Bioinspired Scaffolds for Osteochondral Regeneration

Abstract

Osteochondral defects are difficult to treat because the articular cartilage and the subchondral bone have dissimilar characteristics and abilities to regenerate. Bioinspired scaffolds are designed to mimic structural and biological cues of the native osteochondral unit, supporting both cartilaginous and subchondral bone repair and the integration of the newly formed osteochondral matrix with the surrounding tissues. The aim of this review is to outline fundamental requirements and strategies for the development of biomimetic scaffolds reproducing the unique and multifaceted anatomical structure of the osteochondral unit. Recent progress in preclinical animal studies using bilayer and multilayer scaffolds, together with continuous gradient scaffolds will be discussed and placed in a translational perspective with data emerging from their clinical application to treat osteochondral defects in patients.

Introduction

Osteochondral defects, the type of articular cartilage defects extending deep into the subchondral bone, remain a considerable problem in reconstructive cartilage surgery.¹ If untreated, they do not heal and osteoarthritis (OA) may develop over time.² Yet, such osteochondral defects are difficult to treat because the subchondral bone and the articular cartilage are tissues with very dissimilar intrinsic healing capacities.^3–5 Structural changes of the subchondral bone resulting from inferior subchondral bone repair⁶ translate into altered biomechanical properties of the entire osteochondral unit and influence the long-term performance of the cartilaginous repair tissue.⁵ This underlines the importance of the subchondral bone as the key foundation for successful cartilage repair.⁷ The surgical therapy of osteochondral defects therefore needs to consider in unison the articular cartilage and the subchondral bone.⁸

Biomimetic scaffolds are engineered to reflect anatomical and physiological hierarchical structures.^9–14 In theory, this aim can be achieved by a defined structure or chemical composition of a material. For osteochondral repair to occur, highly specialized scaffolds mimicking the hierarchical anatomical architecture of the natural osteochondral unit are needed.¹⁴ While classically biphasic materials consisting of a single cartilage and bone part have been described, it is now clear that many other anatomical aspects of these both tissues combined in the osteochondral unit need to be reproduced.

The aim of this review is to outline fundamental requirements and strategies for the development of biomimetic scaffolds reproducing the unique and multifaceted anatomical structure of the osteochondral unit. Recent progress in preclinical animal studies using bilayer and multilayer scaffolds, together with continuous gradient scaffolds will be discussed and placed in a translational perspective with data emerging from their clinical application to treat osteochondral defects in patients.

Clinical Aspects of Osteochondral Repair

By definition, osteochondral defects disrupt the integrity of both the articular cartilage and the subchondral bone, in contrast to chondral defects, where the subchondral bone is not damaged (partial-thickness), or merely exposed (full-thickness) (Fig. 1). Pain is often the key clinical symptom, together with an impaired joint function and quality of life. Moreover, untreated osteochondral lesions can lead to OA¹⁵ resulting from the joint incongruence and the abnormal biomechanical loading patterns of the adjacent articular cartilage.¹⁶ From a clinicopathological standpoint, the underlying problem causing an osteochondral defect is of high importance.⁸ The subchondral bone lesions that are the hallmark of osteochondral defects mainly occur in the course of diseases of the subchondral bone such as osteochondritis dissecans (OCD) or as a result of osteochondral fractures (Fig. 2).^17,18 As these defects are well defined and often deep, they represent the best indications to implant bioinspired osteochondral scaffolds. In general, the paramount goal is to reconstruct an anatomical joint surface, resembling as closely as possible the normal structure of the osteochondral unit. Currently, small osteochondral lesions may be treated using osteochondral transplants, while the sandwich technique (comprising the grafting of compacted autologous cancellous bone into the subchondral bone defect together with the implantation of articular chondrocytes (ACI) in a three-dimensional bioresorbable matrix) is an excellent option for large defects.^19,20 Ideally, a single type of bioinspired osteochondral scaffold would be applicable to both small and large defects, although fixation requirements may differ: small osteochondral lesions could be treated with a press-fit approach, while larger lesions might necessitate an additional fixation.

FIG. 1.

Classification of articular cartilage defects. In chondral defects, the subchondral bone is not damaged (partial-thickness), or only exposed (full-thickness). Osteochondral defects, in contrast, disrupt the integrity of both the articular cartilage and the subchondral bone. Color images available online at www.liebertpub.com/tea

FIG. 2.

Macroscopic view of an osteochondral defect resulting from osteochondritis dissecans (OCD) in a 30-year-old man. The osteochondral defect (arrow) is located in the lateral aspect of the medial femoral condyle, a typical location. The circular articular cartilage defect has a diameter of about 35 mm. The subchondral lesion reaches about 20 mm deep into the subarticular spongiosa. Color images available online at www.liebertpub.com/tea

Applied Anatomy of the Osteochondral Unit

The osteochondral unit is composed of the articular cartilage and the underlying subchondral bone.⁸ This vital region has a multifaceted structure,²¹ connecting the articular cartilage through the calcified cartilage with the subchondral bone.

Articular cartilage

Articular cartilage is a highly specialized connective tissue that provides joints with a low friction environment. Lack of vascularization, low cellularity, and the limited metabolic activity of mature chondrocytes, which are the unique cellular component resident within cartilage, are key features of this tissue.²² Type-II collagen is the main type of collagen present.²³ Proteoglycans are another fundamental component of the cartilage extracellular matrix (ECM). Because of their negative charges, proteoglycans are able to retain water molecules (65–80% of wet weight) within cartilage. This allows the load-dependent deformation of the cartilage.²⁴

Articular cartilage has a highly anisotropic architecture, being composed of multiple layers that differ in terms of cell and collagen fibril orientation. The superficial zone contains small flattened chondrocytes and collagen fibrils tangentially oriented to the articular surface that provide resistance to compressive loads and the gliding surface. The transition zone encloses rounded chondrocytes that secrete type-II collagen and aggrecan and includes obliquely oriented collagen fibrils. The deep radial zone of articular cartilage presents chondrocytes arranged in columns and collagen fibrils perpendicular to the articular surface and in parallel to each other.^24,25 The layer of calcified cartilage²⁶ connects the articular cartilage with the subchondral bone plate and is about 20–250 μm thick.²⁷ It is separated from the articular cartilage by the tidemark, a basophilic line on histological sections.^28,29 The calcified cartilage contains also type-X collagen. Type-II collagen fibrils extend from the noncalcified articular cartilage into the calcified cartilage. As organization of the ECM is fundamental to grant functionality of articular cartilage, one of the main challenges for cartilage tissue engineering remains the high fidelity reproduction of these layers with proper orientation of collagen fibers.⁴

Subchondral bone plate and subarticular spongiosa

The subchondral bone is composed of the subchondral bone plate and the subarticular spongiosa. Cancellous bone plates join together in the subchondral bone plate to enclose few narrow intervening spaces. While denser than the subarticular spongiosa, the subchondral bone plate is relatively thin in normal human subchondral bone.³⁰ It is broader and denser in osteoarthritic joints. The intervening spaces are gradually enlarged and become elongated in a direction parallel to the diaphysis in deeper regions of the subchondral bone, forming the subarticular spongiosa.⁸ Thus, the subchondral bone plate has a low total porosity, while the porosity of the subarticular spongiosa is much larger.

