Part I: Scientific Basis for Design Considerations of Bioscaffolds

Cartilage, like most other tissues, is comprised of two components, the extracellular matrix (ECM), composed of various macromolecules and water, and the cells contained within the ECM, which produce and maintain the former. Cartilage repair requires restoration of both components to produce a tissue that is biomechanically and biochemically able to withstand the demands of repetitive joint loading without early degeneration and failure. In addition, as an organ, the joint is composed of and depends on the integration and interplay of various building block materials. Without transgressing into a discussion of the interaction of the menisci, ligaments, capsule, synovium, and cartilage, it is necessary to couple cartilage with the underlying bone. Without complete basilar integration through the calcified cartilage layer, a supposedly perfect cartilage construct will fail through delamination. Classic cell-based approaches, such as microfracture (marrow stimulation) and autologous chondrocyte implantation (ACI), rely on a cellular component to produce ECM, thus filling the lesion and achieving basilar and marginal integration. These procedures require activity restrictions to protect the immature tissue and are associated with long recovery times and complex postoperative rehabilitation, mainly because of the slow production and maturation of the ECM component by the cells. Many current and future approaches have the objective of modifying the classic cell-based techniques through the addition of bioscaffolds, with the goal of simplifying the surgical technique, decreasing postoperative restrictions on the patient, speeding up recovery and return to full activity, and improving outcomes. This exciting area is fluid and in constant change, so this section can only provide a snapshot of current scientific knowledge, with an attempt to delineate the characteristics of an ideal scaffold for cartilage repair.

Scaffold Requirements

Scaffold function can generally be divided into two roles, cell delivery and structural support for cell migration or stratification with composite maturation. The former uses the scaffold as a carrier substrate to help deliver cells into the defect and maintain them in situ until the new cartilage construct can achieve marginal and basilar integration. Theoretically, after the scaffold has fulfilled its purpose, it could be removed or, more realistically, resorb on its own. This function is comparatively simple, with the demands in terms of mechanical properties minimal, and therefore a number of materials have been found to be suitable for this, including the fibrin clot from microfracture, fibrin glue, alginate or agarose, collagen, hyaluronic acid, and artificial polymers, such a polylactic acid (PLA) and polyglycolic acid (PGA) and their modifications.

The second role, to act as a support structure during cell and ECM maturation, is far more complex and may require more advanced engineering to produce a scaffold that optimizes the physical and biochemical structure of the ECM while providing adequate porosity to allow cell invasion and growth (Table 26-1).²⁷ The ideal scaffold should allow early or even immediate weight bearing; thus, it would require mechanical properties strong enough to protect the cells while at the same time not being so stiff as to completely shield the cells from all stresses, which are important signals for tissue maturation. The scaffold should provide secure fixation and enhance basilar integration with the subchondral bone and circumferential integration with the surrounding cartilage. To allow cell growth, the scaffold must consist of a system of interconnected pores; the material should be hydrophilic to ease cell seeding, penetration, and adhesion. Furthermore, certain modifications can improve cell adhesion to a scaffold, such as binding of adhesion ligands to the scaffold material. It should slowly resorb with time, at a pace that allows gradual replacement through host tissue, and this process should not generate degradation products that are toxic or inflammatory.

Table 26-1 Desirable Attributes for Bioscaffolds Used in Cartilage Repair

Structure and Chemistry	Mechanical Properties, Strength, and Integrity	Clinical Application
Biocompatible synthetic versus naturally derived	Mechanical properties comparable to hyaline cartilage	Preformed intraoperatively versus custom shape or contour versus injectable
Porosity-permeability—optimal porosity with three-dimensional architecture	High porosity (does not apply to gel-type scaffolds)	Ease of intraoperative handling and fixation
Optimized geometry to regenerate native matrix (ECM) orientation	Composite structure with varying properties throughout its thickness	Delivery attributes—arthroscopic (air or liquid) versus miniopen versus formal open
Resorption without local or systemic adverse effect versus benign particulate breakdown scavenging	Biocompatible	Chondral versus osteochondral defects
Resorption temporal profile follows new cartilage deposition	No toxic degradation products	Reproducibility
Surface chemistry (protein absorption-deposition, enabling cell adhesion, migration, and outgrowth)	Assists in cell and tissue differentiationResorbable	Regulatory approval pathway and final indicationsCost, value

Basic Science

The following section will review basic aspects and concepts, including the physical and biologic characteristics of bioscaffolds for cartilage repair. More specific information on individual membranes currently in clinical practice or under development will be provided in the second part of this chapter.

