Aging is a significant concern in the field of cartilage regeneration due to its deleterious effects on stem cell density and activity, and the increase in cellular senescence with age.47,48 Cellular senescence can affect the differentiation potential, immunomodulatory abilities, and migratory capabilities of both stem cells and chondrocytes. Thus, older patients with cartilage lesions may require additional treatment measures to enhance stem cell or chondrocyte availability and activity at the lesion site. The MACI technique seeks to address these concerns by expanding autologous chondrocytes and seeding them in media designed to favor differentiation towards mature chondrocytes.49 Cell-free scaffolds (AMIC) rely on the patient’s native cells to infiltrate the graft during implantation, and are thus at an even higher risk of failure in older patients. Thus, researchers have investigated the use of growth factors to increase cell recruitment, improve cell chondrogenesis, and optimize chondrocyte populations for cartilage regeneration therapies. For example, stromal cell-derived factor 1 alpha (SDF-1α) doubled the recruitment of chondrogenic progenitor cells in a hyaluronate-fibrin hydrogel, which formed hyaline cartilage when cultured in a bovine cartilage explant model.50 Local SDF-1α release increased the recruitment of systemically infused bone marrow-derived MSCs by 700% in a mouse model of myocardial infarction (MI),51 suggesting that cartilage repair scaffolds releasing SDF-1α may perform synergistically with MSCs from marrow recruitment or intra-articular injections. Similarly, transforming growth factor-beta 3 (TGF-β3) released from cartilage repair scaffolds improved chondrogenesis in vitro and in large animal models.52,53 Additional examples of bioactive factors used in pre-clinical and clinical trials are included in Table 1, all of which suggest that tailored growth factor delivery may improve cartilage repair in aged patients.

Table 1 Growth factors used to enhance cartilage restoration procedures in recent pre-clinical and clinical trials Full size table

Another technique to enhance the regenerative capacity of cells is to remove neighboring senescent cells. A recent study by Jeon and colleagues showed that senescent chondrocytes accumulate around traumatic cartilage lesions and are associated with the development of arthritis; clearance of these senescent cells, via intra-articular injection of a senolytic molecule, attenuated the development of arthritis in a mouse ACL transection model.54 Another recent study found that rejuvenating aged MSCs with SRT1720, an activator of SIRT1, significantly improved heart function and angiogenesis in a rat MI model compared to control MSCs.55 These potential therapeutics targeted towards rejuvenating, optimizing, and recruiting endogenous stem cells will likely increase the efficacy of cartilage tissue engineering techniques in the older patient population.

For patients with a single cartilage lesion greater than 8 cm2, multiple lesions, or joint wide cartilage degeneration, whole or hemi-joint tissue engineering is an attractive alternative to metal/plastic joint replacement. These ‘living’ implants could potentially last a lifetime, remodeling in response to applied load and continuously generating new matrix. Recent advances in three dimensional (3D) printing56 and rapid prototyping now allow researchers to produce anatomic 3D tissue engineered constructs.57,58 Size matching between native and engineered cartilage is critical to maintain joint mobility and function. Computer assisted design (CAD) programs can translate patient scans, via micro-computed tomography (μCT) or magnetic resonance imaging (MRI), into personalized 3D in silico molds.59 Using layer-by-layer bioprinting, Mao and colleagues’ generated an entire rabbit humeral head using a poly(ɛ-caprolactone) (PCL) and hydroxyapatite scaffold.60 These scaffolds were infused with TGF-β3 to recruit endogenous cells, and inclusion of this growth factor increased cell infiltration into the scaffold by 130%. Another anatomic tissue engineering approach by Moutos and colleagues developed a woven PCL hemispherical scaffold 22 mm in diameter, similarly shaped to the cartilage of the femoral head.61 While Mao and colleagues relied on native cells to populate their scaffold for the rabbit shoulder, Moutos and colleagues seeded their hemispherical scaffold with adipose-derived stem cells (ASCs). ASCs in these woven scaffolds released anti-cytokine factors in a sustained manner to reduce joint inflammation, and showed remarkable mechanical features, with tensile, compressive, and shear properties in the native tissue range.62 This approach could be particularly useful in cases that a large cartilage surface needs to be replaced, and there are signs of joint inflammation. In a third study, Saxena and colleagues used porcine µCT scans to create negative anatomic molds.63 Stem cells encapsulated in a hyaluronic acid hydrogel filled the negative mold, and the resulting tissue was cultured for up to 12 weeks in vitro, exhibiting excellent viability and shape retention. Overall, these studies exemplify how rapid prototyping techniques may be used to generate patient-specific tissue constructs capable of replacing expansive cartilage surfaces.

