Bioinspired scaffolds for osteochondral regeneration.

TISSUE ENGINEERING: Part A Volume 20, Numbers 15 and 16, 2014 ª Mary Ann Liebert, Inc. DOI: 10.1089/ten.tea.2013.0356

Bioinspired Scaffolds for Osteochondral Regeneration Silvia Lopa, PhD,1 and Henning Madry, MD 2,3

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

O

steochondral 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 1 2 3

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 (fullthickness) (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

Cell and Tissue Engineering Laboratory, IRCCS Galeazzi Orthopaedic Institute, Milan, Italy. Center of Experimental Orthopaedics, Saarland University, Homburg/Saar, Germany. Department of Orthopaedic Surgery, Saarland University Medical Center, Saarland University, Homburg/Saar, Germany.

2052

BIOMIMETIC OSTEOCHONDRAL REPAIR

2053

FIG. 2. Macroscopic view of an osteochondral defect resulting from osteochondritis dissecans (OCD) in a 30-yearold 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 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. 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

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 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

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

2054

LOPA AND MADRY

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 mm 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

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.

Subchondral bone plate and subarticular spongiosa

Requirements to restore the articular cartilage

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.

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

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,

Requirements to restore the calcified cartilage

This part of the scaffold should allow mineralization. Specific material compositions can direct bone marrowderived 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


2055

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 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 over-

come 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

2056

LOPA AND MADRY

Table 1. Scaffold Requirements for an Anatomic Reconstruction of the Osteochondral Unit Anatomical layer Superficial zone of articular cartilage Transitional zone of articular cartilage Radial zone of articular cartilage Tidemark Calcified zone of articular cartilage Cement line Subchondral bone plate Subarticular spongiosa

Scaffold requirement(s) Superficial layer. This surface protects the deeper layers from the stresses applied within the joint. Needs to ease cartilage gliding. Transitional layer. Porous structure to allow for cartilaginous repair tissue formation. Biomaterial fibrils are placed tangentially. May be seeded with autologous cells. 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. 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 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. Zone of integration. Strong bonding mandatory between the articular cartilage and subchondral bone layers. Needs to reflect the dense cancellous subchondral bone, may need to shield the subarticular spongiosa from the synovial fluid. Highly porous structure representing the natural subarticular spongious bone structure.

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 osteogenesis64,71,74–76 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 formation78,79 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. 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,

2057

PGA

Biomaterial

—

—

Articular Collagen I/HA/ chondrocytes TCP (Collagraft)

Fresh bone marrow absorbed into Collagraft

Rib chondroPLGA (75:25, cytes scaffold A) PLGA (75:25)/ 20% PLA (scaffold B) PLGA (55:45)/ 20% Bioglass (scaffold C) PLGA (75:25)/ calcium sulfate (scaffold D)

Porous titaniumfiber mesh

—

Cell type

Subchondral bone layer

Articular HA (Endobon ) chondrocytes

Cell type

Dense film of Rib chondroPLGA (PLA:Pcytes GA 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)

Polyvinyl alcohol (PVA) hydrogel

Fibrin glue (Tissuecol)

Biomaterial

Cartilage layer

—

Preculture

Goat

Dog

Goat

Chondral and 4–6 weeks, Rabbit bony phase suculture of tured before chondral implantation. layer to obtain engineered cartilage

PVA solution in— filtrated into the superficial pores of titanium and gelled. Chondral and 2 days bony layers glued with a solvent.

Fibrin glue placed onto the bony layer.

Type of bonding between cartilage and bone layers

Trochlear 6 weeks, groove; 6 months rectangular, 7 · 5 mm, 5 mm deep

Medial femo- 1, 2, 3, 6 ral condyle; months rectangular, 5 · 10 mm, 6 mm deep Medial femo- 4 months ral condyle and patellar groove; circular, Ø 2.8 mm, 4 mm deep

Medial femo- 2 weeks; 1, 3, ral condyle; 6 months, 1 circular, Ø year 10 mm

Osteochondral defect Animal location(s) model and size Time point(s) Histological scoring

Methods applied to assess osteochondral repair

Main results and comments

82

99

98

97

Ref.

(continued)

Cartilaginous repair tissue degraded by 1 year. Fibrin glue completely degraded at 3 months. Instable subchondral bone phase. Empty defect Histological Bone ingrowth into observation the titanium mesh. Microradiography PVA hydrogel layer intact even after 6 months. Cell-free scaffold Macroscopic Implants with PLGA scoring associated to BioHistological glass and calcium sulfate yielding the scoring best histological Polarized-light score. Stiffness of microscopy cartilaginous repair Biomechanical tissue based on analysis these implants only slightly inferior than normal cartilage. Implanted cells had no effect on osteochondral repair. Empty defect Histological Engineered cartilage Cell-free scaffold scoring remodeled in 6 Immunohistomonths into osteochemistry chondral repair (Types-II and X tissue with physiocollagen) logic Young’s Biomechanical modulus. Suboptianalysis mal integration with Biochemical adjacent cartilage. analysis Implants combining (GAG, total tissue-engineered collagen) cartilage and Collagraft outperformed cell-free scaffolds. Bone marrow adsorption had no effect.

—

Control group(s)

Table 2. Preclinical Studies Applying Bioinspired Biphasic Osteochondral Scaffolds

2058

Fibrin glue (Tisseel)

BMSC

—

OPF hydrogel with TGF-b1loaded gelatin microparticles (scaffold A, B)

PCL

OPF hydrogel with gelatin microparticles (scaffold A) OPF hydrogel (scaffold B)

PLA/Hyal (scaffold A, B) Hyal added only in the superficial pores of the bone layer

—

Hyal/Chitosan hydrogel (scaffold A) Collagen matrix (scaffold B)

Biomaterial

Injectable calcium phospate

Cell type

BMSC (cell seeding by fibrin glue embedding)

—

—

—

Cell type


BMSC Hyal sponge: ACP (scaffold A) HYAFF-11 (scaffold B)

Biomaterial

Cartilage layer

PCL with fibringlue/BMSC placed into the defect. Fibrin glue/BMSC placed on top of the PCL layer.

Hyal/Chitosan hydrogel pressfitted on PLA (scaffold A) Collagen matrix press-fitted on PLA and lyophilized (scaffold B) Cross-linking between chondral and bony phases.

Calcium phosphate injected into the defect and Hyal sponge pressfitted on the top.


—

—

—

—

Preculture

Rabbit

Rabbit

Rabbit

Rabbit

Medial femo- 3, 6 months ral condyle; circular, Ø 4 mm, 5.5 mm deep

Medial femo- 1, 3 months ral condyle; circular, Ø 3 mm, 3 mm deep

Medial femo- 24 weeks ral condyle; circular, Ø 3 mm, 2.8 mm deep

Medial femo- 1 month ral condyle; (scaff. A) circular, Ø 1, 3 months 3 mm, 3 mm (scaff. B) deep

Osteochondral defect Animal location(s) model and size Time point(s)

Table 2. (Continued)

Histological scoring Immunohistochemistry (Type-II collagen)

Cell-free scaffold Histological observation Immunohistochemistry (Type-II collagen) Micro-CT

OPF hydrogel Histological with gelatin miscoring croparticles/ OPF hydrogel (chondral/ bony layer)

Empty defect

Cell-free scaffold Histological observation

Control group(s)


Ref.