Physical Characteristics

Mechanical Strength

Mechanical characteristics of hyaline cartilage vary with the joint in question, as well as the specific location within the respective joint. A bioscaffold allowing early or even immediate weight bearing is desirable and should closely mirror the mechanical properties of hyaline cartilage until it has been replaced by mature repair tissue. The scaffold functions to protect the growing tissue while ensuring an appropriate level of physiologic loading to enhance the reparative process,⁴⁷ an effect first described by Pauwels,⁴⁸ who recognized the influence of physical stimuli on cell differentiation pathways of mesenchymal stem cells.

Elasticity (Young’s modulus) of human hyaline cartilage has been reported as between 1 and 20 Mpa, depending on the layer and location, several orders of magnitude lower than that of immature (1000 MPa) or cortical (17,000 MPa) bone.³⁴ It appears from computer modeling that an inhomogeneous three-dimensional scaffold with higher stiffness in the superficial layer, which gradually decreases toward the base of the defect, might be best suited to encourage cartilage, rather than fibrous tissue, regeneration. This theory has been substantiated by findings of a tensile modulus 6 to 20 times higher in the superficial regions than in the deeper regions, whereas permeability demonstrated a reverse distribution, increasing with increasing depth. The higher stiffness at the surface better protects the immature tissue from the high shear forces experienced at this level and the lower stiffness at the base allows sufficient strain rates to encourage chondrogenic differentiation.

Structure

Studies have investigated effects of the overall three-dimensional structure of scaffolds on cells and tissue production. Although chondrocytes attach and grow even on flat nonphysiologic surfaces (two-dimensional growth, such as in a Petri dish), they gradually dedifferentiate into a more fibroblastic phenotype with increased type I collagen production. Conversely, chondrocytes maintain their spherical appearance when grown in three-dimensional culture, such as open-pored scaffolds or alginate beads, and matrix production is improved quantitatively and qualitatively with increased type II collagen.⁴¹ Cartilage ECM consists of a mesh of collagen fibers 10 to 140 nm in diameter⁵³ and studies have demonstrated improved cell adherence to fibers of submicron size.⁶⁵ Many studies have therefore investigated the use of spun or woven nanofibers of various materials for use in bioscaffolds.²⁶

A system of interconnected open pores facilitates cell seeding of bioscaffolds to produce a three-dimensional structure. The normal pore area of hyaline cartilage has been reported as 5 to 33 nm,⁵² but this is not directly comparable to the requirements of a bioscaffold. The former reflects the size of a lacuna, but pores in a bioscaffold have to be large enough to allow cell seeding, penetration, and proliferation, followed by production of ECM. However, increased pore size beyond a threshold value has been demonstrated to decrease attachment for a variety of cells, whereas increased specific surface area (a measure of overall porosity) was found to have a positive effect.⁴⁵ In general terms, a material porosity of 80% to 90% has been found to be beneficial in terms of quantity and quality of regenerated tissue.²⁸ In addition to the pore size, which allows cell migration, the nanostructure of the material must allow cell adherence during migration.