Connection of the articular cartilage with the subchondral bone

The junction of the calcified cartilage with the subchondral bone is called cement line.⁸ This connection is of major importance for maintaining the osteochondral integration. Here, no collagen fibrils extend from the calcified cartilage (containing mainly type-II and -X collagen) into the subchondral bone plate (containing mainly type-I collagen). This junction is also a site of active remodeling.⁸ Of note, a surprisingly high number of arteries, veins, and nerves send minute branches through canals in the subchondral bone plate into the calcified cartilage. Nutrients can therefore reach chondrocytes in the calcified and articular cartilage.³¹ Thus, the osteochondral connection is not impermeable, but a structural and functional unit allowing both mechanical and biochemical interactions.⁸

Requirements of Biomimetic Scaffolds for Osteochondral Repair

Biomimetic scaffolds need to provide the multifaceted signals in a spatial and temporal pattern allowing the regeneration of the entire osteochondral unit. The presence of the articular cartilage and the subchondral bone as the two major tissues commands the design of biphasic, monolithic scaffolds. Here, the cartilage part supports chondrogenesis, while the bone part serves as a template for osteogenesis. Although such scaffolds diminish limitations of homogeneous single phase scaffolds that lack the structure required to regenerate the entire osteochondral unit,³² they do not resemble its complex structure, necessitating biomimetic scaffolds composed of multiple integrated layers corresponding to the different components of the osteochondral unit (Fig. 3). The design of a unique scaffold reflecting both the complexity of the osteochondral unit and satisfying all requirements is particularly challenging, since the subchondral bone and articular cartilage are characterized by distinct features of matrix composition and organization, vascularization, and metabolic requirements (Table 1). Interestingly, many of these needs have been individually addressed so far and even found entry into the clinics, for example by using bone substitutes to fill large bone defects and by applying bioresorbable matrices seeded with autologous chondrocytes in the case of ACI for large cartilage defects.^19,33,34 In contrast, only case reports are available for the treatments of osteochondral defects.

FIG. 3.

Bioinspired design of osteochondral multiphasic scaffolds. (a) Schematic of a possible design of a multiphasic scaffold resembling the anatomical complexity of the osteochondral unit. The superficial layer of the scaffold is thought to shield the layers below from the stresses applied within the joint, allowing for an undisturbed formation of the cartilaginous repair tissue. The underlying layer should mimic both the transitional and the radial zone of articular cartilage with tangentially oriented fibers in the upper part and vertically oriented fibers in the deeper part. The scaffold layer resembling the calcified cartilage could contain biomineralization cues to allow for the calcification of this region and for the deposition of type-X collagen. The central zone of integration represents the cement line. Because this interface combines the cartilage and bone layers, significant biomechanical strength is needed to avoid delamination in the presence of biomechanical stimuli related to joint function. Then, the layer mimicking the subchondral bone plate should resemble the dense cancellous bone plates that join together to enclose narrow intervening spaces. This part of the scaffold shields the subarticular spongiosa from the synovial fluid whose presence might result in the formation of subchondral bone cysts and/or sclerosis. Finally, a highly porous structure with large pores elongated in a direction parallel to the diaphysis should be included to resemble the subarticular spongiosa. Note that this schematic drawing does not reflect the original dimensions of each part of the human osteochondral unit in the knee joint. (b) Integration of osteochondral scaffolds with osteochondral defects. A fundamental requirement is that the subchondral part of the scaffold is able to bond with the surrounding bone. Moreover, the osteochondral construct needs to be resorbed in a rate that is adapted to the new bone and cartilage formation. Since these phenomena do not occur with the same rate, materials with different resorption rates might be needed for the cartilage and subchondral bone layers. Color images available online at www.liebertpub.com/tea

Table 1.

Scaffold Requirements for an Anatomic Reconstruction of the Osteochondral Unit

Anatomical layer	Scaffold requirement(s)
Superficial zone of articular cartilage	Superficial layer. This surface protects the deeper layers from the stresses applied within the joint. Needs to ease cartilage gliding.
Transitional zone of articular cartilage	Transitional layer. Porous structure to allow for cartilaginous repair tissue formation. Biomaterial fibrils are placed tangentially. May be seeded with autologous cells.
Radial zone of articular cartilage	Radial layer. Porous structure to allow for cartilaginous repair tissue formation. Biomaterial fibrils are placed perpendicular to the articular surface and in parallel to each other. May be seeded with autologous cells.
Tidemark	Strong bonding needed when separate tangential and calcified layers are included in a scaffold. Biomaterial fibrils from radial layer may cross and extend into the calcified cartilage layer.
Calcified zone of articular cartilage	Calcified cartilage layer. Could contain cues to allow for calcification. Porous structure to allow for cartilaginous repair tissue formation. Biomaterial fibrils from radial layer extend into this layer. May be seeded with autologous cells.
Cement line	Zone of integration. Strong bonding mandatory between the articular cartilage and subchondral bone layers.
Subchondral bone plate	Needs to reflect the dense cancellous subchondral bone, may need to shield the subarticular spongiosa from the synovial fluid.
Subarticular spongiosa	Highly porous structure representing the natural subarticular spongious bone structure.

Requirements to restore the articular cartilage

Requirements for scaffolds solely aimed at restoring the articular cartilage have been the subject of excellent reviews.^35–37 Toward the joint surface, a smooth gliding part needs to protect the opposing cartilage, to allow for gliding of the joint and to protect to some extent the cartilaginous repair tissue that is developing within the scaffold. Next, a layer resembling more or less the transitional zone may follow. Then, a layer mimicking the columnar orientation of the radial zone with scaffold fibers that are aligned rectangular to the articular surface is needed. Here, it remains to be seen whether the commonly used type-I collagen scaffolds could not be further improved, for example by incorporating type-II collagen.³⁸ Very recent studies have demonstrated that a distinct superficial layer in engineered cartilage can be generated applying oriented nanofibrous scaffolds³⁹ or hydrodynamic stimulation mimicking the flow motion between the articulating surfaces in the synovial joint.⁴⁰ Moreover, Thorpe et al. have reported that the radial confinement of agarose hydrogel-based scaffolds and the simultaneous application of a dynamic compressive load generate an oxygen gradient and increase strains across the top of the construct, resulting in a depth-dependent zonal variation in both the biochemical composition and compressive properties of the engineered tissue.⁴¹

Requirements to restore the calcified cartilage

This part of the scaffold should allow mineralization. Specific material compositions can direct bone marrow-derived mesenchymal stem cells (BMSC) into distinct types of chondrocytes⁴² and layer-by-layer organization of these materials results in a gradient of type-II and -X collagen that translates into an increased compressive modulus from the superficial to bottom layer.⁴³ The group of Rita Kandel mimicked the zonal organization of the osteochondral unit in biphasic constructs composed of cartilaginous tissue anchored to the top surface of porous calcium polyphosphate with a calcified interface as a bone substitute in vitro.⁴⁴ Of note, the shear properties of the cartilage–subchondral bone interface were enhanced as a result of the presence of a calcified cartilage layer, highlighting the importance of such a mineralized zone.

Requirements to restore the subchondral bone–cartilage interphase

The union between the chondral and subchondral bone layers needs to be stable enough to withstand the shear stress related to joint movement, to allow optimal load bearing⁴⁵ and to prevent a possible delamination of the cartilage layer that would be deleterious in vivo.^46,47 The design of heterogeneous scaffolds composed of a single material displaying differential growth factor enrichment, porosity, or composition in the two integrated layers, or the generation of scaffolds with continuous interfaces may help to overcome this limitation.^48–51 It has also been suggested that the bilayer interface should be capable of preventing any upward migration of the subchondral bone plate⁵² into the chondral layer.⁵³ The insertion of a mineralized compact and dense phase⁵⁴ or the use of a functional barrier enriched with anti-angiogenic growth factors⁵⁵ may represent viable strategies to limit the advancement of the subchondral bone plate.

Requirements to restore the subchondral bone

Fundamental requirements such as the de novo formation of the subchondral bone plate and subarticular spongiosa, the integration without formation of a sclerotic rim or subchondral bone cysts are of key importance and need to be met by the scaffold. Particularly for large subchondral bone defects, implant vascularization plays a crucial role to achieve this goal.^56–59 The delivery of angiogenic or growth factors may represent a valid approach,⁶⁰ even though their activity needs to be confined to the subchondral bone layer to avoid hypertrophy and endochondral ossification in the chondral layer.⁶¹ Recently, magnetic biohybrid devices composed of bioactive factors conjugated to magnetic nanoparticles have been suggested to obtain a targeted delivery,⁶² a strategy that could be exploited to improve subchondral bone neo-vascularization.