(continued)

Histological observa- 100 tions suggested that cell-seeded implants resulted in more homogeneous repair tissue and better integration with the adjacent cartilage. Better cartilage and 101 bone repair and integration in Hyal/ chitosan implants. Cartilage repair tissue positive for safranin O staining and type-II collagen in both scaffolds. 48 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 TGFb1 loaded gelatin particles Qualitative evidence 83 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.


2059

PGA

Type-I and -III collagen

PCL

Biomaterial

Cartilage layer

BMSC

—


Cell type

HA

b-TCP

PCL/TCP

Biomaterial

BMSC

—


Cell type


—

—

Preculture Medial femo- 6 weeks, ral condyle; 3, 6 months circular, Ø 4 mm, 5 mm deep

Trochlear 4, 8 months groove; circular, Ø 3.2 mm, 6 mm deep

Minipig Trochlear 6, 12 weeks groove; cir- 1 year cular, Ø 5.4 mm, 8 mm deep

Rabbit

Chondral and 3 days, chon- Rabbit bony layers dral and glued through bony layer fibrin glue prior separately to implantation. cultured

Collagen gel sucked in the superficial pores of TCP by vacuum and crosslinked. Growthfactor mixture applied into the implant site.

PCL/TCP with fibrin-glue/ BMSC placed into the defect. PCL with fibringlue/BMSC placed on top of PCL/TCP.




Histological scoring Biomechanical analysis

Histological scoring Immunohistochemistry (Type-II collagen) Biomechanical analysis

Empty defect Histological Cell-free scaffold scoring Immunohistochemistry (Type-II collagen)

Empty defect Scaffold w/o growth factor mixture

Cell-free scaffold

Control group(s)


Ref.

(continued)

Improved bone and 102 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. 103 Application of growthfactors (mixture of BMP-2, -3, -4, -6, -7, TGF-b1, - b2, - b3, 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. Cartilaginous repair 104 tissue based on cellseeded 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 cellfree implants, while complete integration observed in cell-seeded implants.


2060

Periosteal flap (scaffold A, B)

—

Porous tantalum (scaffold A) PLC (scaffold B)

Articular b-TCP/HA chondrocytes

Hyal/Atelocollagen hydrogel

Type I collagen/ HA/TCP (Collagraft)

Biomaterial

Synoviumderived stem cells (cell seeding by fibrin glue embedding)

Cell type

—

—

—

Cell type


PGA

Biomaterial

Cartilage layer Preculture

Periosteal flap sutured onto porous tantalum or PCL prior to implantation. —

Frozen cartilage 2 weeks hydrogel placed onto prewet bone scaffold and freezedried.

Rabbit

Rabbit

Collagraft placed 4 weeks, bio- Rabbit into the osteoreactor culchondral defect ture of the and in vitro enchondral gineered tissue layer to placed upon it. obtain engineered cartilage


Medial and 15 weeks lateral femoral condyles; circular, Ø 5 mm, 6 mm deep

Trochlear 3 months groove; circular, Ø 6 mm, 5 mm deep

Medial femo- 3 weeks ral condyle; 6 months circular, Ø 4 mm, 5 mm deep



Empty defect

Histological scoring

Empty defect Macroscopic Cell-free scaffold scoring Osteochondral Histological autograft scoring Immunohistochemistry (Type-I and -II collagen)

Empty defect Histological Cell-free scaffold scoring Immunohistochemistry (Type-I and -II collagen)

Control group(s)


Ref.

(continued)

113 Cartilaginous repair tissue positive for Safranin O and type-II collagen only in implants with the composite scaffold containing engineered cartilage at 6 months. Cellfree scaffold yielded no cartilage repair. Collagraft induced long-term inflammation interfering with osteochondral regeneration. Cartilage repair char47 acterized 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). Better histological 105 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.


2061

Collagen I hydro- BMSC gel (CaReS)

b-TCP (CERA- BMSC SORB)

—

b-TCP/HA

Hyal/Atelocollagen hydrogel

Articular chondrocytes

—

PCL/TCP

—

Cell type

Cartilage resurfa- BMSC (cell cing through seeding by PCL/Collagen fibrin glue electrospun embedding) mesh Porous PCL

Biomaterial

Porous tantalum (scaffold A) Bioactive glass (scaffold B)

Cell type


PEG hydrogel BMSC (scaffold A, B)

Biomaterial

Cartilage layer

—

—

Preculture

A mixture of BMSC and EDTA plasma used to fix chondral and bony layers.

Medial femo- 6 months ral condyle and trochlear groove; circular, Ø 8 mm, 8 mm deep

Medial femo- 6, 12 weeks ral condyle and trochlear groove; circular, Ø 3.2 mm, 4 mm deep

Medial femo- 6, 12 months ral condyle; circular Ø 6.4 mm, 12 mm deep

Minipig Medial and 5 months lateral femoral condyles; circular, Ø 6 mm, 7 mm deep

Pig

Rabbit

2 weeks, Sheep chondral and bony layers cultured in separate conditions

Frozen cartilage 2 weeks hydrogel placed onto prewet bone scaffold and freezedried.

Bone scaffold placed on top of the hydrogel layer. PEG solution added and cross-linked to bind the two layers. PCL/Collagen mesh sutured to the implant and fixed through fibrin glue. No detail available on bony and chondral layer bonding.




Osteochondral au- Histological tograft scoring Immunohistochemistry (Type-I and -II collagen) Biomechanical analysis Micro-CT

Osteochondral Macroscopic autograft scoring Autologous chon- Histological drocyte implanscoring tation (ACI) ImmunohistoCell-free scaffold chemistry (Type-I and -II collagen) Biomechanical analysis

Native tissue Histological Cell-seeded scafscoring fold w/o PCL/ Immunohistocollagen mesh chemistry Cell-free scaffold (Types-I and II collagen) Biomechanical analysis Micro-CT

Empty defect Histological PEG/bone scoring allograft ImmunohistoCell-free scaffold chemistry (A, B) (Type-I and -II collagen)

Control group(s)


106

Ref.

(continued)

96 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. Abundant subchondral 107 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. Sinking of implants 108 into the subchondral bone (2–3 mm below subchondral bone plate). Repair tissue in collagen/bTCP implants comparable to osteochondral autograft in terms of histological scoring and biomechanical properties.

Better subchondral bone integration in PEG/tantalum implants compared to bioactive glass or allograft implants.


2062

BMSC

Chitosan/Gelatin loaded with TGF-b1 plasmid

HA/Chitosan/ Gelatin loaded with BMP-2 plasmid

TCP

Biomaterial

BMSC

—

Osteoblasts

Cell type


Decellularized BMSC (chonDecellularized cartilage matrix drogenic precancellous (DCM) differentiated, bone matrix 14d) (DCBM)


Cell type

PLGA

Biomaterial

Cartilage layer Preculture

Dog

2 weeks, sep- Rabbit arate culture for 1 week, assembly, culture for 1 week

Cross-linking by 4 days dehydrothermal treatment using a carbodiimide

Chondral and bony layers combined with fibrin glue.

