Table 1.

Scaffold-Based Interface Tissue Engineering Strategies for Osteochondral Regeneration

Study type	Objective	Fabrication method	Osteochondral scaffold layers	Scaffold constituents	Cells	Mechanical properties reported	Observations	References
In vitro	Optimize the effects of chondrocyte density on matrix production and on mechanical properties of the cartilage region. Evaluate the effects of the BG phase of the PLGA-BG composite on chondrocyte mineralization potential and the formation of a calcified cartilage region	Casting	Hyaline cartilage (cartilage region). Calcified cartilage (interface region). ScB (bone region)	Agarose 45S5 BG PLGA	Bovine articular chondrocytes. Bovine osteoblasts	Compressive modulus	The stratified scaffold supported the region-specific coculture of chondrocytes and osteoblasts that promotes the formation of three distinct yet continuous regions of cartilage, calcified cartilage, and bone-like matrices. Higher cell density improved chondrogenesis and enhanced the scaffold's mechanical properties. PLGA-BG microspheres were found to increase chondrocyte mineralization potential and were required for the formation of a calcified cartilage and bone layers of the scaffold	Jiang et al.54
In vitro	Compare impact of hydroxyapatite on hypertrophic and non-hypertrophic chondrocytes in a composite scaffold Optimize the particle size and concentration of hydroxyapatite in composite scaffolds	Casting	Calcified cartilage (monophasic construct)	Agarose. Hydroxyapatite	Bovine articular (deep zone) chondrocytes	Complex shear modulus. Compressive modulus. Phase angle	Hypertrophic chondrocytes exhibited higher levels of matrix deposition and mineralization potential with addition of hydroxyapatite, without affecting deep zone chondrocyte biosynthesis and hypertrophy Higher matrix content corresponded to significant increases in both compressive and shear mechanical properties. Matrix deposition was higher only with the addition of micron-sized ceramic particles, while cell hypertrophy was independent of ceramic size	Khanarian et al.55
In vitro	Determine the response of chondrocytes to a composite alginate-HA scaffold and identify chondrocyte density that optimizes calcified cartilage formation	Casting	Calcified cartilage	Alginate. Hydroxyapatite	Bovine articular chondrocytes	Complex shear modulus. Compressive modulus	Composite HA/alginate scaffolds promoted the formation of a proteoglycan and type II collagen rich matrix when seeded with chondrocytes from the deep zone of cartilage. Composite scaffold exhibited increased compressive and shear moduli	Khanarian et al.56
In vitro In vivo (rabbit)	Evaluate the impact of a compact layer in a biphasic scaffold in promoting osteochondral tissue regeneration	Chondral phase: modified temperature gradient-guided TIPS technique. Compact layer: phase separation. Bony phase: rapid prototyping technique	Hyaline cartilage (chondral phase). Calcified cartilage (compact layer). ScB (bony phase)	Bovine decellularized cartilage ECM. Poly(lactic-co-glycolic acid) (PLGA) β-TCP Type I collagen	Rabbit bone marrow MSCs	Maximal shear strength (N/m2). Maximal tensile strength (N/m2)	Scaffolds with a compact layer displayed superior glycosaminoglycan and collagen content over controls without the compact layer in vivo. Anti-tensile and anti-shear properties were significantly enhanced within scaffolds containing the compact layer	Da et al.57
In vitro	Design a bilayered scaffold using ACECM and hydroxylapatite. Assess the impact of scaffold permeability on cartilage defect healing and cell behavior	Casting via liquid-phase cosynthesis technique and TIPS technique	Hyaline cartilage (porous upper layer). Calcified cartilage (dense mineralized lower layer)	ACECM. Hydroxyapatite	Rabbit chondrocytes	Compressive modulus. Interface shear stiffness	Gradual interfacial region was formed with pore sizes varying from 128.2 ± 20.3 μm in the mineralized component and 21.2 ± 3.1 μm in the nonmineralized component. Scaffold permeability decreased with increasing compressive strain and hydroxyapatite content while shear stiffness was higher in scaffolds containing lower concentration of hydroxylapatite. Chondrocytes could not penetrate the interface but were responsible for abundant matrix deposition in the upper layer	Wang et al.58