Important structural parameters to grant bone tissue ingrowth and vascularization include porosity, pore size, and interconnectivity.^63–66 These features need to be balanced with other key parameters, such as mechanical properties, material composition, and degradation rate, which need to be compatible with functional requirements of the implant.^67–69 Over time, scaffold parameters are modified by the healing process and scaffold pores are filled with newly formed bone matrix that integrates the scaffold with the subarticular spongiosa.⁷⁰

A variety of both natural and synthetic materials have been studied in animal models for subchondral bone repair.^45,71–73 In the clinical situation, however, the critical requirement of subchondral bone substitutes to induce and support osteogenesis^{64,71,74–76} is often not fulfilled.^74,75 Therefore, a key challenge that remains yet to be solved is the elucidation of the effect of distinct scaffold parameters on subchondral bone and articular cartilage repair.

Although great attention has been paid to the zonal organization of articular cartilage, the different components of the subchondral bone are often neglected when designing scaffolds. Indeed, bone scaffolds are usually characterized by an isotropic macroporous structure mimicking the architecture of trabecular bone. Very recently, Despang et al.⁷⁷ have designed an anisotropic bone scaffold with parallel-aligned pores mimicking the microstructure of cortical bone. A similar bioinspired approach could be applied to design multilayer scaffolds resembling the structure of the subchondral bone. Keeping the structure of the subchondral bone in mind, a layer mimicking the laminated sheets of the subchondral bone plate (with a high bone volume) that also seals the subarticular spongiosa from the synovial fluid and thus may prevent cyst formation^78,79 is mandatory. A second layer resembling the porous structure of the subarticular spongiosa, perhaps with trabeculae aligned in parallel to the diaphysis, is additionally needed.

Integration of the scaffold with the surrounding osteochondral tissue

The lateral integration of the implant with the surrounding osteochondral tissue continues to be difficult.^80,81 Several preclinical studies have reported an efficient bonding of the subchondral bone layer, but complete integration of the chondral layer to the surrounding articular cartilage remains a challenging goal.^82,83

Bioinspired Strategies for Osteochondral Repair in Preclinical Models

The increasing number of preclinical studies evaluating the in vivo performances of osteochondral scaffolds attests to the growing interest in regenerating the entire osteochondral unit, as widely reviewed.^{32,45,72,84–86} In general, such bioinspired osteochondral scaffolds are composite structures composed either of synthetic or natural materials or representing combinations thereof (Tables 2 and 3). This section first gives a general overview of the principles of translational animal models to study osteochondral repair. Next, the results of studies focusing on bioinspired bilayer scaffolds are given (Table 2). Considering the complexity of the osteochondral unit, multilayer scaffolds (Table 3) and continuous gradient scaffolds representing a step forward toward the design of bioinspired osteochondral scaffolds are also discussed.

Table 2.

