniscal replacement have been reported with variable outcomes. Although meniscus allograft transplantation is the only clinically available option for total meniscus replacement, disadvantages to its use include limited donor supply, the need for size matching, the risk of disease transmission and the possibility of an immune response to affect healing and incorporation. Therefore there is an acute need to develop alternative strategies for meniscus replacement and regeneration. This chapter summarises progress in the field and the translational potential of cell-free scaffolds and biomaterials developed for meniscus replacement and regeneration.
Tissue Engineering and the Meniscus
Tissue engineering is a rapidly evolving field in biomedical research aimed at developing biological substitutes that restore, maintain or improve tissue function. In general, tissue engineering is based on a combination of cells, scaffolds and biological or biomechanical signals to stimulate neo-tissue formation. The classic tissue engineering paradigm involves the stimulation of cells, in vitro or in vivo, to migrate and proliferate through a three-dimensional (3D) scaffold, leading to production of functional replacement tissue. The scaffolds should ideally promote uniform cell adhesion, migration, differentiation and proliferation. The microstructure and composition of the scaffold should also promote proper phenotypic expression by infiltrating cells and functional extracellular matrix production.
Although a number of approaches have been reported for tissue engineering of the meniscus, an optimal meniscus replacement has yet to be discovered. Importantly, many of the published tissue engineering studies do not report the mechanical properties of the meniscal constructs or repair tissue, which is a critical outcome measure given the tissue’s intrinsic functions. There is also a lack of standardised and objective outcome measures across studies to compare the effectiveness of different meniscal scaffolds and no consensus on the definitions or benchmarks for success.
An ideal meniscus replacement should have biological and biomechanical properties that are similar to the native meniscus ( Fig. 20.1 ). Importantly, a functional meniscus replacement should ultimately restore the meniscus structure, function and anisotropic properties. The scaffold should also maintain a synergistic relationship with the articular cartilage, provide joint protection and reduce the onset and progression of osteoarthritis. Further, the ideal meniscus replacement should resorb or degrade slowly enough to allow for ingrowth and regeneration of the tissue’s vascular, structural, cellular and extracellular matrix components. The meniscal scaffold should also be nontoxic to the joint and not evoke a foreign body reaction or immune response on degradation. Lastly the optimal scaffold should be readily available for point-of-care clinical use, minimally invasive, implantable with technical ease and reimbursable by federal and private insurance providers.
Scaffolds for Meniscus Replacement
Broad categories of scaffolds investigated for meniscal replacement include synthetic polymers, hydrogels, extracellular matrix components and tissue-derived materials. Natural polymeric scaffolds offer a distinct advantage for scaffold fabrication in that they are easily modified and the biomechanical and biological properties of the construct can be manipulated during the fabrication process, therefore allowing the engineered construct to resemble tissues for a specific application. They are also less likely than synthetic polymers to incite a strong inflammatory response in vivo . The hydrophobic composition of synthetic polymers may also hinder cell growth in a 3D architecture. Thus a natural polymeric scaffold can be designed in such a way that it is biocompatible and biodegradable, allows for diffusion of nutrients and metabolites, provides mechanical support, has appropriate porosity and provides an aqueous environment for cellular encapsulation, proliferation and tissue production.
Hydrogels
Hydrogel scaffolds are fabricated from natural or synthetic polymers, consisting of hydrophilic building blocks that are insoluble because of the presence of cross-links. Examples include alginate, collagen, agarose, chitosan, gelatine and hyaluronic acid. The latter polymers are formed from soluble monomers, multifunctional polymers (macromers) and nano- or microparticles. In addition, a variety of crosslinking processes may be used to affect the chemical and physical properties for a specific application. Potential chemical crosslinking agents include glutaraldehyde, genipin, adipic dihydrazide, and bis(sulfosuccinimidyl) suberate. Thermal, ionic and free radical crosslinking methods may also be employed, depending on the type of polymer to be investigated. Controlling hydrogel density and crosslinking chemistry of the scaffolds can result in subsequent control over mechanical properties and tissue in-growth rates via scaffold resorption. Because hydrogels have functional and structural similarities to native extracellular matrix, they also have the potential to provide cells with a biologically relevant microenvironment that fosters cell proliferation, migration and extracellular matrix production. , Other advantages include the chemical diversity of hydrogels under an array of different environmental factors, including temperature, pH, electric field, ultrasound and salinity.