In vitro	Engineer hyaline and calcified cartilage layers via scaffold-free engineering	Gravity sintering for calcium polyphosphate disks. Sol-gel processing for hydroxyapatite coating	Hyaline cartilage. Calcified cartilage	Calcium polyphosphate. Hydroxyapatite	Sheep bone marrow stromal cells predifferentiated into chondrocytes	Compressive modulus. Interface shear strength	The engineered construct consisted of a hyaline cartilage zone rich in proteoglycans and collagen type II, as well as a highly mineralized calcified cartilage zone with type X collagen. Constructs that included the calcified interface had compressive strength on par with native sheep tissue and higher shear strength than uncalcified constructs	Lee et al.59
In vitro	Investigate the effects of extracellular calcium concentration on hASC chondrogenesis and the potential to use controlled calcium delivery to induce site-specific chondrogenesis and osteogenesis using hASCs in a single osteochondral scaffold	Electrospinning	Hyaline Cartilage layer (three layers). ScB (two layers). Nanofibrous scaffolds stacked together using type I collagen gel	PLA β-TCP Type I collagen	hASCs	Not applicable	Elevated extracellular calcium levels induced osteogenesis of hASCs while inhibiting chondrogenesis. The multilayer stacked nanofibrous construct with regional incorporation of TCP nanoparticles enabled layer-specific hASC differentiation within the same structure	Mellor et al.60
In vitro	Evaluate the efficacy of a dual chambered well system for simultaneously providing osteogenic and chondrogenic stimulation to different layers of an osteochondral scaffold	Freeze-drying	Hyaline cartilage (chondrogenic region). Calcified cartilage (middle region). ScB (osteogenic region)	RADA self-assembly peptide. Silk fibers (Bombyx mori)	Rabbit bone marrow stromal cells	Compressive load	The specially designed two-chambered well could successfully provide specific chemical stimulation to BMSCs located in different regions of a single scaffold, leading to the formation of distinct hyaline cartilage, calcified cartilage, and ScB layers. Cells in the intermediate region were found to be hypertrophic chondrocytes that produced a matrix of GAGs and collagen types I, II and X.	Chen et al.61
In vivo (goat)	Assess the ability of a multilayered collagen-based scaffold to regenerate and repair osteochondral tissue in two surgically created critical sized osteochondral defects within the caprine stifle joint.	Casting. Iterative freeze-drying method	Hyaline cartilage. Calcified cartilage. ScB	Hyaluronic acid. Hydroxyapatite. Type I collagen. Type II collagen	Acellular	Not applicable	Compared with the bilayered synthetic polymer scaffold, the multilayered scaffold improved regeneration upon evaluation of repair up to 12 months, with a zonal architecture comparable to that of native osteochondral tissue. These scaffolds demonstrated increased cartilage thickness and superior levels of ScB formation in the multilayered scaffold group compared with empty and synthetic polymer scaffold groups	Levingstone et al.62
In vitro	Evaluate the impact of calcium phosphate ratio on deep zone chondrocytes within hydrogel-ceramic hybrid scaffolds	Casting	Calcified cartilage (monophasic construct)	Agarose CDA	Bovine articular (deep zone) chondrocytes	Complex shear modulus. Compressive modulus. Phase angle	Higher calcium-phosphorus ratio increased chondrocyte proliferation and glycosaminoglycan production while the group with a lower calcium–phosphorus ratio produced results on par with a ceramic-free control	Boushell et al.63
In vitro	Fabricate and characterize a triphasic, anisotropic scaffold	Freeze casting and lyophilization	Hyaline cartilage (superficial and transition zones). Calcified cartilage (calcified cartilage zone). ScB (osseous zone)	Collagen. Hyaluronic acid. Hydroxyapatite	Acellular	Collapse plateau modulus. Compressive modulus. Elastic compressive strength	Zone-specific localization of hyaluronic acid resembling the depth-dependent increase in glycosaminoglycans of native osteochondral unit. Compressive testing revealed depth-dependent increase in stiffness and that the compressive moduli of the chondral and osseous zones was within the range needed to promote chondrogenic/osteogenic differentiation of MSCs	Clearfield et al.64