Preclinical Studies Applying Bioinspired Biphasic Osteochondral Scaffolds

Cartilage layer		Subchondral bone layer
Biomaterial	Cell type	Biomaterial	Cell type	Type of bonding between cartilage and bone layers	Preculture	Animal model	Osteochondral defect location(s) and size	Time point(s)	Control group(s)	Methods applied to assess osteochondral repair	Main results and comments	Ref.
Fibrin glue (Tissuecol^®)	Articular chondrocytes	HA (Endobon^®)	—	Fibrin glue placed onto the bony layer.	—	Goat	Medial femoral condyle; circular, Ø 10 mm	2 weeks; 1, 3, 6 months, 1 year	—	Histological scoring	Cartilaginous repair tissue degraded by 1 year. Fibrin glue completely degraded at 3 months. Instable subchondral bone phase.	97
Polyvinyl alcohol (PVA) hydrogel	—	Porous titanium-fiber mesh	—	PVA solution infiltrated into the superficial pores of titanium and gelled.	—	Dog	Medial femoral condyle; rectangular, 5×10 mm, 6 mm deep	1, 2, 3, 6 months	Empty defect	Histological observation Microradiography	Bone ingrowth into the titanium mesh. PVA hydrogel layer intact even after 6 months.	98
Dense film of PLGA (PLA:PGA 75:25) for the superficial chondral layer (scaffold A, B, C, D) PLGA (75:25, scaffold A) PLGA (75:25)/10% PLA (scaffold B, C, D)	Rib chondrocytes	PLGA (75:25, scaffold A) PLGA (75:25)/20% PLA (scaffold B) PLGA (55:45)/20% Bioglass^® (scaffold C) PLGA (75:25)/calcium sulfate (scaffold D)	Rib chondrocytes	Chondral and bony layers glued with a solvent.	2 days	Goat	Medial femoral condyle and patellar groove; circular, Ø 2.8 mm, 4 mm deep	4 months	Cell-free scaffold	Macroscopic scoring Histological scoring Polarized-light microscopy Biomechanical analysis	Implants with PLGA associated to Bioglass^® and calcium sulfate yielding the best histological score. Stiffness of cartilaginous repair tissue based on these implants only slightly inferior than normal cartilage. Implanted cells had no effect on osteochondral repair.	99
PGA	Articular chondrocytes	Collagen I/HA/TCP (Collagraft^®)	Fresh bone marrow absorbed into Collagraft^®	Chondral and bony phase sutured before implantation.	4–6 weeks, culture of chondral layer to obtain engineered cartilage	Rabbit	Trochlear groove; rectangular, 7×5 mm, 5 mm deep	6 weeks, 6 months	Empty defect Cell-free scaffold	Histological scoring Immunohistochemistry (Types-II and X collagen) Biomechanical analysis Biochemical analysis (GAG, total collagen)	Engineered cartilage remodeled in 6 months into osteochondral repair tissue with physiologic Young's modulus. Suboptimal integration with adjacent cartilage. Implants combining tissue-engineered cartilage and Collagraft^® outperformed cell-free scaffolds. Bone marrow adsorption had no effect.	82
Hyal sponge: ACP^® (scaffold A) HYAFF-11^® (scaffold B)	BMSC	Injectable calcium phospate	—	Calcium phosphate injected into the defect and Hyal sponge press-fitted on the top.	—	Rabbit	Medial femoral condyle; circular, Ø 3 mm, 3 mm deep	1 month (scaff. A) 1, 3 months (scaff. B)	Cell-free scaffold	Histological observation	Histological observations suggested that cell-seeded implants resulted in more homogeneous repair tissue and better integration with the adjacent cartilage.	100
Hyal/Chitosan hydrogel (scaffold A) Collagen matrix (scaffold B)	—	PLA/Hyal (scaffold A, B) Hyal added only in the superficial pores of the bone layer	—	Hyal/Chitosan hydrogel press-fitted on PLA (scaffold A) Collagen matrix press-fitted on PLA and lyophilized (scaffold B)	—	Rabbit	Medial femoral condyle; circular, Ø 3 mm, 2.8 mm deep	24 weeks	Empty defect	Histological scoring Immunohistochemistry (Type-II collagen)	Better cartilage and bone repair and integration in Hyal/chitosan implants. Cartilage repair tissue positive for safranin O staining and type-II collagen in both scaffolds.	101
OPF hydrogel with TGF-β1-loaded gelatin microparticles (scaffold A, B)	—	OPF hydrogel with gelatin microparticles (scaffold A) OPF hydrogel (scaffold B)	—	Cross-linking between chondral and bony phases.	—	Rabbit	Medial femoral condyle; circular, Ø 3 mm, 3 mm deep	1, 3 months	OPF hydrogel with gelatin microparticles/OPF hydrogel (chondral/bony layer)	Histological scoring	Maturation of cartilaginous repair tissue over time in all scaffold groups. Scaffold A yielded the best histological result at both time points. Significantly increased safranin O staining in cartilaginous repair tissue derived from scaffolds with TGF-β1 loaded gelatin particles	48
Fibrin glue (Tisseel)	BMSC	PCL	BMSC (cell seeding by fibrin glue embedding)	PCL with fibrin-glue/BMSC placed into the defect. Fibrin glue/BMSC placed on top of the PCL layer.	—	Rabbit	Medial femoral condyle; circular, Ø 4 mm, 5.5 mm deep	3, 6 months	Cell-free scaffold	Histological observation Immunohistochemistry (Type-II collagen) Micro-CT	Qualitative evidence of good cartilage repair at 3 months, but degeneration and poor integration with host cartilage after 6 months in the majority of samples. No cartilage formation in cell-free scaffolds.	83
PCL	BMSC (cell seeding by fibrin glue embedding)	PCL/TCP	BMSC (cell seeding by fibrin glue embedding)	PCL/TCP with fibrin-glue/BMSC placed into the defect. PCL with fibrin-glue/BMSC placed on top of PCL/TCP.	—	Rabbit	Medial femoral condyle; circular, Ø 4 mm, 5 mm deep	6 weeks, 3, 6 months	Cell-free scaffold	Histological scoring Immunohistochemistry (Type-II collagen) Biomechanical analysis	Improved bone and cartilage repair in cell-seeded scaffolds. At 6 months cartilaginous repair tissue with Young's modulus similar to native cartilage but lacking zonal organization. Fissures at the interface with host cartilage. Good integration with subchondral bone.	102
Type-I and -III collagen	—	β-TCP	—	Collagen gel sucked in the superficial pores of TCP by vacuum and cross-linked. Growth-factor mixture applied into the implant site.	—	Minipig	Trochlear groove; circular, Ø 5.4 mm, 8 mm deep	6, 12 weeks 1 year	Empty defect Scaffold w/o growth factor mixture	Histological scoring Biomechanical analysis	Application of growth-factors (mixture of BMP-2, -3, -4, -6, -7, TGF-β1, - β2, - β3, FGF-1, osteocalcin and osteonectin derived from bovine bone with unknown concentrations of individual factors) led to improved cartilage repair. Better subchondral bone repair over time.	103
PGA	BMSC	HA	BMSC	Chondral and bony layers glued through fibrin glue prior to implantation.	3 days, chondral and bony layer separately cultured	Rabbit	Trochlear groove; circular, Ø 3.2 mm, 6 mm deep	4, 8 months	Empty defect Cell-free scaffold	Histological scoring Immunohistochemistry (Type-II collagen)	Cartilaginous repair tissue based on cell-seeded PGA/HA scaffolds stained positive for safranin O and collagen II. Mainly fibrous repair tissue when cell-free scaffold was applied. Delayed subchondral bone repair in cell-free implants, while complete integration observed in cell-seeded implants.	104
PGA	Synovium-derived stem cells (cell seeding by fibrin glue embedding)	Type I collagen/HA/TCP (Collagraft^®)	—	Collagraft^® placed into the osteochondral defect and in vitro engineered tissue placed upon it.	4 weeks, bioreactor culture of the chondral layer to obtain engineered cartilage	Rabbit	Medial femoral condyle; circular, Ø 4 mm, 5 mm deep	3 weeks 6 months	Empty defect Cell-free scaffold	Histological scoring Immunohistochemistry (Type-I and -II collagen)	Cartilaginous repair tissue positive for Safranin O and type-II collagen only in implants with the composite scaffold containing engineered cartilage at 6 months. Cell-free scaffold yielded no cartilage repair. Collagraft^® induced long-term inflammation interfering with osteochondral regeneration.	113
Hyal/Atelocollagen hydrogel	Articular chondrocytes	β-TCP/HA	—	Frozen cartilage hydrogel placed onto prewet bone scaffold and freeze-dried.	2 weeks	Rabbit	Trochlear groove; circular, Ø 6 mm, 5 mm deep	3 months	Empty defect Cell-free scaffold Osteochondral autograft	Macroscopic scoring Histological scoring Immunohistochemistry (Type-I and -II collagen)	Cartilage repair characterized by areas of fibrous tissue and incomplete integration with host cartilage. Good subchondral bone repair integration with the host bone. Inefficient bonding between chondral and bony phase (bone layer denudation in 3 implants).	47
Periosteal flap (scaffold A, B)	—	Porous tantalum (scaffold A) PLC (scaffold B)	—	Periosteal flap sutured onto porous tantalum or PCL prior to implantation.	—	Rabbit	Medial and lateral femoral condyles; circular, Ø 5 mm, 6 mm deep	15 weeks	Empty defect	Histological scoring	Better histological score for PCL/periosteum than for tantalum/periosteum. Good integration of scaffolds with the host bone with partial restoration of the tidemark and good subchondral bone repair. Cartilaginous repair tissue formation from periosteum in both scaffolds inconsistent.	105
PEG hydrogel (scaffold A, B)	BMSC	Porous tantalum (scaffold A) Bioactive glass (scaffold B)	—	Bone scaffold placed on top of the hydrogel layer. PEG solution added and cross-linked to bind the two layers.	—	Rabbit	Medial femoral condyle and trochlear groove; circular, Ø 3.2 mm, 4 mm deep	6, 12 weeks	Empty defect PEG/bone allograft Cell-free scaffold (A, B)	Histological scoring Immunohistochemistry (Type-I and -II collagen)	Better subchondral bone integration in PEG/tantalum implants compared to bioactive glass or allograft implants.	106
Cartilage resurfacing through PCL/Collagen electrospun mesh Porous PCL	BMSC (cell seeding by fibrin glue embedding)	PCL/TCP	—	PCL/Collagen mesh sutured to the implant and fixed through fibrin glue. No detail available on bony and chondral layer bonding.	—	Pig	Medial femoral condyle and trochlear groove; circular, Ø 8 mm, 8 mm deep	6 months	Native tissue Cell-seeded scaffold w/o PCL/collagen mesh Cell-free scaffold	Histological scoring Immunohistochemistry (Types-I and II collagen) Biomechanical analysis Micro-CT	Subchondral bone ingrowth into the cartilage layer, perhaps resulting from the use of skeletally immature animals due to joint enlargement. Articular cartilage repair compromised in the absence of cells or of the resurfacing membrane.	96
Hyal/Atelocollagen hydrogel	Articular chondrocytes	β-TCP/HA	—	Frozen cartilage hydrogel placed onto prewet bone scaffold and freeze-dried.	2 weeks	Minipig	Medial and lateral femoral condyles; circular, Ø 6 mm, 7 mm deep	5 months	Osteochondral autograft Autologous chondrocyte implantation (ACI) Cell-free scaffold	Macroscopic scoring Histological scoring Immunohistochemistry (Type-I and -II collagen) Biomechanical analysis	Abundant subchondral bone formation in the bony phase. Articular cartilage repair with biomechanical properties similar to native cartilage both in cell-seeded and cell-free scaffold. No significant improvement related to chondrocyte addition to the biphasic scaffold.	107