Biological Scaffolds for Meniscus Replacement
Collagen
Collagen is a naturally occurring matrix polymer and the most abundant extracellular matrix protein. It contains three polypeptide α chains forming a right-handed collagen triple helix. The meniscus is composed predominantly of type I collagen, but there is variation in the type and structure of collagens between the vascular and avascular zones, as well as between the superficial and deep zones. Type I collagen significantly contributes to normal matrix composition and the biomechanical properties of the meniscus. Collagen fibres are primarily responsible for the tensile strength of the meniscus, and the longitudinal fibres are critical for normal meniscal function and chondroprotection in that they transfer vertical compressive loads into circumferential hoop stress. Consequently a meniscal replacement intended for clinical translation should be able to recapitulate the force transmission properties of the meniscus in vivo .
High-density collagen gels have been investigated for fabrication of cell-seeded meniscal constructs with improved mechanical and biochemical properties compared with alginate gels with or without insulin-like growth factor supplementation. , The Collagen Meniscal Implant (CMI) is a porous meniscus implant derived from purified bovine collagen and will be discussed in another section (see Meniscal Scaffolds in Clinical Use).
Hyaluronic acid
Hyaluronic acid (hyaluronan; HA) is widely distributed throughout the extracellular matrix of normal connective tissues. It is a nonsulphated glycosaminoglycan containing repeating units of N-acetylglucosamine linked to glucuronic acid. HA has shown promise for tissue engineering applications, especially as a composite scaffold, given its excellent biocompatibility, biodegradability and gelation properties. Mechanical properties of HA hydrogels can be enhanced by covalent crosslinking with hydrazide derivatives, esterification and annealing. , Composite scaffolds containing collagen and HA have been synthesised for bone regeneration. A multilayer tissue engineered meniscal substitute composed of collagen and HA has also been reported for potential use as a partial meniscus replacement based on early laboratory evidence. In addition, human meniscus-derived cells have been successfully cultured in vitro on 3D polyglycolic acid–HA scaffolds.
Silk fibroin
Silk proteins are derived from the cocoon or glands of the silkworm and other arthropods. The silk protein fibroin has been processed into scaffolds for various biomedical applications. Advantages of silk compared with other natural polymers include strong mechanical properties, biocompatibility, water-based processing, biodegradability, affordability and the presence of chemical groups that are easily accessible and modified.
FibroFix Meniscus (Orthox Ltd., Abingdon, UK) is a nonresorbable silk fibroin xenograft biomaterial indicated for cartilage and meniscus injuries. In a preclinical study to evaluate the suitability of the scaffold for partial meniscus replacement, 28 sheep underwent partial medial meniscectomy with or without scaffold implantation. The silk scaffold caused no inflammatory reaction in the joint at 6 months, and early data showed promising biomechanical and biological properties. The tribological properties of the scaffold have been tested and suggest possible long-term chondroprotective function. However, further data are required to evaluate the fixation technique and long-term safety and efficacy of silk scaffolds for meniscus replacement. ,
Synthetic Scaffolds for Meniscus Replacement
Synthetic polymer scaffolds have been evaluated for many tissue engineering applications. Synthetic polymers are advantageous in that they can be manufactured into reproducible, custom-designed biomedical implants and devices, with specified geometry, porosity and biomechanical properties. They may also be tailored to a specific tissue type, cellular environment and degradation profile. Potential disadvantages of synthetic polymers include the low cell-adhesive properties and the potential for a foreign body reaction after implantation or on degradation of the material.
As stated, the ideal synthetic biomaterial for meniscus replacement should be biocompatible and biodegradable in vivo . It should allow cell adhesion but have a long enough half-life to not prematurely dissolve after implantation. It should also have chondroprotective effects and not cause damage to the articular cartilage. Many synthetic polymers have been reported for tissue engineering strategies and potential meniscus replacement. These polymers are approved by the US Food and Drug Administration (FDA), readily available and can be processed into biomaterials and devices for a variety of applications. Specifically, aliphatic polyesters are bioresorbable and biocompatible polymers with great potential for regeneration of large tissues.