Clinical study (human RCT)	Evaluate the efficacy of a multiphasic collagen-hydroxyapatite-based scaffold in patients suffering from osteochondral knee defects	Physical combination of separately prepared layers. Freeze-drying	Hyaline cartilage (100% type I collagen with smooth surface). Calcified cartilage (intermediate layer; 40% Mg-HA and 60% type I collagen). ScB (70% Mg-HA and 30% type I collagen)	Hydroxyapatite. Type I collagen	Acellular	Not applicable	Two-year follow-up showed that patients with deep osteochondral lesions or sport active patients that received the coll-HA scaffold responded significantly better than the control group patients treated with BMS. Although there was no statistically significant difference between coll-HA scaffolds and BMS for chondral lesions, the procedure demonstrated potential to treat osteochondral lesions with coll-HA scaffolds	Kon et al.65
In vitro In vivo (murine)	Develop a bizonal scaffold that can induce in vivo regeneration of cartilage while preventing mineralization	Casting	Hyaline cartilage. Calcified cartilage	Heparin StarPEG	Porcine articular chondrocytes in superficial layer. Porcine bone marrow MSCs in calcified cartilage layer	Not applicable	Bizonal StarPEG/heparin scaffold supplemented with cell-type mediated spatiotemporal regulation allowed for growth of bizonal cartilage with stable calcified cartilage layer	Kunisch et al.66
In vitro	Assess the impact of different formulations of ScB scaffold degradation parameters including mass loss, change in environmental pH, bioactivity, compressive mechanics. Improve the material homogeneity and compressive mechanics of ScB construct. Construct multilayered osteochondral scaffold by combining the ScB construct with a cartilage analog	Casting	Hyaline cartilage Calcified cartilage ScB	Hydroxyapatite PEG PLGA	Human bone marrow derived stromal cells	Hysteresis Complex, storage, and loss modulus Compressive modulus Phase angle	ScB construct formed the foundation of a cytocompatible, multi-layered osteochodnral constructed that supported a mechanically competent cartilage layer Optimized ScB construct did not alter pH upon degradation, exhibited bioactivity, and had significantly greater compressive mechanics compared to other constructs	Marionneaux et al.67
In vitro In vivo (rabbit)	Investigate how Ica-HA/Col hydrogel scaffolds respond to the different culture conditions, stimulate the chondrogenic and osteogenic differentiation of BMSCs, and facilitate the deposition of calcified layer matrix in different inductive media	Casting	Monophasic construct	Ica-HA. Type I collagen (Col)	Rabbit bone marrow MSCs	Compressive modulus	Ica-HA/Col hydrogel enhanced the osteogenic and chondrogenic differentiation of BMSCs in vitro. Ica-HA/Col hydrogel promoted the synthesis of type X collagen and deposition of calcium salt in mixed chondrogenic/osteogenic inductive media. In vivo rabbit model study demonstrated that Ica-HA/Col constructs had a potential to facilitate the reconstruction of the osteochondral interface	Yang et al.68
In vivo (porcine)	Determine whether hydrogel-filled PCL-constructs with a chondrocyte-seeded upper layer and an MSC-seeded bottom layer deemed to induce calcified cartilage can improve cartilage regeneration of superficial osteochondral defects in vivo	Casting (hydrogel). 3D printing (PCL mesh)	Hyaline cartilage (upper layer). Calcified cartilage and ScB (bottom layer)	Heparin PCL StarPEG or StarPEG-MMP-conjugates	Porcine articular chondrocytes. Porcine bone marrow mesenchymal stromal cells	Hardness	Grafts showed comparable hardness at implantation and did not cause visible signs of inflammation. After 6 months, μCT analysis revealed significant bone loss in both treatment groups compared with the control. Some parts of the PCL mesh and hydrogel were retained in all defects, but most implants were pressed into the ScB	Bothe et al.69
In vitro In vivo (goat)	Assess the potential of osteochondral tissue-specific ECM-derived scaffolds to spatially direct MSC differentiation in vitro. Determine the mechanics by which the scaffolds can direct joint repair in vivo following their cell-free implantation in critically sized osteochondral defects	Iterative freeze-drying process	Hyaline cartilage. ScB	ACECM (cartilage layer). Growth plate ECM (ScB layer)	Porcine bone marrow derived MSCs (for in vitro evaluation). Acellular (in vivo)	Not applicable	Scaffolds could spatially direct stem cell differentiation in vitro, promoting the formation of graded cartilage tissue transitioning from calcified cartilage to hyaline cartilage. Over 12 months in vivo, the bilayered ECM derived scaffolds promoted the regeneration of hyaline cartilage with a collagen fiber architecture recapitulating the native tissue compared to commercially available control scaffolds	Cunniffe et al.70