Collagen I hydrogel (CaReS^®)	BMSC	β-TCP (CERASORB^®)	BMSC	A mixture of BMSC and EDTA plasma used to fix chondral and bony layers.	2 weeks, chondral and bony layers cultured in separate conditions	Sheep	Medial femoral condyle; circular Ø 6.4 mm, 12 mm deep	6, 12 months	Osteochondral autograft	Histological scoring Immunohistochemistry (Type-I and -II collagen) Biomechanical analysis Micro-CT	Sinking of implants into the subchondral bone (2–3 mm below subchondral bone plate). Repair tissue in collagen/β-TCP implants comparable to osteochondral autograft in terms of histological scoring and biomechanical properties.	108
PLGA	Articular chondrocytes	TCP	Osteoblasts	Chondral and bony layers sutured 24h before implantation.	2 weeks, bioreactor culture for chondral layer and static culture for bony layer	Minipig	Medial femoral condyle; circular, Ø 8 mm, 8 mm deep	6 months	Empty defect Cell-seeded chondral layer implanted in a full-thickness chondral defect, (Ø 8 mm, 2 mm deep)	Macroscopic scoring Histological scoring Immunohistochemistry (Type-II collagen) Biomechanical analysis Biochemical analysis (GAG)	Articular cartilage repair tissue integrated into host cartilage and subchondral bone. In 5 defects implanted with the biphasic scaffold subchondral bone region partly filled with cartilaginous tissue. Cartilaginous repair tissue formed from the biphasic scaffold with significantly higher biomechanical properties and GAG content compared to the chondral layer alone.	109
Chitosan/Gelatin loaded with TGF-β1 plasmid	BMSC	HA/Chitosan/Gelatin loaded with BMP-2 plasmid	BMSC	Chondral and bony layers combined with fibrin glue.	2 weeks, separate culture for 1 week, assembly, culture for 1 week	Rabbit	Medial and lateral femoral condyles; circular, Ø 4 mm, 5 mm deep	4, 8, 12 weeks	Empty defect Cell-seeded DNA-free scaffold Cell-seeded chondral layer with TGF-β1 plasmid/ Cell-seeded bony layer with BMP-2 plasmid	Histological observation Immunohistochemistry (Type-II collagen)	Based on histological images, simultaneous repair of subchondral bone and articular cartilage observed only in TGF-β1/BMP-2 plasmid-loaded biphasic implants.	110
Decellularized cartilage matrix (DCM)	BMSC (chondrogenic predifferentiated, 14d)	Decellularized cancellous bone matrix (DCBM)	—	Cross-linking by dehydrothermal treatment using a carbodiimide	4 days	Dog	Medial and lateral femoral condyles; circular, Ø 4.2 mm, 6 mm deep	3, 6 months	Cell-free scaffold	Macroscopic scoring Histological scoring Biomechanical analysis Micro-CT	Better histological score with cell-seeded scaffolds. Fibrocartilaginous repair tissue in cell-free implant group and better repair in cell-seeded implants after 6 months. Mature subchondral bone at 3 and 6 months. Cartilage and subchondral bone stiffness inferior to native tissue (70–75%).	111
Macroporous PLGA	BMSC	Microporous PLGA	BMSC	Unique synthesis with differential sized porogens. A BMSC cell-sheet was wrapped around the scaffold prior to implantation	—	Rabbit	Medial femoral condyle; circular, Ø 4 mm, 3 mm deep	6, 12 weeks	Cell-free scaffold Cell-seeded scaffold without BMSC cell-sheet	Macroscopic scoring Histological observation Histomorphometry	The incorporation of BMSC sheet to PLGA/BMSC scaffold promoted cartilage repair and integration. Cartilaginous repair tissue stained positive for Safranin O observed only in implants enriched with cells after 12 weeks.	49
Hyal (HYAFF^®-11)	Articular chondrocytes	HA/Hyal (HYAFF^®-11)	—	Bony scaffold placed into the defect. Engineered cartilage placed on the top of the subchondral bone phase	2 days, 2 weeks and 6 weeks culture of the chondral layer to obtain engineered cartilage	Goat	Trochlear groove; circular, Ø 4 mm, 5 mm deep	8 weeks 8 months	Empty defect Cell-free scaffold	Histological scoring Biochemical analysis (GAG, type-I and -II collagen)	At 8 weeks all defects filled with fibrocartilaginous tissue. At 8 months, poor cartilage architecture in cell-free implants. Composite scaffold with 2 weeks precultured engineered cartilage yielded the best results in terms of cartilage architecture and integration with the surrounding cartilage. Subchondral bone remodelling in all the experimental groups.	112
Alginate with PLGA microspheres containing: 50 ng TGF-β1 (scaffold A) 2.5 μg BMP-2 (scaffold B) 5 μg BMP-2(scaffold C)	—	PLGA (Scaffold A, B, C)	—	PLGA microspheres dispersed in alginate. Alginate solution poured onto the bone layer and cross-linked. Freeze-drying of the composite scaffold.	—	Rabbit	Femoral condyles; circular, Ø 4.5 mm, 4 mm deep	2, 6, 12, 24 weeks	Empty defect Scaffold without growth factors	Histological scoring Immunohistochemistry (Type-I and -II collagen, aggrecan)	Better histological scores in growth factor-enriched scaffolds, with relevant short-term outcomes with 50 ng TGF-β1 and 5 μg BMP-2. Improved integration and type II collagen and aggrecan staining in 5 μg BMP-2 implants. Effect of growth factors maintained after 24 weeks.	114
Gelatin/Chondroitin sulphate/Sodium Hyal (GCH)	Articular chondrocytes	Gelatin/Ceramic bovine bone	BMSC (osteogenic predifferentiated for 3d)	GCH solution poured into molds to form a cartilage layer. Bone scaffold inserted into the GCH hydrogel. Composite scaffold frozen, lyophilized and then cross-linked again	—	Rabbit	Large osteochondral defect distal femur; 15×10×5 mm, rectangular	6, 12, 24 weeks	Empty defect Cell-free scaffold	Histological scoring Immunohistochemistry (Type-I and -II collagen) Gene expression analysis (Type-II collagen)	Cell-seeded scaffold positive for type-II collagen at 12 weeks. Subchondral bone scaffold replaced by bone matrix. Fibrous tissue with surface fibrillation in cell-free implants.	115
Alginate hydrogel binding TGF-β1	—	Alginate hydrogel binding BMP-4	—	BMP-4/affinity-binding alginate solution injected in situ and gelled. The top layer was generated in the same way.	—	Rabbit	Trochlear groove; circular, Ø 3 mm, 3 mm deep	2, 4 weeks	Empty defect	Histological observation Immunohistochemistry (Type-II collagen) Micro-CT	Evidence for cartilaginous matrix positive for type-II collagen in the superficial layer. Subchondral bone repaired after 4 weeks.	116
Collagen/GAG (Chondromimetic™, scaffold A) PLGA/PGA (Trufit™, scaffold B)	—	Calcium phosphate (Chondromimetic™, scaffold A) Calcium sulfate (Trufit™, scaffold B)	—	N/A	—	Goat	Medial femoral condyle and lateral trochlea, circular, Ø 5.8 mm, 6 mm deep	12, 26 weeks	Empty defect	Macroscopic scoring Histological scoring Biomechanical analysis	Collagen-GAG scaffold led to better histological repair compared to the biphasic PLGA material at 26 weeks with fewer of subchondral bone cysts. Increasing stiffness from 12 to 26 weeks observed only in collagen-GAG implants.	117
Hyal/Platelet Rich Plasma (HYAFF-11^®/PRP)	—	Hyal/Platelet Rich Plasma (HYAFF-11^®/PRP)	—	Chondral and bony layer joined through fibrin glue and/or cartilage fragments	—	Rabbit	Trochlear groove; circular, Ø 4.5 mm, 4 mm deep	1, 3, 6 months	Empy defect Biphasic scaffold without intermediate layer	Histological scoring Immunohistochemistry (Type-I and -II collagen)	Pilot study with maximal 4 animals per group (unilateral design). Inflammatory responses in subchondral bone at 3 months with poor subchondral bone regeneration and inferior histological scores for all scaffolds compared to empty control. At 6 months, cartilage fragment-loaded scaffolds better histological repair compared to other groups.	117
Type-I and -III collagen (scaffold A, B)	Articular chondrocytes	HA/Collagen (scaffold A) Allogeneic bone (scaffold B)	—	Type-I and–III collagen solution dropped onto the surface of the bone phase. Composite scaffold was then lyophilized.	—	Sheep	Medial and lateral femoral condyles; circular, Ø 9.4 mm, 11 mm deep	6 weeks	Cell-free scaffold (only for scaffold A)	Macroscopic scoring Histological observation Immunohistochemistry (Type-I and -II collagen) Gene expression analysis (Type-I and -II collagen, SOX-9)	Incomplete filling with repair tissue defect in scaffold A group with consistent recruitment of immune cells around implant. No difference between cell-free and cell-seeded implants. Defect height restored in scaffold B group but no integration with the layer of native cartilage surrounding the implant. Partial integration with the subchondral bone. Repair tissue in the chondral layer mainly consisting of bony and fibrous tissue.	92
Modified coralline aragonite: Drilled channels/Hyal (scaffold A1) Drilled channels (scaffold A2) Hyal (scaffold B1, C1) No modifications (scaffold B2, C2)	—	Modified coralline aragonite: No modifications (scaffold A1, A2, C1, C2) Drilled channels (scaffold B1, B2)	—	Unique scaffold of coralline aragonite differentially modified in the chondral and/or bony phase through impregnation with Hyal and drilling of channels	—	Goat	Medial and lateral femoral condyles; circular, Ø 6 mm, 8 mm deep	6 months	Empty defect	Macroscopic scoring Histological scoring Immunohistochemistry (Type-I and II collagen)	Implants with drilled channels and Hyal impregnation in the chondral phase outperformed all other implants. Differential distribution of type-I and -II collagen in the repaired cartilage and subchondral bone layers. Implants well integrated with native cartilage and subchondral bone.	119
PLGA with different porosity: 50–100 μm (scaffold A) 100–200 μm (scaffold B) 200–300 μm (scaffold C) 300–450 μm (scaffold D) 300–450 μm (scaffold E)	BMSC	PLGA with different porosity: 300–450 μm (scaffold A) 300–450 μm (scaffold B) 200–300 μm (scaffold C) 100–200 μm (scaffold D) 50–100 μm (scaffold E)	—	Two mixtures with porogens of different pore sizes (corresponding to the two layers) were glued together with a small amount of dichloromethane	1 week, culture in expansion medium	Rabbit	Medial and lateral femoral condyles; circular, Ø 4 mm, 5 mm deep	6,12 weeks	Empty defect Autologous osteochondral graft Cell-free scaffold	Histological scoring Immunohistochemistry (Types-I and II collagen) Gene expression analysis (Types-I and II collagen, aggrecan)	At 6 and 12 weeks, significantly higher histological scores for cell-seeded scaffolds compared to the corresponding cell-free scaffolds. The bilayer scaffold with 100–200 μm pores in the chondral phase and 300–450 mm in the bony phase yielded the highest histological score among the tested combinations and showed matrix stained positive for type-II collagen with structural organization similar to native cartilage.	120
Type I collagen (scaffold A, B)	—	Dense collagen layer (scaffold A) PLA electrospun nanofibers (scaffold B)	—	Collagen solution placed upon the bony layer. Composite scaffolds obtained by freeze-drying	—	Rabbit	Patellar groove; cylindrical, Ø 4 mm, 3.5–4 mm deep	6, 12 weeks	Empty defect	Macroscopic scoring Histological scoring Micro-CT Biomechanical analysis	Scaffold A yielded significantly better results compared to scaffold B. Cartilaginous repair tissue positive for safranin O and well integrated with host cartilage. Restoration of subchondral bone in the collagen/PLA group, while mainly fibrous tissue observed in the collagen group.	121