Polyglycolic acid
Polyglycolic acid (PGA) is a biodegradable aliphatic polyester reported for biomedical applications. It was first described in the 1970s as a degradable suture material and found to have other advantages, including its biocompatibility and strong mechanical properties. For the meniscus, PGA scaffolds promoted cell survival and attachment of fibrochondrocytes. PGA scaffolds seeded with meniscal fibrochondrocytes had increased extracellular matrix production compared with agarose constructs. In another study PGA scaffolds seeded with meniscal fibrochondrocytes were implanted into the subcutaneous space of nude mice and supported new fibrous matrix production at 16 weeks after implantation.
In a rabbit total meniscectomy model, PGA scaffolds seeded with meniscal fibrochondrocytes had increased fibrocartilage production over a 10-week period. In that study the material properties of PGA were increased by physically bonding adjacent PGA fibres with 75:25 poly(lactic-co-glycolic acid) (PLGA). Based on these results, bonded polymer scaffolds demonstrated more optimal compressive moduli compared with nonbonded scaffolds, which could have important implications for scaffold design intended for meniscal tissue engineering. Overall, PGA is a potentially beneficial biomaterial for meniscus regeneration given its biomechanical and biological properties and potentially chondroprotective effects.
Poly- l -lactic Acid and poly- d -lactic acid
PLA is a slow-crystallising, semicrystalline polymer that has been reported for soft tissue and orthopaedic tissue engineering applications. PLA is a biodegradable, thermoplastic polyester that typically consists of l – and d -lactate units, and the stereoisometry can profoundly affect the thermal and mechanical properties of the polymer. Control during processing of PLA scaffolds can result in desired pore structure, porosity and scaffold thickness for various biomedical applications. The high mechanical strength of PLA is potentially advantageous for the meniscus given the tissue’s innate material properties.
Porous poly( l -co- d,l -lactic acid)/poly(caprolactone-triol) (PLDLA/PCL-T) (90%/10%) scaffolds are seeded with meniscal fibrochondrocytes in the lapine total meniscectomy model. There is formation of fibrocartilaginous tissue after 24 weeks, without apparent rejection, infection, or chronic inflammatory response. In another study, a bioresorbable polymer scaffold made of Poly- l -lactic acid (PLLA) and poly( p -dioxanone) (PPD) blend was used as a temporary meniscal prosthesis to stimulate meniscal regeneration in rabbits. The scaffold fostered tissue ingrowth and fibrocartilage formation 12 weeks postimplantation.
Conversely, in an ovine total medial meniscectomy model, a collagen-hyaluronan sponge reinforced with PLLA fibres resulted in unanticipated cartilage degeneration in the knee joint. The authors reported that the PLLA implant could not withstand load transmission in the joint, possibly because of a build-up of lactic acid and/or rapid PLLA degradation. These data suggest that the PLLA fibres as produced in this study cannot be used as reinforcement for a meniscus replacement scaffold.
There are currently no commercially available PLA-based biomaterials indicated for meniscus repair or scaffolds used for large meniscal defects in humans. However, PLA is a potentially advantageous biomaterial given its versatility and ability to be processed for a number of tissue-specific applications. Further studies are necessary to investigate the utility of PLA-based biomaterials as potential scaffolds for meniscus replacement and regeneration.
Composite polymer scaffolds
Composite scaffolds containing combinations of natural and synthetic polymers have also been reported for the meniscus. Polycaprolactone (PCL) is a biodegradable polyester that is easy to manufacture and manipulate into a variety of biomedical implants and devices given its superior viscoelastic and rheological properties. Organised alignment of PCL nanofibers supports better neo-tissue organisation and mechanical properties of the scaffolds. PCL is also FDA approved and biocompatible, highly manufacturable and slow to degrade.
For the meniscus, hyaluronan (HYAFF)–PCL scaffolds (Fidia Advanced Biopolymers, Padua, Italy) with and without cells were assessed after partial and total meniscal replacement in an ovine model. The biomaterial was surgically implanted and had excellent properties in terms of tissue ingrowth and mechanical stability. Electrospun PCL scaffolds have also shown promise as meniscal scaffolds, achieving comparable or superior integration with native tissue. The electrospinning process can also be modified to create scaffolds with varying structural organisation and to optimally recapitulate the native cellular microenvironment. , A composite scaffold composed of decellularised meniscus extracellular matrix and electrospun PCL (DMECM/PCL) has been fabricated and demonstrated in vitro cytocompatibility with cellular attachment and meniscus repair tissue production in a lapine total meniscectomy model.