Biologic Characteristics

Biocompatibility

More commonly an issue with synthetic bioscaffolds, biocompatibility refers to tissue reactivity toward the implanted material. Biocompatibility can be improved by surface modification of the material to improve cell adhesion—for example, the wettability of hydrophobic polymers, such as the polyesters PGA and PLA, can be improved by gas plasma treatment to polymerize specific monomers to the scaffold surface.⁴⁶ Biomolecules can also be attached, such as arginine-glycine-aspartic acid (RGD), which interact with integrin receptors to anchor the cell cytoskeleton to the ECM.⁵⁷ However, even within the group of biologic scaffold materials, such as collagen and hyaluronic acid, subtle variations exist that influence cell adhesion. In a review of several collagen membranes, type II collagen appeared to be better suited to enhance cell attachment than type I collagen membranes.^23,³⁶

Part II: Scaffolds in Development

Part I of this chapter established the scientific basis for the use of scaffolds in the repair of articular cartilage defects of the knee. However, at the time of this writing, none are clinically available in the United States. Although the list of desirable attributes for an articular cartilage repair scaffold represents realistic goals, attempting to achieve all the attributes in one scaffold remains elusive.⁵⁹ As with all aspects of medicine, if there were a true best method or best scaffold, then all physicians would adopt that single technique. However, in this relatively new field, the reality is that many approaches remain under evaluation,¹¹ because none has provided the stated end goal: to produce a true stratified hyaline cartilage, with full basilar and marginal integration implanted, using a minimally invasive technique with minimal inconvenience to the patient and cost to society. Nevertheless, from the view point of demand matching, the laudable but possibly unobtainable stated goal may not be necessary for many knee lesions. Consider that many first-generation cartilage repair techniques appear to work satisfactorily in up to 70% of patients. Therefore, the goal may need to be restated from a patient function and pain perspective, and not from a histologic perspective. That is, the cartilage repair goal may be the most cost-effective and acceptably durable treatment for a specific patient and specific cartilage lesion, rather than a fully integrated hyaline cartilage. This on the ground clinical approach should not deter basic science research, but illustrates the difference between preclinical results and clinical applications. It is important to keep the patient’s knee in mind while exploring the newer scaffold cartilage repair options.⁴³

The first clinical application of a scaffold for knee cartilage repair was reported in 1998 by Behrens.⁸ The two-stage technique was an extension of the original ACI. After an autologous biopsy was cultured, the chondrocytes were seeded onto a porcine collagen I-III scaffold (Chondro-Gide, Geistlich Biomaterials, Wolhusen, Switzerland) and allowed to grow on the scaffold before implantation. It was termed matrix-associated autologous chondrocyte implantation (MACI; Genzyme, Cambridge, Mass). Shortly thereafter, in 1999, Hyalograft C was introduced. The scaffold was a benzylic ester of hyaluronic acid (HYAFF 11, Fidia Advanced Biopolymers Laboratories, Padova, Italy).⁶⁹ Like the two-stage MACI, the autologous cartilage was harvested from the patient, followed by expansion and seeding onto the scaffold, where they are allowed to grow. Both of these three-dimensional scaffolds have been shown to improve the maintenance of a chondrocyte-differentiated phenotype when compared with two-dimensional culturing. These initial biodegradable polymers remain in active clinical use in Europe, with many reports of efficacy over time, and have been joined by several other seeded, cultured scaffold applications. In addition, these scaffolds allowed arthroscopic implantation in certain regions of the knee, typically the femoral condyles and trochlea, which was not possible with first-generation ACI. Because this is a rapidly changing field, our goal here is to show current scaffold applications in a general sense, with the understanding that the initiated reader will review current literature and conference presentations before making any clinical decisions. In addition, it is necessary for the reader to fully understand the regulatory process in his or her respective country because allowed clinical use may vary over time.

As an overview, it is important to reemphasize that not all cutting edge cartilage techniques use scaffolds. This is well documented elsewhere in this text. In fact, there is a certain degree of overlap of topics. For example, in the subset of cell therapies, scaffolds are obviously a further subset. Understanding the importance of marrow stimulation, osteochondral autograft, and allograft as stand-alone techniques, the subset of cell therapies may be classified by the following: (1) cells alone; (2) scaffolds without cells at time of implantation (host cell source); (3) scaffolds with seeded cells; and (4) scaffolds with seeded and cultured cells. This section will focus only on those applications using scaffolds.