BMSC, bone marrow-derived mesenchymal stem cells; BMP, bone morphogenetic protein; CT, computed tomography; GAG, glycosaminoglycans, Hyal, hyaluronic acid; HA, hydroxyapatite; OPF, oligo(polyethylene-glycol)fumarate; PCL, polycaprolactone; PEG, polyethylene glycol; PGA, polyglycolic acid; PLA, polylactic acid; PLGA, polylactic-co-glycolic acid, TGF, transforming growth factor; TCP, tricalcium phosphate.

Table 3.

Preclinical Studies Applying Bioinspired Multiphasic Osteochondral Scaffolds

Cartilage layer		Intermediate layer		Subchondral bone layer
Biomaterial	Cell type	Biomaterial	Cell type	Biomaterial	Cell type	Type of bonding between layers	Preculture	Animal model	Osteochondral defect location(s) and size	Time point(s)	Control group(s)	Methods applied to assess osteochondral repair	Main results and comments	Ref.
Type-I collagen	—	Type-I collagen/HA (60:40)	—	Type-I collagen/HA (30:70)	—	The three layers were physically assembled and freeze-dried.	—	Horse	Fetlock joint; medial condyle of the distal epiphysis of the third metacarpal bone; circular, Ø 10 mm, 8–10 mm deep	6 months	—	Macroscopic scoring Histological scoring Polarized light microscopy	Pilot study on two horses. Newly formed bone integrating with the host bone. Resurfacing of the defect with cartilaginous repair tissue. Formation of a tidemark within the newly formed osteochondral unit. No subchondral bone plate advancement.	123
Type-I collagen	Articular chondrocytes (cell seeding by fibrin glue embedding)	Type-I collagen/HA (60:40)	—	Type-I collagen/HA (30:70)	—	The three layers were physically assembled and freeze-dried.	5 days	Sheep	Medial and lateral femoral condyles, circular, Ø 7 mm, 9 mm deep	6 months	Empy defect Cell-free scaffold	Macroscopic scoring Histological scoring Immunohistochemistry (Type-I and -II collagen) Microradiography	Cartilaginous repair tissue positive for type-II collagen and safranin O. Good integration of repaired subchondral bone. No subchondral bone plate advancement. No significant difference between cell-seeded and cell-free scaffolds in terms of cartilage and subchondral bone repair.	124
Type-I collagen	—	Type-I collagen/HA (60:40)	—	Type-I collagen/HA (30:70)	—	The three layers were physically assembled and freeze-dried. The entire scaffold was soaked into (PRP).	—	Sheep	Medial and lateral femoral condyles, circular, Ø 7 mm, 9 mm deep	6 months	Empy defect PRP-free scaffold	Macroscopic scoring Histological scoring Immunohistochemistry (Type-I and -II collagen) Microradiography	Superior osteochondral repair in the group treated with the scaffold alone. Incomplete subchondral bone repair and irregular cartilage integration in PRP-soaked scaffolds suggestive of a inhibitory effect of PRP on osteochondral repair.	125
Type-I collagen/Chitosan	ASC	Collagen membrane	ASC	Collagen/ Bovine cancellous bone grafts	ASC	Type-I collagen/Chitosan solution poured on the collagen membrane/bony layer. Composite construct frozen, lyophilized, and cross-linked.	—	Rabbit	Trochlear groove; circular, Ø 4 mm, depth unknown	8, 12 weeks	Empy defect Cell-free scaffold	Histological scoring	Composite scaffold enriched with a mixture of BMPs. Fibrous tissues within all the defects at 8 weeks. After 12 weeks cell-based group yielding the best histological score with formation of repair tissue. Toluidine blue staining inferior to native cartilage. No immunohistochemical staining included.	126
Bovine cartilage ECM-derived scaffold	BMSC (condrogenic differentiated, 21 days)	PLGA/β-TCP (compact layer)	—	PLGA/β-TCP/Collagen I	BMSC (osteogenic differentiated, 21 days)	Chondral and bony phases bonded by the compact layer through dissolving-conglutination method.	—	Rabbit	Trochlear groove; circular, Ø 5 mm, 6 mm deep	3, 6 months	Empy defect Biphasic scaffold w/o intermediate layer	Histological scoring Immunohistochemistry (Type-I and -II collagen) Biochemical analysis (GAG, collagen)	Presence of a compact layer increased anti-tensile and anti-shear properties of the scaffold. Superior repair of cartilage and subchondral bone in triphasic scaffolds compared to the group without compact layer with safranin O-positive cartilaginous repair tissue and subchondral bone trabeculae oriented perpendicularly to the joint surface.	54

ASC, adipose-derived stem cells; ECM, extracellular matrix; PRP, platelet-rich plasma.