Other synthetic polymers and composite scaffolds reported for the meniscus include polyurethane-PCL and polyvinyl alcohol–hydrogel (PVA-H) scaffolds. Both biomaterials have been implanted into the knee for total meniscal replacement in preclinical models with variable success based on outcome measures including tissue regeneration, safety to the articular cartilage, long-term durability, and ease of implantation. A porous, polyurethane-based polymer scaffold (Actifit) has been commercialised for clinical use and will be discussed in a later section (see Meniscal Scaffolds in Clinical Use).
PVA is a linear synthetic polymer used for medical and nonmedical devices. It is an attractive biomaterial because of its biocompatibility and low protein adsorption properties. PVA hydrogels are also permeable, hydrophilic and have low frictional properties. PVA hydrogels have been investigated in animals as an artificial meniscus replacement after lateral meniscectomy. However, problems with biocompatibility, wear and inferior material properties have led researchers to explore alternative strategies for the development of a safe and effective scaffold for partial or complete meniscus replacement.
3D-Printed Meniscus
Three-dimensional–printed meniscal scaffolds represent a novel approach for meniscus replacement in an era of personalised and precision medicine. These 3D scaffolds can be replicated to match patient-specific geometries and to restore the overall shape of the meniscus. In addition, 3D-printed scaffolds may be fabricated to create off-the-shelf implants, which could become readily available in clinical practice. Szojka et al. have designed and produced a biomimetic 3D-printed meniscal scaffold composed of 43 to 50 kDa molecular weight PCL. The scaffold was designed to reproduce the circumferential type I collagen and radial-tie fibres of the native meniscus. Suture tabs were also included in the design to facilitate surgical fixation of the scaffold. As such, the scaffolds produced in this study demonstrate a promising strategy for meniscus replacement and warrant further investigation.
Similarly, a 3D-printed PCL meniscus scaffold has been previously developed and seeded with PLGA microspheres encapsulating spatially released human connective tissue growth factor (CTGF) and transforming growth factor-β3 (TGF-β3). The microspheres release spatially delivered growth factors to direct cell differentiation and zone-specific extracellular matrix production. The scaffolds were evaluated in a preclinical ovine model and led to new tissue formation in meniscal defects. Histologically, meniscuses treated with the scaffold and growth factors promoted neo-tissue formation with an organised extracellular matrix rich in collagen and proteoglycans. There was also less severe articular cartilage degeneration in the treatment group compared with the control group.
MeniscoFix (NovoPedics, Inc., Princeton, NJ, USA) is a patented, 3D-printed scaffold currently under investigation for total meniscal replacement. The scaffold is composed of a biodegradable polymer composed of poly(desaminotyrosyl-tyrosine dodecyl ester dodecanoate) (p(DTD DD)) infused with a collagen-hyaluronate sponge. The scaffold is an acellular, off-the-shelf device intended to restore knee mechanics after partial meniscectomy. In a preclinical pilot study, the scaffold was cytocompatible and supported tissue ingrowth and integration into the host tissue in an ovine meniscectomy model. Subsequent studies are in progress to further assess the fixation technique and safety and efficacy of the scaffold at longer time points.
Meniscal Scaffolds in Clinical Use
Collagen Meniscal implant
The Collagen Meniscal Implant (ReGen Biologics, Hackensack, NJ, USA; formerly known as Menaflex ) is a collagen-based scaffold developed by Stone, Rodkey and Steadman. The scaffold is composed of purified bovine Achilles tendon collagen type I fibres with proteoglycan that is cross-linked using aldehyde vapour. , Briefly, the tendon tissue is minced, and the collagen fibres are purified with various chemical treatments to remove the noncollagenous proteins and other materials. The purified collagen fibres are then swollen in HA and chondroitin sulphate and homogenised. The fibres are then dehydrated, moulded, lyophilised, chemically cross-linked and sterilised with γ radiation ( Fig. 20.2 ).