Other approaches to treat large-scale cartilage defects involve shape-filling chondro-inductive biomaterials. For example, particulated and desiccated allograft tissue, referred to as ‘BioCartilage’,64 has been used to repair cartilage in vivo in an equine cartilage large-defect model. A combination of BioCartilage, microfracture, and platelet-rich plasma showed no signs of synovial inflammation, and had superior histological scoring compared to microfracture controls. Others have also utilized devitalized tissue for cartilage repair, including Detamore and colleagues, who showed that stem cells, in the presence of devitalized cartilage microparticles, produced mechanically robust cartilage tissue.65 Taken together, there has been continued progress in the field of large-scale cartilage repair, with multiple approaches now being tested in clinically relevant large animal models. Table 2 provides examples of the various fabrication methods discussed. These approaches have the potential to address patients with large lesions that are often excluded from cartilage repair trials.

Table 2 Fabrication methods for large cartilage tissue engineering and regeneration therapies in pre-clinical and translational stages Full size table

Cartilage restoration procedures have typically avoided small-size defects (<1 cm2), likely due to relatively positive outcomes with microfracture in defects of this size.66,67 However, the fibrocartilage that forms in these defects is still susceptible to long-term degeneration due to its mechanical insufficiency.6,68 Furthermore, treating partial thickness cartilage defects with microfracture may compromise the healthy intact basal layer of cartilage under the defect. Recent biomaterial technologies have been developed to address small full and partial-thickness lesions, with an emphasis on injectable therapeutics for defect-specific filling and ease of application.69,70 These injectable scaffolds typically revolve around a material solution that is either photo-polymerized71,72 or chemically cross-linked,73,74,75 allowing it to completely fill a defect before solidifying. Combining these scaffold-based injectable formulations with stem cells can also improve the treatment of small focal cartilage injuries. Since cell migration from neighboring cartilage into these small partial-thickness defects is limited,76 encapsulating cells within the injected matrix77,78 may initiate remodeling and extracellular matrix (ECM) formation immediately, facilitating and improving cartilage regeneration.

An array of exclusion criteria (BMI > 35, partial meniscectomy, ligamentous instability, malalignment) are related to mechanical comorbidities that current treatments do not address. Due to these mechanical circumstances, the cartilage in these knees is subject to complex stresses that are significantly greater in magnitude than those the healthy “green” knee would experience. Cartilage repair strategies must account for these stresses early in recovery to avoid failure following implantation and patient remobilization. Therapies that rely on scaffold-free cellular approaches, such as ACI, are not designed to handle these mechanical burdens early post-surgery, and thus, utilizing scaffolds with improved bulk mechanical properties could increase efficacy of treatment and promote earlier return to normal activity in these knees. To review the mechanical properties of cartilage scaffolds over the years, a systematic review was performed (Supplemental Methods). Initial scaffold attempts used simple sponges and hydrogels with moduli (both instantaneous and equilibrium) in the tens of kilopascals (kPa), while the modulus of native cartilage is 20–50 times higher. More recently, groups have utilized a variety of fabrication (weaving,62,79 3D printing,56,80,81 casting57,63) and cross-linking (UV photoinitiator,82,83 EDC crosslinking,83,84,85 dehydrothermal treatment83) techniques to improve the mechanical properties of the time-zero scaffold (Fig. 4—red squares and triangles). For example, Valonen and colleagues developed a 3D-woven poly(ε-caprolactone) (PCL) scaffold with an aggregate modulus of 550 kPa, well within the range of native articular cartilage.86 Other groups have created fiber-reinforced hydrogels, and achieved stiffness values greater than 400 kPa,80,87 resulting in replacement scaffolds for defects that require greater mechanical support. An important consideration is balancing the mechanics of these scaffolds with their resorption and ability to form new cartilage tissue.88,89 A scaffold should provide architecture and support for initial load-bearing, but exhibit a degradation profile that allows infiltrated cells to respond and form neo-cartilage tissue. Alternatively, in vitro culture of constructs52,90 can promote increased deposition and organization of ECM, further elevating mechanical properties of these scaffolds (Fig. 4—green squares and triangles). While more expensive, these lab-grown in vitro engineered replacements have the potential to withstand loadbearing immediately upon implantation, as long as adequate fixation of the grafts can be achieved.