General translational aspects of animal models for osteochondral repair

Small animal models (e.g., rabbits) are primarily used to translate promising in vitro developments.⁸⁷ Next, promising approaches are tested in larger animals (e.g., mini-pigs, goat, or sheep).⁸⁸ Differences in the composition of the osteochondral unit have to be kept in mind, such as the cartilage thickness and its relation to the thickness of the subchondral bone plate.⁸⁹ Observation times that are long enough to account for possible pathological events such as the degeneration of the cartilaginous repair tissue, the advancement of the subchondral bone plate, and the formation of intralesional osteophytes are needed.^52,90 A thorough analysis of cartilage repair is mandatory, including semi-quantitative macroscopic scoring,⁹¹ molecular biology,⁹² biochemical,⁹³ histological, and immunohistochemical⁹⁴ analyses. The subchondral bone is depicted and quantified by micro-computed tomography (micro-CT).^38,90 Biomechanical testing reveals whether the restored osteochondral unit is similar to the normal situation.^67,95 Although skeletally immature animals reproduce the problem of children and adolescents suffering from OCD, skeletally mature animals are preferred in preclinical studies to reflect the clinical problem of osteochondral repair in adults.⁸⁷ Interestingly, possible complications may occur in skeletally immature animals, for example a subchondral depression of the implant due to joint enlargement.⁹⁶

Bioinspired biphasic scaffolds

The most common strategy tested so far in preclinical models mimics the osteochondral structure through biphasic materials combining synthetic or natural hydrogels with biomaterials used as bone substitute, as highlighted by the list of preclinical studies reported in Table 2.^{47–49,82,83,92,96–121} Natural hydrogels have been the most used materials to generate the chondral layer of preclinically tested biphasic scaffolds. In particular, natural materials such as fibrin, hyaluronic acid (Hyal), collagen, chitosan, chondrotin sulphate, alginate, and their combinations have been applied in a wide range of osteochondral scaffolds.^{47,83,92,97,100,101,103,107,108,110,112,114–117,121} One of the main advantages of natural hydrogels is their enhanced biological interaction with cells compared with synthetic materials. This feature makes natural hydrogels very appealing candidates for the fabrication of biphasic osteochondral scaffolds although their synthesis and their final properties are less controllable compared to synthetic materials. From a clinical standpoint, an initial high mechanical strength of the articular cartilage compartment might not be immediately needed, as demonstrated by the clinical success of ACI to cover very large defects.¹²² Here, bioresorbable soft matrices are commonly used, indicating that in the absence of high loads (as achieved by postoperative nonweight bearing), the chondral layer does not need to have a high mechanical strength.³⁴

Polymeric materials such as polylactic acid, polyglycolic acid (PGA), polylactic-co-glycolic acid (PLGA), and polycaprolactone have been used to generate both the chondral and the subchondral bone layer of biphasic osteochondral scaffolds. The advantage of using synthetic polymers consists in the possibility to up-scale the scaffold manufacturing at the industrial level and in the high control over their synthetic parameters. For example, different porosities in the two layers can be obtained by using different-sized porogens during the synthetic process, thus generating an integrated PLGA bilayer scaffold.⁴⁹ The effect of the differential porosity in the chondral and subchondral bone layer has been also investigated in vivo by Duan et al.¹²⁰ using PLGA biphasic scaffolds. Chondral and subchondral bone phases characterized by 100–200 μm and 300–450 μm pores, respectively, led to superior osteochondral repair compared with other combinations of porosity. Thus, the use of highly controllable polymers can be exploited not only for the production of novel scaffolds, but also to test parameters that affect the repair process, thus gaining better insights for a rational scaffold design.

More frequently, biocompatible and degradable ceramic materials, such as hydroxyapatite (HA) and/or tricalcium phosphate (TCP), are used for the subchondral bone phase. These materials are able to support bone ingrowth into the implant, leading in most of the cases to satisfying regeneration of the subchondral bone.^{47,103,104,107–109} Metallic materials for subchondral bone reconstruction have also been proposed, among which porous titanium and tantalum that have been combined with synthetic hydrogels for chondral repair.^98,105,106 However, issues based on their lack of degradation, which hamper the substitution of the scaffold with native bone matrix and may thus interfere with the regeneration of the osteochondral unit, have to be kept in mind. Indeed, the degradation rate of materials used for both chondral and subchondral bone layer a key parameter in the healing process and it should be compatible with the regeneration rate of the corresponding tissue to prevent inefficient osteochondral repair. Also, being the stable union of chondral and subchondral bone layers a major challenge when the scaffold is subjected to mechanical stress related to joint function, suturing, press-fitting, or simple apposition of the chondral layer upon the bone layer that are reported in most of the analyzed preclinical studies, still appear a suboptimal solution, and major efforts need to focus on the development of new strategies to face this limitation.

Among the biphasic scaffolds, Chondromimetic™ and Trufit™ are currently clinically used to treat osteochondral defects. The first one combines type-I collagen and chondroitin-6-sulfate for the chondral phase and calcium phosphate for the subchondral bone phase, whereas Trufit™ is a bilayered porous scaffold containing PLGA and PGA fibers in the chondral phase and calcium sulphate in the subchondral bone phase. These biphasic implants have been recently compared in a goat model.¹¹⁷ After 6 months, a significantly higher histological score and lower incidence of subchondral bone cysts were seen for the type-I collagen/GAG-based biphasic scaffold, thus supporting the concept that natural composites better support osteochondral repair. Rapid translation to the clinical situation of these results in well-designed studies over a long period of time will determine the optimal strategy.

Incorporation of growth factors into biphasic scaffolds

The spatially defined incorporation of growth factors represents a bioinspired strategy that provides the scaffold with fundamental cues to induce progenitor cells toward chondro- or osteogenesis. Only few studies have investigated the effect of biphasic scaffold enrichment with growth factors on the in vivo osteochondral regenerative process. Holland et al.⁴⁸ have shown that the incorporation of transforming growth factor (TGF)-β1-loaded microparticles in the chondral phase of a oligo(poly(ethylene glycol)fumarate) bilayer scaffold promotes the formation of a continuous cartilage layer yielding a superior histological score compared with TGF-β1-free scaffolds. Recently, an alginate matrix (representing the chondral phase of a bilayer scaffold), has been combined with PLGA microspheres loaded with either TGF-β1 or BMP-2 and used to treat osteochondral defects in the rabbit model.¹¹⁴ A pilot study (three animals per experimental group) showed better histological repair at 24 weeks when TGF-β1 and BMP-2 implants were applied. The differential inclusion of growth factors in the two layers has been proposed by Re'em et al. who bound alginate to TGF-β1 and BMP-4 in the chondral and subchondral bone layer, respectively and implanted the scaffold into lapine subchondral defects.¹¹⁶ Although no histological scoring and no control group with a growth factor-free implant was included, this study may serve as a starting point to further evaluate the simultaneous incorporation of different growth factors for osteochondral repair.

Bioinspired multiphasic scaffolds

Compared to biphasic scaffolds, multiphasic scaffolds have been less studied in vivo so far, as reported in Table 3.^54,123–126 A bioinspired approach was applied by Da et al.⁵⁴ who designed a multiphasic scaffold with the calcified cartilage zone mimicked by a compact phase fabricated from PLGA/β-TCP. The chondral phase was derived from bovine decellularized articular cartilage matrix with an oriented structure designed to resemble the vertical orientation of the fibers in the deep zone. The subchondral bone phase consisted in a PLGA/β-TCP skeleton wrapped in type-I collagen obtained by computer-controlled rapid prototyping technique. Interestingly, the presence of the compact layer significantly increased the biomechanical properties of the multiphasic scaffold in terms of tensile and shear strength. Further, when used to treat rabbit osteochondral defects and compared to the compact layer-free scaffold group, the triphasic scaffold led to improved macroscopic and histological scores with neo-formed cartilaginous and bone tissue integrating with the host tissues. However, before moving toward a clinical application, it will be fundamental to reproduce these impressive results in a large animal model more reflective of the clinical situation.

Tampieri et al. developed a three-layered scaffold composed of type-I collagen and nanostructured HA that supported cartilage and bone formation.^127,128 This osteochondral scaffold is composed of a type-I collagen layer with a smooth surface for cartilaginous repair, an intermediate layer based on a combination of type-I collagen and HA (60:40) reflecting the calcified zone, and a lower layer composed of a mineralized blend of type-I collagen and HA (30:70) to replicate the subchondral bone. The intermediate and lower layers were obtained by nucleating HA into self-assembling collagen fibers through a bioinspired synthetic process mimicking the natural ossification process. Remarkably, this triphasic scaffold (MaioRegen^®) has been tested by Marcacci and co-workers in pilot studies in a sheep and horse model.^123–125 Macroscopic, histological, and immunohistochemical observations, and high resolution X-rays showed that the multiphasic scaffold induced osteochondral repair with an ordered architecture.¹²⁴ Interestingly, the addition of cells did not significantly improve the repair process, indicating that the scaffold itself is able to support the intrinsic ability of progenitor cells recruited at the implant site to produce specific matrix.