In vitro In vivo (rabbit)	Fabricate and assess regenerative performance of gradient osteochondral scaffold	Electrospinning (single-layer fibrous mesh). Gradient 3D scaffold constructed by stacking different cell/mesh complexes layer-by-layer	Hyaline cartilage (four layers). Calcified cartilage (interface; four layers). ScB (four layers)	Gelatin PLA	Rabbit bone marrow MSCs that were predifferentiated in three different culture conditions (chondrogenic, mixed, and osteogenic)	Not applicable	The multilayered osteochondral scaffolds regenerated distinct osteochondral layers better than homogenous constructs within rabbit knee defect model	Jin et al.71
In vitro	Assess the regenerative potential of triphasic osteochondral scaffolds fabricated via an iterative layering process with biomimetic ratios of natural ECM components	Hyaline and calcified cartilage layers: thermal gelation. ScB layer: freeze-drying method All layers combined by iteratively overlaying each layer on top of the other	Hyaline cartilage. Calcified cartilage. ScB	Chitosan. Hydroxyapatite. Type I collagen. Type II collagen	Murine chondrocytes (ATDC5). Murine preosteoblasts (MC3T3-E1)	Not applicable	Final stratified scaffold demonstrated compact structure and no separation upon fabrication. Coculture of preosteoblasts and chondrocytes within the scaffold showed high viability and potential for selective maintenance of layer-specific cells without the addition of growth factors. ECM production was enhanced upon 21-day culture	Korpayev et al.72
In vitro In vivo (rabbit)	Investigate the role of a calcified cartilage layer and adipose tissue derived stem cells in promoting osteochondral regeneration in a rabbit model	Paraffin-sphere leaching and modified temperature gradient-guided TIPS technique	Hyaline cartilage. Calcified cartilage. ScB	Hydroxyapatite. Silk fibroin	Rabbit adipose tissue-derived MSCs	Mechanical stiffness (N/m)	Scaffolds containing both adipose tissue-derived stem cells and a calcified cartilage layer outperformed other scaffolds in terms of biomechanics and promoting osteochondral regeneration within rabbit osteochondral defects	Zhao et al.73
In vivo (porcine)	Explore the use of natural calcified cartilage zone in trilayer osteochondral scaffolds. Elucidate the role of the calcified cartilage zone in the osteochondral repair process	Type II collagen sponge grafted onto decellularized scaffolds consisting of ScB with or without native calcified cartilage zone, and then lyophilized	Hyaline cartilage (chondral phase). Calcified cartilage. ScB	Porcine decellularized calcified cartilage and/or ScB. Type II collagen	Porcine bone marrow stem cells	Not applicable	Implants that contained natural calcified cartilage layer were superior in promoting osteochondral repair in a minipig model compared with the control group treated with scaffolds without the calcified cartilage layer	Huang et al.74
In vivo (sheep)	Evaluate the healing process of osteochondral defects in the sheep stifle joint supported by an acellular porous PHB/chitosan-based implant	Casting	Single layer	CHIT PHB	Acellular	Not applicable	The healing osteochondral defect was comparable with the intact cartilage at the surface of the defect upon MRI evaluation 6 months after surgery. CT scan showed low regenerative potential of the implant at the osseous zone of defect compared with chondral zone. Hyaline-like cartilage was observed in most of the treated animals, except for one that healed with fibrocartilage formation	Petrovova et al.75

μCT, micro-computed tomography; β-TCP, β-tricalcium phosphate; 3D, three-dimensional; ACECM, articular cartilage extracellular matrix; BG, bioactive glass; BMS, bone marrow stimulation; BMSCs, bone marrow stromal cells; CDA, calcium-deficient apatite; CHIT, chitosan; ECM, extracellular matrix; GAGs, glycosaminoglycans; hASC, human adipose-derived stem cell; Ica-HA, icariin-conjugated hyaluronic acid; MRI, magnetic resonance imaging; MSC, mesenchymal stem cell; PCL, polycaprolactone; PEG, polyethylene glycol; PHB, polyhydroxybutyrate; PLA, polylactic acid; PLGA, poly(lactic-co-glycolic acid); RCT, randomized clinical trial; ScB, subchondral bone; StarPEG, star-shaped poly(ethylene glycol); TIPS, thermal-induced phase separation.