Empty defect

Control group(s)



Ref.

(continued)

Macroscopic Articular cartilage re- 109 scoring pair tissue integrated into host Cell-seeded chon- Histological cartilage and subdral layer scoring chondral bone. In 5 implanted in a Immunohistodefects implanted full-thickness chemistry with the biphasic chondral defect, (Type-II scaffold subchon(Ø 8 mm, 2 mm collagen) dral bone region deep) Biomechanical partly filled with analysis cartilaginous tissue. Biochemical Cartilaginous repair analysis (GAG) tissue formed from the biphasic scaffold with significantly higher biomechanical properties and GAG content compared to the chondral layer alone. 4, 8, 12 weeks Empty defect Histological Based on histological 110 Medial and Cell-seeded DNAobservation images, simultalateral femoral confree scaffold Immunohistoneous repair of dyles; circuCell-seeded chemistry subchondral bone lar, Ø 4 mm, chondral layer (Type-II and articular carti5 mm deep with TGF-b1 collagen) lage observed only plasmid/ in TGF-b1/BMP-2 Cell-seeded bony plasmid-loaded layer with biphasic implants. BMP-2 plasmid Medial and 3, 6 months Cell-free scaffold Macroscopic Better histological 111 lateral femscoring score with celloral conHistological seeded scaffolds. dyles; circuscoring Fibrocartilaginous lar, Ø Biomechanical repair tissue in cell4.2 mm, analysis free implant group 6 mm deep Micro-CT 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%).

Chondral and 2 weeks, bio- Minipig Medial femo- 6 months bony layers sureactor culral condyle; tured 24h before ture for circular, Ø implantation. chondral 8 mm, 8 mm layer and deep static culture for bony layer




2063

—


Hyal (HYAFF11)

Alginate with PLGA microspheres containing: 50 ng TGF-b1 (scaffold A) 2.5 mg BMP-2 (scaffold B) 5 mg BMP-2(scaffold C)

BMSC

Cell type

Macroporous PLGA

Biomaterial

Cartilage layer

PLGA (Scaffold A, B, C)

HA/Hyal (HYAFF-11)

Microporous PLGA

Biomaterial BMSC

—

—

Cell type


—

Preculture Rabbit

PLGA microspheres dispersed in alginate. Alginate solution poured onto the bone layer and cross-linked. Freeze-drying of the composite scaffold. —

Rabbit

Bony scaffold 2 days, 2 Goat placed into the weeks and defect. Engi6 weeks neered cartilage culture of placed on the the chontop of the subdral layer chondral bone to obtain phase engineered cartilage

Unique synthesis with differential sized porogens. A BMSC cellsheet was wrapped around the scaffold prior to implantation


Femoral con- 2, 6, 12, 24 dyles; circu- weeks lar, Ø 4.5 mm, 4 mm deep

Trochlear 8 weeks groove; 8 months circular, Ø 4 mm, 5 mm deep

Medial femo- 6, 12 weeks ral condyle; circular, Ø 4 mm, 3 mm deep




Ref.

(continued)

Cell-free scaffold Macroscopic The incorporation of 49 Cell-seeded scafscoring BMSC sheet to fold without Histological PLGA/BMSC scafBMSC cellobservation fold promoted carsheet Histomorphometry tilage repair and integration. Cartilaginous repair tissue stained positive for Safranin O observed only in implants enriched with cells after 12 weeks. Empty defect Histological At 8 weeks all defects 112 Cell-free scaffold scoring filled with fibrocarBiochemical tilaginous tissue. analysis At 8 months, poor (GAG, type-I and cartilage architec-II collagen) ture 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. 114 Empty defect Histological Better histological Scaffold without scoring scores in growth growth factors Immunohistofactor-enriched chemistry scaffolds, with rele(Type-I and vant short-term -II collagen, outcomes with aggrecan) 50 ng TGF-b1 and 5 mg BMP-2. Improved integration and type II collagen and aggrecan staining in 5 mg BMP-2 implants. Effect of growth factors maintained after 24 weeks.

Control group(s)


2064

Cell type

—

—

—

Alginate hydrogel binding TGF-b1

Collagen/GAG (Chondromimetic, scaffold A) PLGA/PGA (Trufit, scaffold B)

Hyal/Platelet Rich Plasma (HYAFF-11/ PRP)

Gelatin/Chondroi- Articular chontin sulphate/ drocytes Sodium Hyal (GCH)

Biomaterial

Cartilage layer Cell type


Hyal/Platelet Rich Plasma (HYAFF-11/ PRP)

—

Chondral and bony layer joined through fibrin glue and/ or cartilage fragments

Gelatin/Ceramic BMSC (osteo- GCH solution bovine bone genic predifpoured into ferentiated molds to form for 3d) a cartilage layer. Bone scaffold inserted into the GCH hydrogel. Composite scaffold frozen, lyophilized and then crosslinked again Alginate hydro— BMP-4/affinitygel binding binding alginate BMP-4 solution injected in situ and gelled. The top layer was generated in the same way. Calcium phos— N/A phate (Chondromimetic, scaffold A) Calcium sulfate (Trufit, scaffold B)

Biomaterial


—

—

—

—

Preculture

Rabbit

Goat

Rabbit

Rabbit



Ref.

Empty defect

Empty defect

Histological scoring Immunohistochemistry (Type-I and -II collagen)

Macroscopic scoring Histological scoring Biomechanical analysis

Histological observation Immunohistochemistry (Type-II collagen) Micro-CT

(continued)

Evidence for cartilag- 116 inous matrix positive for type-II collagen in the superficial layer. Subchondral bone repaired after 4 weeks. Collagen-GAG scaf117 fold 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 collagenGAG implants. Pilot study with max- 117 imal 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.

Empty defect Histological Cell-seeded scaffold 115 Cell-free scaffold scoring positive for type-II Immunohistocollagen at 12 chemistry weeks. Subchondral (Type-I and bone scaffold re-II collagen) placed by bone maGene expression trix. Fibrous tissue analysis with surface fibril(Type-II collagen) lation in cell-free implants.

Control group(s)

Trochlear 1, 3, 6 months Empy defect groove; cirBiphasic scaffold cular, Ø without inter4.5 mm, mediate layer 4 mm deep

Medial femo- 12, 26 weeks ral condyle and lateral trochlea, circular, Ø 5.8 mm, 6 mm deep

Trochlear 2, 4 weeks groove; circular, Ø 3 mm, 3 mm deep

Large osteo- 6, 12, 24 chondral de- weeks fect distal femur; 15 · 10 · 5 mm, rectangular



2065

Modified coralline aragonite: Drilled channels/ Hyal (scaffold A1) Drilled channels (scaffold A2) Hyal (scaffold B1, C1) No modifications (scaffold B2, C2)

Type-I and -III collagen (scaffold A, B)

Biomaterial

Cartilage layer

—


Cell type

Modified coralline aragonite: No modifications (scaffold A1, A2, C1, C2) Drilled channels (scaffold B1, B2)

HA/Collagen (scaffold A) Allogeneic bone (scaffold B)

Biomaterial

—

—

Cell type


Unique scaffold of coralline aragonite differentially modified in the chondral and/or bony phase through impregnation with Hyal and drilling of channels

Type-I and–III collagen solution dropped onto the surface of the bone phase. Composite scaffold was then lyophilized.