Fig. 4 Modulus values (kPa) as a function of time (Jan 2001 to Jan 2018). Squares and triangles represent instantaneous and equilibrium moduli, respectively. Red, green, and blue points represent time-zero scaffold, cultured construct, and mechanical assessments from in vivo studies, respectively. Survey of PubMed literature utilizing search terms “Cartilage”, “Scaffold”, and “Modulus”. Studies with inadequate description of testing methods were excluded Full size image

Fig. 5 Summary figure showing emerging translational therapies for potential application in the ‘Red Knee’, including therapies that address large lesions, mechanical demands, subchondral damage, small lesions, aging, disease, and inflammation Full size image

Another important mechanical exclusion criterion in cartilage repair procedures is the presence of kissing lesions. Due to the continuous contact and articulation between two kissing implants, surface frictional and shear properties are of vital importance. Likewise, ligamentous instability and malalignment can exacerbate stresses parallel to the articular surface, increasing shear forces experienced by the implant and repair tissue.91,92,93 Therefore, along with bulk compressive mechanical properties, any cartilage replacement should minimize friction and maximize shear resistance to prevent wear or implant dislodgement. One study successfully increased lubricin (lubricating protein) concentration in superficial zone chondrocytes;94 this advance could be applied to cartilage scaffolds in order to reduce the coefficient of friction at the articulating surface. Another modality to decrease friction, that also provides shear resistance, is fabricating scaffolds with an aligned superficial zone.95,96 For example, Accardi and colleagues97 found that varying the alignment of electrospun nanofibers had a positive effect on implant shear properties. The group additionally varied electrospinning speed during fabrication to tune fiber organization through the scaffold depth to provide a shear-resistant superficial layer. These adaptations could be considered in knees requiring supplemental mechanical stability.

A previous attempt at cartilage repair is often an exclusion criterion for a subsequent cartilage regenerative procedure. This is driven by the likelihood of subchondral bone changes as a consequence of failed repair, particularly if the first attempt involved disruption of the tidemark for marrow recruitment. Without sufficient subchondral load support, subsequent cartilage treatment approaches may be predisposed to failure, and thus, the entire osteochondral unit must be considered in this cohort of patients. One potential intervention, currently used clinically, is osteochondral allograft transplantation (OATS).98,99 These grafts can provide symptom relief and success as a salvage procedure following failed cartilage repair. However, issues with graft survivability, disease transmission, and integration have motivated tissue engineers to develop newer composite scaffolds that guide localized regeneration of the cartilage and bone layers of an osteochondral unit.100,101 Clinically, the TruFit Plug (Smith & Nephew, San Antonio TX) is one of the only synthetic osteochondral scaffolds that has been evaluated in patients,102,103 with a subchondral phase containing calcium sulfate for bone regeneration and an articulating phase that relied on marrow stimulation for cartilage regeneration. While short-term results showed clinical improvement in patient MRI scores, improvement over conventional microfracture/OATS procedures has not been proven. In order to improve outcomes, biphasic scaffolds can be created with a softer upper layer containing chondrogenic factors (e.g., TGF-β, chondroitin sulfate) and a stiffer lower layer, often loaded with calcium phosphate, hydroxyapatite, or bone morphogenetic protein (BMP), to provide structural support of the above cartilage layer and to promote osteogenic tissue formation and boney integration.53,104,105,106 Bi-layered scaffolds derived from articular cartilage ECM and growth plate ECM can be used to regenerate osteochondral tissue with better recapitulation of the native architecture.107