Continuous gradient osteochondral scaffolds

Continuous gradient scaffolds are a principal alternative to bilayer and multilayer scaffolds, displaying intermediate features in terms of material composition and growth factors in the transitional region of the scaffold. These scaffolds may overcome the problem of inefficient binding between different layers. The group of Michael Detamore has obtained promising results with continuous gradient scaffolds. Here, an osteochondral scaffold with continuous opposing gradients of TGF-β1 and BMP-2 was generated using growth factor-loaded PLGA microspheres.^129,130 This scaffold, with opposing gradients of microspheres encapsulating TGF-β1 or BMP-2 without or with HA nanoparticles, led to optimal rabbit osteochondral repair after 3 months compared to control groups, demonstrating that the simultaneous spatial patterning of bioactive signals and materials is advantageous compared with a single gradient of bioactive signals.⁵¹

Cell-free versus cell-based approaches

Do additional autologous cells need to populate osteochondral scaffolds? Although some studies report no significant improvement when using cell-seeded scaffolds,^{92,99,107,124} most of the preclinical studies have reported a positive effect of transplanted cells in comparison to cell-free scaffolds.^{49,82,83,100,102,104,112,115,120} The effect of cells implantation is particularly evident when cells are precultured in the chondral layer to obtain in vitro engineered cartilaginous tissue prior to implantation, leading to improved cartilage repair in vivo.^82,112,113 In particular, Miot et al.¹¹² demonstrated that the maturation state of in vitro engineered cartilaginous tissue obtained culturing articular chondrocytes into a hyaluronic acid mesh (HYAFF-11^®) affects the healing outcomes, when implanted as chondral layer in association to HA/HYAFF-11 subchondral bone layer.

As the basis for implantation of autologous articular chondrocytes and BMSC is well established, it would be easy to seed either cell type into the cartilaginous scaffold part, either before or during implantation. Both articular chondrocytes and BMSCs seeded in the layers of biphasic scaffolds have been tested in rabbit and equine osteochondral defect models.^115,131 Similarly, co-culture of osteoblasts and chondrocytes has been investigated both in vitro¹³² and in vivo.¹⁰⁹ However, as the subchondral bone part will always be exposed to BMSC and osteoblasts, already existing osteoinductive scaffolds might omit the need for additionally implanted cells enabling subchondral bone repair.¹³³ Considerations about the need for cell transplantation should also take in account additional procedures related to cell harvesting, safety requirements, and the overall increase in costs, necessitating high-quality clinical studies to answer this important question.

Clinical Application of Bioinspired Osteochondral Scaffolds

Although several invigorating avenues are being followed in the field of in vitro and preclinical in vivo tissue engineering, no optimal strategy has been described that is clinically accepted. Here, only a few scaffolds for osteochondral regeneration are currently commercially available for clinical application to the best knowledge of the authors.

The biphasic scaffold Chondromimetic™¹¹⁷ is currently under clinical investigation (ClinicalTrials.gov identifier: NCT01209390) in a prospective postmarketing study to confirm the clinical efficacy and safety outcome of its application for the treatment of osteochondral defects over a 3 year postimplantation follow-up period.

The biphasic scaffold Trufit™ is tested in a prospective randomized, multicenter, clinical trial, evaluating its effectiveness for the treatment of single cartilage defects in the knee compared to the microfracture technique (ClinicalTrials.gov identifier: NCT01246635). First short-term results of Trufit are inconclusive.⁷⁵ For example, Barber et al. demonstrated by CT that no bone ingrowth, osteoconductivity, or integration with the adjacent subchondral bone occurred.⁷⁴ In contrast, a sclerotic rim often surrounded the lesion and subchondral cyst formation was frequent and recently confirmed in another study.⁷⁶ Unfavorable short-term magnetic resonance imaging (MRI) results have been reported,⁷⁵ although it has been suggested that the clinical outcome may improve over time.^134,135 Nevertheless, the evidence for delayed subchondral incorporation of the implants warrants further long-term investigation.

MaioRegen^®, an osteochondral biomimetic scaffold with a trilayered structure containing only type-I collagen in the chondral layer and a mixture of type-I collagen and HA combined in different ratios to mimic the tidemark zone (60:40) and the subchondral layer (30:70),^123–125 was recently tested in a pilot study involving 28 patients with chondral and osteochondral lesions and sizes ranging from 1.5 to 6.0 cm².⁷⁶ In another study, 27 patients affected by knee OCD (average defect size: 3.4±2.2 cm²) were treated.¹³⁶ Good clinical outcomes were seen at 2-year follow-up in both studies. Specifically, MRI evaluation revealed a complete filling of the cartilage defect and complete integration of the scaffold in most of the cases. Of note, the subchondral bone was never completely restored, and subchondral bone changes such as cysts and sclerosis were often present.^76,136 This multilayer scaffold is currently under evaluation in a prospective, randomized, multicenter clinical trial (ClinicalTrials.gov identifier: NCT01282034) to compare its effectiveness with standard marrow stimulation techniques for the treatment of both chondral and osteochondral lesions.

Altogether, these clinical reports suggest that an insufficient integration with the subchondral bone and a limited overall restoration of this tissue are the major technical limitations of current scaffolds. Interestingly, this important finding is in contrast with the observations derived from the preclinical studies, where the subchondral bone part was reported to be well integrated with the native tissue. More research is therefore needed to elucidate the reasons for these differences between the human applications and the animal models and to build more knowledge to support better clinical decisions in the future. The development of novel bone scaffolds^137,138 that permit an enhanced de novo subchondral bone formation is therefore of considerable importance to design bioinspired scaffolds with improved potential for osteochondral integration and repair.

Outlook

Innovative biofabrication techniques will be of great value to design scaffolds with a complex pattern mimicking the structural organization of the osteochondral unit. Computer-aided rapid prototyping techniques allow the fabrication of 3D scaffold with a highly reproducible architecture in terms of pore size, geometry, and interconnectivity. Material composition in the different layer of the scaffold can also be highly controlled through these techniques, generating compositional variation within a single scaffold. Further, as recently reported by Fedorovich et al.¹³⁹ these techniques can be exploited to achieve a controlled incorporation of different cell types in different layers of the construct, thus creating osteochondral grafts that can recapitulate the complex distribution of cells and matrix observed in native tissues. Also, the combination of rapid prototyping techniques with advanced high resolution imaging analysis, such as CT, enables the generation of biomimetic scaffolds on the basis of microanatomical data. Recently, bioprinting has been used to precisely deliver chondrocytes combined with polyethylene glycol (PEG) hydrogel into a cartilage defect created within an osteochondral plug, thus demonstrating the feasibility of in situ thermal inkjet-based cartilage bioprinting.¹⁴⁰ These results suggest that further advances in the field of biofabrication techniques could allow the design of complex bioinspired osteochondral scaffolds and possibly the development of novel treatments where the osteochondral defect could be treated by the in situ assembly of cell-laden biomaterials.

As indicated by the promising results obtained in preclinical studies, future avenues might also be achieved by combining bioactive molecules supporting bone and/or cartilage repair either through the use spatially controlled incorporation of specific growth factors in the different layers or through the generation of gradients of bioactive signals that can be sensed by resident cells.

From a clinical point of view, it will be important to address recently emerged problems of the subchondral bone such as the formation of intralesional osteophytes, the advancement of the subchondral bone plate, formation of subchondral bone cysts, or lack of integration with the adjacent subchondral bone.⁶ Thus, continued monitoring through noninvasive imaging methods of cartilage and subchondral bone repair in patients treated with multiphasic implants that are currently in clinical use is vital to evaluate the long-term outcomes of each scaffold. Precise information on the physiological response to the different osteochondral constructs may help to understand which of these undesired events is more often associated with each treatment and possibly reduce their frequency through the improved design of novel scaffolds.

The successful application of biomimetic multiphasic scaffolds for osteochondral repair will continue to require a combined effort of orthopedic, clinical, and basic science investigators, among which are biomaterial scientists, cell, molecular, and developmental biologists, biochemists, biotechnologists, and biomechanical engineers. To bring such individuals together, it will be critical to supplement the promising preclinical and clinical accomplishments with the scientific rigor of well-designed long-term randomized controlled trials.

Footnotes

Disclosure statement

No competing financial interests exist for all authors.

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