The resultant meniscal scaffold is porous and acellular and is designed to support cell migration and attachment of migrating cells from the meniscus, synovium and synovial fluid. The CMI resembles the size and shape of the normal human meniscus and allows in-growth of functional meniscus repair tissue over time. The implant can be trimmed and adapted to a specific meniscal defect during arthroscopic knee surgery ( Fig. 20.3 ). The CMI has demonstrated safety in terms of cytotoxicity, pyrogenicity and carcinogenicity. Further, the product is bioresorbable, and most of the scaffold will be resorbed over a 1- to 2-year period.
Indications for clinical use of CMI include medial or lateral meniscal defects that involve greater than 25% of the meniscus, but include an intact peripheral rim, and anterior and posterior horn attachments. In early clinical studies the CMI showed no adverse effects and supported new tissue formation, and there was short-term improvement in clinical scores in patients with irreparable meniscal tears. In another study 8 patients showed improvement in pain and function, and all patients underwent second-look arthroscopy with biopsy of the repair tissue at either 6 or 12 months postoperatively. Histological analysis confirmed new fibrocartilage matrix formation at the site of implantation.
Based on feasibility studies, the CMI was noted to be implantable, biocompatible, and resorbable and to support new tissue production. The repair tissue was also noted to maintain its structure and function after 6 to 8 years follow-up in a prospective case series of 8 patients. In the latter study all the patients were able to return to activities of daily living after 3 months, with all but one patient showing improvement in Cincinnati Knee Rating Scale (CKRS) and International Knee Documentation Committee (IKDC) scores.
In a prospective cohort study 33 male patients with meniscal injuries were treated with either a medial CMI or partial medial meniscectomy (PMM), with a minimum of 10 years follow-up. Patients were assessed for pain and function with several outcome scores, and the CMI group showed significantly lower pain scores and improved clinical function. There was also significantly less medial joint space narrowing on radiographs in the CMI group compared with the PMM group. The magnetic resonance imaging (MRI) evaluation of CMI patients revealed 11 cases with myxoid degeneration, 4 with normal signal, 4 with normal signal and reduced size and 2 with no recognisable implant. The authors concluded that randomised controlled trials in larger cohorts of patients are necessary to confirm the benefits of CMI at longer time points.
In the largest CMI study, a multicentre clinical trial, 311 patients with an irreparable injury of the medial meniscus or a previous PMM were randomly assigned to either receive the CMI or to serve as a control subject treated with PMM only. Patients were followed over 2 years and completed validated outcomes assessments over 7 years. CMI patients were also required to undergo second-look arthroscopy at 1 year to assess new tissue ingrowth with histopathological testing. Based on the results of this study, the CMI was found to be safe and to support meniscus repair tissue production and integration as the scaffold was resorbed. The risk of reoperation 5 years after surgery was 2.7 times greater in the PMM group compared with the CMI group. Interestingly, patients with chronic meniscus injuries showed more clinical improvement compared with patients with acute injuries (no prior surgery on the involved meniscus). Thus the CMI did not provide significant clinical benefits to patients with acute meniscus injuries 5 years after implantation but was beneficial to patients with chronic injuries. Limitations of this study included the short follow-up time of 5 years and a lack of validated and objective patient outcome measures. In addition, radiological outcomes were not included.
Importantly, only a few studies have followed the MRI-based findings of the CMI after implantation. There is reportedly a frequent and progressive decrease in the size of the scaffold over time, as well as differences in size and signal intensity between the medial and lateral meniscus. The most commonly used MRI scoring system after partial meniscus substitution with CMI was described by Genovese et al. This MRI-based score is intended to analyse the size and signal intensity of the CMI after implantation and to correlate imaging data with clinical and histological data from patients undergoing second-look arthroscopy ( Table 20.1 ). In one study the Genovese grading scores for morphology, size and signal intensity of the meniscal implant had only slight to moderate inter- and intraobserver reliability. Only the criteria for bone marrow oedema and meniscal extrusion had strong inter- and intraobserver reliability. Despite these potential limitations, MRI remains the gold standard for imaging of the meniscus, and it should be included as a primary outcome measure in studies to longitudinally assess the safety and functionality of meniscal scaffolds in patients.
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