—

—

Preculture

Goat

Sheep

Medial and 6 months lateral femoral condyles; circular, Ø 6 mm, 8 mm deep

Medial and 6 weeks lateral femoral condyles; circular, Ø 9.4 mm, 11 mm deep




Ref.

(continued)

Cell-free scaffold Macroscopic Incomplete filling 92 (only for scafscoring with repair tissue fold A) Histological defect in scaffold A observation group with consisImmunohistotent recruitment of chemistry immune cells (Type-I and -II around implant. No collagen) difference between Gene expression cell-free and cellanalysis (Type-I seeded implants. and -II collagen, Defect height reSOX-9) stored 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. Empty defect Macroscopic Implants with drilled 119 scoring channels and Hyal Histological impregnation in the scoring chondral phase outImmunohistoperformed all other chemistry implants. Differen(Type-I and II tial distribution of collagen) type-I and -II collagen in the repaired cartilage and subchondral bone layers. Implants well integrated with native cartilage and subchondral bone.

Control group(s)


2066

Dense collagen layer (scaffold A) PLA electrospun nanofibers (scaffold B)

PLGA with different porosity: 300–450 mm (scaffold A) 300–450 mm (scaffold B) 200–300 mm (scaffold C) 100–200 mm (scaffold D) 50–100 mm (scaffold E)

Biomaterial

—

—

Cell type

Subchondral bone layer Preculture

Collagen solution placed upon the bony layer. Composite scaffolds obtained by freezedrying —

Two mixtures with 1 week, porogens of dif- culture in ferent pore sizes expansion (corresponding medium to the two layers) were glued together with a small amount of dichloromethane


Rabbit

Rabbit

Patellar 6, 12 weeks groove; cylindrical, Ø 4 mm, 3.5– 4 mm deep

Medial and 6,12 weeks lateral femoral condyles; circular, Ø 4 mm, 5 mm deep


Ref.

120 Empty defect Histological At 6 and 12 weeks, Autologous osteoscoring significantly higher chondral graft Immunohistohistological scores for cell-seeded Cell-free scaffold chemistry scaffolds compared (Types-I and II to the correspondcollagen) ing cell-free scafGene expression folds. The bilayer analysis scaffold with 100– (Types-I and II 200 mm pores in the collagen, chondral phase and aggrecan) 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. Empty defect Macroscopic Scaffold A yielded 121 scoring significantly better Histological results compared to scoring scaffold B. CartilaMicro-CT ginous repair tissue Biomechanical positive for safranin analysis 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.

Control group(s)


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.

Type I collagen (scaffold A, B)

—

Cell type

PLGA with differ- BMSC ent porosity: 50–100 mm (scaffold A) 100–200 mm (scaffold B) 200–300 mm (scaffold C) 300–450 mm (scaffold D) 300–450 mm (scaffold E)

Biomaterial

Cartilage layer



2067

Type-I collagen

Type-I collagen

Type-I collagen

Biomaterial

—

Cell type

Type-I collagen/ HA (60:40)

Biomaterial

—


—

—

—

Cell type

Intermediate layer

Articular Type-I chondrocollagen/ cytes (cell HA (60:40) seeding by fibrin glue embedding)

Cartilage layer




Biomaterial

—

—

—

Cell type


—

The three layers were physically assembled and freeze-dried. The entire scaffold was soaked into (PRP). —

Sheep

Sheep

Horse

Animal Preculture model

The three layers 5 days were physically assembled and freeze-dried.

The three layers were physically assembled and freeze-dried.

Type of bonding between layers —

Control group(s)



Ref.

(continued)

Macroscopic Pilot study on two 123 scoring horses. Newly Histological formed bone intescoring grating with the Polarized light host bone. Resurfamicroscopy cing of the defect with cartilaginous repair tissue. Formation of a tidemark within the newly formed osteochondral unit. No subchondral bone plate advancement. 124 Medial and lateral 6 months Empy defect Macroscopic Cartilaginous repair femoral conCell-free scoring tissue positive for dyles, circular, scaffold Histological type-II collagen and safranin O. Good Ø 7 mm, 9 mm scoring integration of redeep Immunohistopaired subchondral chemistry bone. No subchon(Type-I and -II dral bone plate adcollagen) vancement. No Microradio significant differgraphy ence between cellseeded and cell-free scaffolds in terms of cartilage and subchondral bone repair. Medial and lateral 6 months Empy defect Macroscopic Superior osteochon125 femoral conPRP-free scoring dral repair in the dyles, circular, scaffold Histological group treated with Ø 7 mm, 9 mm scoring the scaffold alone. deep ImmunohistoIncomplete subchemistry chondral bone re(Type-I and pair and irregular -II collagen) cartilage integration Microradiograin PRP-soaked phy scaffolds suggestive of a inhibitory effect of PRP on osteochondral repair.

Time point(s)

Fetlock joint; me- 6 months dial condyle of the distal epiphysis of the third metacarpal bone; circular, Ø 10 mm, 8–10 mm deep

Osteochondral defect location(s) and size

Table 3. Preclinical Studies Applying Bioinspired Multiphasic Osteochondral Scaffolds

2068

Collagen membrane

Biomaterial

BMSC PLGA/b-TCP (condro(compact genic differlayer) entiated, 21 days)

Cell type

—

ASC

Cell type

Intermediate layer

ASC

Cell type Type-I collagen/ Chitosan solution poured on the collagen membrane/bony layer. Composite construct frozen, lyophilized, and crosslinked.

Type of bonding between layers

Chondral and PLGA/b-TCP/ BMSC bony phases Collagen I (osteogenic bonded by the differenticompact layer ated, 21 through dissoldays) ving-conglutination method.

Collagen/ Bovine cancellous bone grafts

Biomaterial


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

Bovine cartilage ECMderived scaffold

Type-I ASC collagen/ Chitosan

Biomaterial

Cartilage layer

—

—

Time point(s) Empy defect Histological Cell-free scoring scaffold

Control group(s)



Ref.

Composite scaffold 126 enriched with a mixture of BMPs. Fibrous tissues within all the defects at 8 weeks. After 12 weeks cellbased group yielding the best histological score with formation of repair tissue. Toluidine blue staining inferior to native cartilage. No immunohistochemical staining included. Rabbit Trochlear groove; 3, 6 Empy defect Histological Presence of a compact 54 circular, Ø months Biphasic scoring layer increased anti5 mm, 6 mm scaffold Immunohistotensile and antideep w/o interchemistry shear properties of mediate (Type-I and -II the scaffold. Superlayer collagen) ior repair of cartiBiochemical lage and analysis subchondral bone in (GAG, collatriphasic scaffolds gen) compared to the group without compact layer with safranin O-positive cartilaginous repair tissue and subchondral bone trabeculae oriented perpendicularly to the joint surface.