To avoid the interfacial shear stress that are experienced between two such distinct layers, recent studies have developed gradient scaffolds, using the same growth factors and scaffold materials as a biphasic scaffold, but with a smooth transition between layers.108,109 For example, Di Luca et al.109 used a brush functionalization technique to create a gradient of TGF-β3, decreasing in concentration from the articulating surface to the subchondral region, and likewise created a reverse gradient of BMP-2. Other studies have utilized growth factor gradients, for example via microsphere incorporation.108,110 Furthermore, a transitional zone between cartilage and bone layers has been achieved via dispersion mixing with syringe pump systems, selective laser sintering, and pore shape gradients.111,112,113,114 The field of osteochondral tissue engineering has matured substantially and progress towards clinically applicable replacements will be vital for the large portion of the population currently excluded from clinical trials.

Patients with inflammation of the joint are often excluded from cartilage repair attempts given that catabolic cytokines and proteinases (MMPs) present in the synovial fluid degrade the native ECM, and would similarly degrade any implanted tissue, predisposing treatment failure. Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNFα) are two important inflammatory cytokines that not only lead to cartilage matrix destruction, but also prevent chondrogenic differentiation of mesenchymal stem cells.115 These cytokines can be systemically upregulated, or produced by synoviocytes, chondrocytes, or meniscal cells.116 Also, co-morbidities, such as obesity, can elevate levels of pro-inflammatory cytokines, exacerbating the effects of a joint injury.117

To control the inflammatory environment and reduce proteolysis, intra-articular injection is clinically appealing. Such a treatment could be administered following a traumatic joint injury to prevent cartilage ECM proteolysis in the presence of pro-inflammatory cytokines (IL-1, IL-6, IL-8, and TNF-ɑ) and MMPs. To address this possibility, in a number of animal118,119 and human studies,120 high doses of anti-catabolic glucocorticoids, which inhibit the activation of MMPs121 and the expression inflammatory cytokines,122 have been administered. Dexamethasone (DEX) inhibits inflammation and cartilage damage by influencing both synoviocytes and chondrocytes.119,123 However, given the dynamic environment of the knee and the low residence time of small molecules in the synovial space, delivery and retention of molecules to positively impact cartilage regeneration before being cleared remains a challenge. Targeted intra-articular delivery of DEX can be achieved by using small positively charged nanoparticles bound to DEX to form electrostatic interactions between the cationic particle and the anionic cartilage matrix.124 Dendrimer-based nanocarriers were also recently shown to penetrate full-thickness cartilage explants, and be retained in a native joint environment.125 These nanoparticles may be powerful carriers for a glucocorticosteroid treatment.

In addition to localized glucocorticosteroid delivery, IL-1 receptor antagonist (IL-1Ra), a naturally occurring inhibitor of IL-1 activity, is another potential avenue for intra-articular anti-inflammatory therapeutics. There are currently multiple IL-1 targeting drugs on the market including Anakinra, a modified version of the human IL-Ra protein, and Canakinumab, a monoclonal antibody targeted at IL-1β. However, neither of these drugs has improved OA symptoms in human clinical trials to date, likely because of the dynamic joint environment.126,127 For either glucocorticosteroid or IL-1Ra delivery to be successful, the delivery mechanism (nanoparticles, scaffolds, etc.) is an extremely important design consideration. To improve the efficacy of IL-1Ra, researchers have tethered the protein to nanoscale block polymers to target cartilage.128,129 The IL-1Ra tethered polymeric nanoparticles were stable, non-toxic, and effective at blocking the IL-1 signaling pathway. In another study, IL-1Ra transgenes were incorporated into a woven scaffold.61 As a result, the stem cells in the scaffold released IL-1Ra in a sustained fashion over the course of 28 days in culture. Gene delivery of IL-1Ra through a regenerative scaffold or direct delivery of IL-1Ra via nanoparticle carriers are both promising options for cartilage repair in patients with post-surgical inflammation and other chronic inflammatory conditions.