Osteochondral defect location(s) and size

Rabbit Trochlear groove; 8, 12 circular, Ø weeks 4 mm, depth unknown

Animal Preculture model



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 semiquantitative 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

2069

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 mm and 300–450 mm 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)-b1-loaded microparticles in the chondral phase of a oligo(poly(ethylene

2070

glycol)fumarate) bilayer scaffold promotes the formation of a continuous cartilage layer yielding a superior histological score compared with TGF-b1-free scaffolds. Recently, an alginate matrix (representing the chondral phase of a bilayer scaffold), has been combined with PLGA microspheres loaded with either TGF-b1 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-b1 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-b1 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/b-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/b-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 Xrays showed that the multiphasic scaffold induced osteochondral repair with an ordered architecture.124 Interestingly, the addition of cells did not significantly improve

LOPA AND MADRY

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-b1 and BMP-2 was generated using growth factor-loaded PLGA microspheres.129,130 This scaffold, with opposing gradients of microspheres encapsulating TGF-b1 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 Chondromimetic117 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 followup 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 scaffolds137,138 that permit an enhanced de novo subchondral bone formation is therefore of considerable

2071

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

2072

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. Disclosure statement

No competing financial interests exist for all authors. References

1. Panseri, S., Russo, A., Cunha, C., Bondi, A., Di Martino, A., Patella, S., and Kon, E. Osteochondral tissue engineering approaches for articular cartilage and subchondral bone regeneration. Knee Surg Sports Traumatol Arthrosc 20, 1182, 2012. 2. Michael, J.W., Wurth, A., Eysel, P., and Konig, D.P. Long-term results after operative treatment of osteochondritis dissecans of the knee joint-30 year results. Int Orthop 32, 217, 2008. 3. Hunziker, E.B. The elusive path to cartilage regeneration. Adv Mater 21, 3419, 2009. 4. Huey, D.J., Hu, J.C., and Athanasiou, K.A. Unlike bone, cartilage regeneration remains elusive. Science 338, 917, 2012. 5. Hunziker, E.B. Articular cartilage repair: are the intrinsic biological constraints undermining this process insuperable? Osteoarthritis Cartilage 7, 15, 1999. 6. Orth, P., Cucchiarini, M., Kohn, D., and Madry, H. Alterations of the subchondral bone in osteochondral repair—translational data and clinical evidence. Eur Cell Mater 25, 299, 2013. 7. Madry, H. The subchondral bone: a new frontier in articular cartilage repair. Knee Surg Sport Tr A 18, 417, 2010. 8. Madry, H., van Dijk, C.N., and Mueller-Gerbl, M. The basic science of the subchondral bone. Knee Surg Sports Traumatol Arthrosc 18, 419, 2010. 9. Shin, H., Jo, S., and Mikos, A.G. Biomimetic materials for tissue engineering. Biomaterials 24, 4353, 2003. 10. Tampieri, A., Celotti, G., and Landi, E. From biomimetic apatites to biologically inspired composites. Anal Bioanal Chem 381, 568, 2005. 11. Grayson, W.L., Martens, T.P., Eng, G.M., Radisic, M., and Vunjak-Novakovic, G. Biomimetic approach to tissue engineering. Semin Cell Dev Biol 20, 665, 2009. 12. Boccaccio, A., Ballini, A., Pappalettere, C., Tullo, D., Cantore, S., and Desiate, A. Finite element method (FEM), mechanobiology and biomimetic scaffolds in bone tissue engineering. Int J Biol Sci 7, 112, 2011. 13. Owen, S.C., and Shoichet, M.S. Design of three-dimensional biomimetic scaffolds. J Biomed Mater Res A 94, 1321, 2010. 14. Tampieri, A., Sprio, S., Sandri, M., and Valentini, F. Mimicking natural bio-mineralization processes: a new tool for osteochondral scaffold development. Trends Biotechnol 29, 526, 2011. 15. Linden, B. The incidence of osteochondritis dissecans in the condyles of the femur. Acta Orthop Scand 47, 664, 1976. 16. Shirazi, R., and Shirazi-Adl, A. Computational biomechanics of articular cartilage of human knee joint: effect of osteochondral defects. J Biomech 42, 2458, 2009. 17. Steinhagen, J., Kurz, B., Niggemeyer, O., and Bruns, J. The pathophysiology of cartilage diseases. Ortop Traumatol Rehabil 3, 163, 2001.

LOPA AND MADRY

18. Menetrey, J., Unno-Veith, F., Madry, H., and Van Breuseghem, I. Epidemiology and imaging of the subchondral bone in articular cartilage repair. Knee Surg Sports Traumatol Arthrosc 18, 463, 2010. 19. Madry, H., Grun, U.W., and Knutsen, G. Cartilage repair and joint preservation: medical and surgical treatment options. Dtsch Arztebl Int 108, 669, 2011. 20. Marcacci, M., Filardo, G., and Kon, E. Treatment of cartilage lesions: what works and why? Injury 44 Suppl 1, S11, 2013. 21. Lyons, T.J., McClure, S.F., Stoddart, R.W., and McClure, J. The normal human chondro-osseous junctional region: evidence for contact of uncalcified cartilage with subchondral bone and marrow spaces. BMC Musculoskelet Disord 7, 52, 2006. 22. Newman, A.P. Articular cartilage repair. Am J Sports Med 26, 309, 1998. 23. Bhosale, A.M., and Richardson, J.B. Articular cartilage: structure, injuries and review of management. Br Med Bull 87, 77, 2008. 24. Minas, T. A primer in cartilage repair. J Bone Joint Surg Br 94, 141, 2012. 25. Becerra, J., Andrades, J.A., Guerado, E., Zamora-Navas, P., Lopez-Puertas, J.M., and Reddi, A.H. Articular cartilage: structure and regeneration. Tissue Eng Part B Rev 16, 617, 2010. 26. Wang, F., Ying, Z., Duan, X., Tan, H., Yang, B., Guo, L., Chen, G., Dai, G., Ma, Z., and Yang, L. Histomorphometric analysis of adult articular calcified cartilage zone. J Struct Biol 168, 359, 2009. 27. Hoemann, C.D., Lafantaisie-Favreau, C.H., LascauComan, V., Chen, G., and Guzman-Morales, J. The cartilage-bone interface. J Knee Surg 25, 85, 2012. 28. Broom, N.D., and Poole, C.A. A functional-morphological study of the tidemark region of articular cartilage maintained in a non-viable physiological condition. J Anat 135, 65, 1982. 29. Lyons, T.J., Stoddart, R.W., McClure, S.F., and McClure, J. The tidemark of the chondro-osseous junction of the normal human knee joint. J Mol Histol 36, 207, 2005. 30. Hunziker, E.B. Articular cartilage repair: basic science and clinical progress. A review of the current status and prospects. Osteoarthritis Cartilage 10, 432, 2002. 31. Pan, J., Zhou, X., Li, W., Novotny, J.E., Doty, S.B., and Wang, L. In situ measurement of transport between subchondral bone and articular cartilage. J Orthop Res 27, 1347, 2009. 32. Nukavarapu, S.P., and Dorcemus, D.L. Osteochondral tissue engineering: Current strategies and challenges. Biotechnol Adv 31, 706, 2013. 33. Szpalski, C., Wetterau, M., Barr, J., and Warren, S.M. Bone tissue engineering: current strategies and techniques— part I: Scaffolds. Tissue Eng Part B Rev 18, 246, 2012. 34. Brittberg, M. Cell carriers as the next generation of cell therapy for cartilage repair: a review of the matrixinduced autologous chondrocyte implantation procedure. Am J Sports Med 38, 1259, 2010. 35. van Osch, G.J., Brittberg, M., Dennis, J.E., BastiaansenJenniskens, Y.M., Erben, R.G., Konttinen, Y.T., and Luyten, F.P. Cartilage repair: past and future—lessons for regenerative medicine. J Cell Mol Med 13, 792, 2009. 36. Reddi, A.H., Becerra, J., and Andrades, J.A. Nanomaterials and hydrogel scaffolds for articular cartilage regeneration. Tissue Eng Part B Rev 17, 301, 2011.


37. Raghunath, J., Rollo, J., Sales, K.M., Butler, P.E., and Seifalian, A.M. Biomaterials and scaffold design: key to tissue-engineering cartilage. Biotechnol Appl Biochem 46, 73, 2007. 38. Pulkkinen, H.J., Tiitu, V., Valonen, P., Jurvelin, J.S., Rieppo, L., Toyras, J., Silvast, T.S., Lammi, M.J., and Kiviranta, I. Repair of osteochondral defects with recombinant human type II collagen gel and autologous chondrocytes in rabbit. Osteoarthritis Cartilage 21, 481, 2013. 39. Wise, J.K., Yarin, A.L., Megaridis, C.M., and Cho, M. Chondrogenic differentiation of human mesenchymal stem cells on oriented nanofibrous scaffolds: engineering the superficial zone of articular cartilage. Tissue Eng Part A 15, 913, 2009. 40. Chen, T., Hilton, M.J., Brown, E.B., Zuscik, M.J., and Awad, H.A. Engineering superficial zone features in tissue engineered cartilage. Biotechnol Bioeng 110, 1476, 2013. 41. Thorpe, S.D., Nagel, T., Carroll, S.F., and Kelly, D.J. Modulating Gradients in Regulatory Signals within Mesenchymal Stem Cell Seeded Hydrogels: A Novel Strategy to Engineer Zonal Articular Cartilage. PLoS One 8, e60764, 2013. 42. Nguyen, L.H., Kudva, A.K., Guckert, N.L., Linse, K.D., and Roy, K. Unique biomaterial compositions direct bone marrow stem cells into specific chondrocytic phenotypes corresponding to the various zones of articular cartilage. Biomaterials 32, 1327, 2011. 43. Nguyen, L.H., Kudva, A.K., Saxena, N.S., and Roy, K. Engineering articular cartilage with spatially-varying matrix composition and mechanical properties from a single stem cell population using a multi-layered hydrogel. Biomaterials 32, 6946, 2011. 44. Allan, K.S., Pilliar, R.M., Wang, J., Grynpas, M.D., and Kandel, R.A. Formation of biphasic constructs containing cartilage with a calcified zone interface. Tissue Eng 13, 167, 2007. 45. O’Shea, T.M., and Miao, X. Bilayered scaffolds for osteochondral tissue engineering. Tissue Eng Part B Rev 14, 447, 2008. 46. Driesang, I.M., and Hunziker, E.B. Delamination rates of tissue flaps used in articular cartilage repair. J Orthop Res 18, 909, 2000. 47. Ahn, J.H., Lee, T.H., Oh, J.S., Kim, S.Y., Kim, H.J., Park, I.K., Choi, B.S., and Im, G.I. Novel hyaluronate-atelocollagen/beta-TCP-hydroxyapatite biphasic scaffold for the repair of osteochondral defects in rabbits. Tissue Eng Part A 15, 2595, 2009. 48. Holland, T.A., Bodde, E.W., Baggett, L.S., Tabata, Y., Mikos, A.G., and Jansen, J.A. Osteochondral repair in the rabbit model utilizing bilayered, degradable oligo(poly(ethylene glycol) fumarate) hydrogel scaffolds. J Biomed Mater Res A 75, 156, 2005. 49. Qi, Y., Du, Y., Li, W., Dai, X., Zhao, T., and Yan, W. Cartilage repair using mesenchymal stem cell (MSC) sheet and MSCs-loaded bilayer PLGA scaffold in a rabbit model. Knee Surg Sports Traumatol Arthrosc 2012. DOI: 10.1007/s00167-012-2256-3. 50. Harley, B.A., Lynn, A.K., Wissner-Gross, Z., Bonfield, W., Yannas, I.V., and Gibson, L.J. Design of a multiphase osteochondral scaffold III: Fabrication of layered scaffolds with continuous interfaces. J Biomed Mater Res A 92, 1078, 2010. 51. Mohan, N., Dormer, N.H., Caldwell, K.L., Key, V.H., Berkland, C.J., and Detamore, M.S. Continuous gradients

2073

52.

53. 54.

55. 56.

57. 58.

59.

60. 61. 62.

63. 64. 65.

66. 67.

of material composition and growth factors for effective regeneration of the osteochondral interface. Tissue Eng Part A 17, 2845, 2011. Orth, P., Cucchiarini, M., Kaul, G., Ong, M.F., Graber, S., Kohn, D.M., and Madry, H. Temporal and spatial migration pattern of the subchondral bone plate in a rabbit osteochondral defect model. Osteoarthritis Cartilage 20, 1161, 2012. Hunziker, E.B., Driesang, I.M., and Saager, C. Structural barrier principle for growth factor-based articular cartilage repair. Clin Orthop Relat Res 391 Supp, S182, 2001. Da, H., Jia, S.J., Meng, G.L., Cheng, J.H., Zhou, W., Xiong, Z., Mu, Y.J., and Liu, J. The impact of compact layer in biphasic scaffold on osteochondral tissue engineering. PLoS One 8, e54838, 2013. Hunziker, E.B., and Driesang, I.M. Functional barrier principle for growth-factor-based articular cartilage repair. Osteoarthritis Cartilage 11, 320, 2003. Correia, C., Grayson, W.L., Park, M., Hutton, D., Zhou, B., Guo, X.E., Niklason, L., Sousa, R.A., Reis, R.L., and Vunjak-Novakovic, G. In vitro model of vascularized bone: synergizing vascular development and osteogenesis. PLoS One 6, e28352, 2011. Laschke, M.W., Vollmar, B., and Menger, M.D. Inosculation: connecting the life-sustaining pipelines. Tissue Eng Part B Rev 15, 455, 2009. Rivron, N.C., Raiss, C.C., Liu, J., Nandakumar, A., Sticht, C., Gretz, N., Truckenmuller, R., Rouwkema, J., and van Blitterswijk, C.A. Sonic Hedgehog-activated engineered blood vessels enhance bone tissue formation. Proc Natl Acad Sci U S A 109, 4413, 2012. Nguyen, L.H., Annabi, N., Nikkhah, M., Bae, H., Binan, L., Park, S., Kang, Y., Yang, Y., and Khademhosseini, A. Vascularized bone tissue engineering: approaches for potential improvement. Tissue Eng Part B Rev 18, 363, 2012. Hunziker, E.B., Enggist, L., Kuffer, A., Buser, D., and Liu, Y. Osseointegration: the slow delivery of BMP-2 enhances osteoinductivity. Bone 51, 98, 2012. Bonnet, C.S., and Walsh, D.A. Osteoarthritis, angiogenesis and inflammation. Rheumatology (Oxford) 44, 7, 2005. Panseri, S., Russo, A., Giavaresi, G., Sartori, M., Veronesi, F., Fini, M., Salter, D.M., Ortolani, A., Strazzari, A., Visani, A., Dionigi, C., Bock, N., Sandri, M., Tampieri, A., and Marcacci, M. Innovative magnetic scaffolds for orthopedic tissue engineering. J Biomed Mater Res A 100, 2278, 2012. Hollister, S.J. Porous scaffold design for tissue engineering. Nat Mater 4, 518, 2005. Karageorgiou, V., and Kaplan, D. Porosity of 3D biomaterial scaffolds and osteogenesis. Biomaterials 26, 5474, 2005. Wu, C., Luo, Y., Cuniberti, G., Xiao, Y., and Gelinsky, M. Three-dimensional printing of hierarchical and tough mesoporous bioactive glass scaffolds with a controllable pore architecture, excellent mechanical strength and mineralization ability. Acta Biomater 7, 2644, 2011. Bertoldi, S., Fare, S., and Tanzi, M.C. Assessment of scaffold porosity: the new route of micro-CT. J Appl Biomater Biomech 9, 165, 2011. Schlichting, K., Schell, H., Kleemann, R.U., Schill, A., Weiler, A., Duda, G.N., and Epari, D.R. Influence of scaffold stiffness on subchondral bone and subsequent

2074

68.

69.

70.

71.

72. 73. 74.

75.

76.

77.

78.

79. 80.

81.

LOPA AND MADRY

cartilage regeneration in an ovine model of osteochondral defect healing. Am J Sports Med 36, 2379, 2008. Layton, M.W., Goldstein, S.A., Goulet, R.W., Feldkamp, L.A., Kubinski, D.J., and Bole, G.G. Examination of subchondral bone architecture in experimental osteoarthritis by microscopic computed axial tomography. Arthritis Rheum 31, 1400, 1988. Lajeunesse, D., and Reboul, P. Subchondral bone in osteoarthritis: a biologic link with articular cartilage leading to abnormal remodeling. Curr Opin Rheumatol 15, 628, 2003. Phipps, M.C., Clem, W.C., Grunda, J.M., Clines, G.A., and Bellis, S.L. Increasing the pore sizes of bone-mimetic electrospun scaffolds comprised of polycaprolactone, collagen I and hydroxyapatite to enhance cell infiltration. Biomaterials 33, 524, 2012. Panseri, S., Russo, A., Cunha, C., Bondi, A., Di Martino, A., Patella, S., and Kon, E. Osteochondral tissue engineering approaches for articular cartilage and subchondral bone regeneration. Knee Surg Sports Traumatol Arthrosc. 2011. Martin, I., Miot, S., Barbero, A., Jakob, M., and Wendt, D. Osteochondral tissue engineering. J Biomech 40, 750, 2007. Sundelacruz, S., and Kaplan, D.L. Stem cell- and scaffoldbased tissue engineering approaches to osteochondral regenerative medicine. Semin Cell Dev Biol 20, 646, 2009. Barber, F.A., and Dockery, W.D. A computed tomography scan assessment of synthetic multiphase polymer scaffolds used for osteochondral defect repair. Arthroscopy 27, 60, 2011. Dhollander, A.A., Liekens, K., Almqvist, K.F., Verdonk, R., Lambrecht, S., Elewaut, D., Verbruggen, G., and Verdonk, P.C. A pilot study of the use of an osteochondral scaffold plug for cartilage repair in the knee and how to deal with early clinical failures. Arthroscopy 28, 225, 2012. Kon, E., Delcogliano, M., Filardo, G., Busacca, M., Di Martino, A., and Marcacci, M. Novel nano-composite multilayered biomaterial for osteochondral regeneration: a pilot clinical trial. Am J Sports Med 39, 1180, 2011. Despang, F., Bernhardt, A., Lode, A., Dittrich, R., Hanke, T., Shenoy, S.J., Mani, S., John, A., and Gelinsky, M. Synthesis and physicochemical, in vitro and in vivo evaluation of an anisotropic, nanocrystalline hydroxyapatite bisque scaffold with parallel-aligned pores mimicking the microstructure of cortical bone. J Tissue Eng Regen Med 2013. [Epub ahead of print]; DOI: 10.1002/ term.1729. Van der Vis, H.M., Aspenberg, P., Marti, R.K., Tigchelaar, W., and Van Noorden, C.J. Fluid pressure causes bone resorption in a rabbit model of prosthetic loosening. Clin Orthop Relat Res 350, 201, 1998. van Dijk, C.N., Reilingh, M.L., Zengerink, M., and van Bergen, C.J. The natural history of osteochondral lesions in the ankle. Instr Course Lect 59, 375, 2010. Khan, I.M., Gilbert, S.J., Singhrao, S.K., Duance, V.C., and Archer, C.W. Cartilage integration: evaluation of the reasons for failure of integration during cartilage repair. A review. Eur Cell Mater 16, 26, 2008. Gilbert, S.J., Singhrao, S.K., Khan, I.M., Gonzalez, L.G., Thomson, B.M., Burdon, D., Duance, V.C., and Archer, C.W. Enhanced tissue integration during cartilage repair in vitro can be achieved by inhibiting chondrocyte death at the wound edge. Tissue Eng Part A 15, 1739, 2009.

82. Schaefer, D., Martin, I., Jundt, G., Seidel, J., Heberer, M., Grodzinsky, A., Bergin, I., Vunjak-Novakovic, G., and Freed, L.E. Tissue-engineered composites for the repair of large osteochondral defects. Arthritis Rheum 46, 2524, 2002. 83. Shao, X.X., Hutmacher, D.W., Ho, S.T., Goh, J.C., and Lee, E.H. Evaluation of a hybrid scaffold/cell construct in repair of high-load-bearing osteochondral defects in rabbits. Biomaterials 27, 1071, 2006. 84. Mano, J.F., and Reis, R.L. Osteochondral defects: present situation and tissue engineering approaches. J Tissue Eng Regen Med 1, 261, 2007. 85. Nooeaid, P., Salih, V., Beier, J.P., and Boccaccini, A.R. Osteochondral tissue engineering: scaffolds, stem cells and applications. J Cell Mol Med 16, 2247, 2012. 86. Keeney, M., and Pandit, A. The osteochondral junction and its repair via bi-phasic tissue engineering scaffolds. Tissue Eng Part B Rev 15, 55, 2009. 87. Laverty, S., Girard, C.A., Williams, J.M., Hunziker, E.B., and Pritzker, K.P. The OARSI histopathology initiative— recommendations for histological assessments of osteoarthritis in the rabbit. Osteoarthritis Cartilage 18 Suppl 3, S53, 2010. 88. Little, C.B., Smith, M.M., Cake, M.A., Read, R.A., Murphy, M.J., and Barry, F.P. The OARSI histopathology initiative—recommendations for histological assessments of osteoarthritis in sheep and goats. Osteoarthritis Cartilage 18 Suppl 3, S80, 2010. 89. Aigner, T., Cook, J.L., Gerwin, N., Glasson, S.S., Laverty, S., Little, C.B., McIlwraith, W., and Kraus, V.B. Histopathology atlas of animal model systems—overview of guiding principles. Osteoarthritis Cartilage 18 Suppl 3, S2, 2010. 90. Orth, P., Goebel, L., Wolfram, U., Ong, M.F., Graber, S., Kohn, D., Cucchiarini, M., Ignatius, A., Pape, D., and Madry, H. Effect of subchondral drilling on the microarchitecture of subchondral bone: analysis in a large animal model at 6 months. Am J Sports Med 40, 828, 2012. 91. Goebel, L., Orth, P., Muller, A., Zurakowski, D., Bucker, A., Cucchiarini, M., Pape, D., and Madry, H. Experimental scoring systems for macroscopic articular cartilage repair correlate with the MOCART score assessed by a high-field MRI at 9.4 T—comparative evaluation of five macroscopic scoring systems in a large animal cartilage defect model. Osteoarthritis Cartilage 20, 1046, 2012. 92. Schleicher, I., Lips, K.S., Sommer, U., Schappat, I., Martin, A.P., Szalay, G., Hartmann, S., and Schnettler, R. Biphasic scaffolds for repair of deep osteochondral defects in a sheep model. J Surg Res 183, 184, 2012. 93. Kiss, A., Cucchiarini, M., Menger, M.D., Kohn, D., Hannig, M., and Madry, H. Enamel matrix derivative inhibits proteoglycan production and articular cartilage repair, delays the restoration of the subchondral bone and induces changes of the synovial membrane in a lapine osteochondral defect model in vivo. J Tissue Eng Regen Med 2012. [Epub ahead of print]; DOI: 10.1002/term.1495. 94. Cucchiarini, M., Orth, P., and Madry, H. Direct rAAV SOX9 administration for durable articular cartilage repair with delayed terminal differentiation and hypertrophy in vivo. J Mol Med (Berl) 91, 625, 2013. 95. Duda, G.N., Maldonado, Z.M., Klein, P., Heller, M.O., Burns, J., and Bail, H. On the influence of mechanical conditions in osteochondral defect healing. J Biomech 38, 843, 2005.


96. Ho, S.T., Hutmacher, D.W., Ekaputra, A.K., Hitendra, D., and Hui, J.H. The evaluation of a biphasic osteochondral implant coupled with an electrospun membrane in a large animal model. Tissue Eng Part A 16, 1123, 2010. 97. van Susante, J.L., Buma, P., Homminga, G.N., van den Berg, W.B., and Veth, R.P. Chondrocyte-seeded hydroxyapatite for repair of large articular cartilage defects. A pilot study in the goat. Biomaterials 19, 2367, 1998. 98. Chang, Y.S., Gu, H.O., Kobayashi, M., and Oka, M. Comparison of the bony ingrowth into an osteochondral defect and an artificial osteochondral composite device in load-bearing joints. Knee 5, 205, 1998. 99. Niederauer, G.G., Slivka, M.A., Leatherbury, N.C., Korvick, D.L., Harroff, H.H., Ehler, W.C., Dunn, C.J., and Kieswetter, K. Evaluation of multiphase implants for repair of focal osteochondral defects in goats. Biomaterials 21, 2561, 2000. 100. Gao, J., Dennis, J.E., Solchaga, L.A., Goldberg, V.M., and Caplan, A.I. Repair of osteochondral defect with tissueengineered two-phase composite material of injectable calcium phosphate and hyaluronan sponge. Tissue Eng 8, 827, 2002. 101. Frenkel, S.R., Bradica, G., Brekke, J.H., Goldman, S.M., Ieska, K., Issack, P., Bong, M.R., Tian, H., Gokhale, J., Coutts, R.D., and Kronengold, R.T. Regeneration of articular cartilage—evaluation of osteochondral defect repair in the rabbit using multiphasic implants. Osteoarthritis Cartilage 13, 798, 2005. 102. Shao, X., Goh, J.C., Hutmacher, D.W., Lee, E.H., and Zigang, G. Repair of large articular osteochondral defects using hybrid scaffolds and bone marrow-derived mesenchymal stem cells in a rabbit model. Tissue Eng 12, 1539, 2006. 103. Gotterbarm, T., Richter, W., Jung, M., Berardi Vilei, S., Mainil-Varlet, P., Yamashita, T., and Breusch, S.J. An in vivo study of a growth-factor enhanced, cell free, twolayered collagen-tricalcium phosphate in deep osteochondral defects. Biomaterials 27, 3387, 2006. 104. Zhou, X.Z., Leung, V.Y., Dong, Q.R., Cheung, K.M., Chan, D., and Lu, W.W. Mesenchymal stem cell-based repair of articular cartilage with polyglycolic acid-hydroxyapatite biphasic scaffold. Int J Artif Organs 31, 480, 2008. 105. Mrosek, E.H., Schagemann, J.C., Chung, H.W., Fitzsimmons, J.S., Yaszemski, M.J., Mardones, R.M., O’Driscoll, S.W., and Reinholz, G.G. Porous tantalum and polyepsilon-caprolactone biocomposites for osteochondral defect repair: preliminary studies in rabbits. J Orthop Res 28, 141, 2010. 106. Bal, B.S., Rahaman, M.N., Jayabalan, P., Kuroki, K., Cockrell, M.K., Yao, J.Q., and Cook, J.L. In vivo outcomes of tissue-engineered osteochondral grafts. J Biomed Mater Res B Appl Biomater 93, 164, 2010. 107. Im, G.I., Ahn, J.H., Kim, S.Y., Choi, B.S., and Lee, S.W. A hyaluronate-atelocollagen/beta-tricalcium phosphatehydroxyapatite biphasic scaffold for the repair of osteochondral defects: a porcine study. Tissue Eng Part A 16, 1189, 2010. 108. Marquass, B., Somerson, J.S., Hepp, P., Aigner, T., Schwan, S., Bader, A., Josten, C., Zscharnack, M., and Schulz, R.M. A novel MSC-seeded triphasic construct for the repair of osteochondral defects. J Orthop Res 28, 1586, 2010. 109. Cui, W., Wang, Q., Chen, G., Zhou, S., Chang, Q., Zuo, Q., Ren, K., and Fan, W. Repair of articular cartilage defects with tissue-engineered osteochondral composites in pigs. J Biosci Bioeng 111, 493, 2011.

2075

110. Chen, J., Chen, H., Li, P., Diao, H., Zhu, S., Dong, L., Wang, R., Guo, T., Zhao, J., and Zhang, J. Simultaneous regeneration of articular cartilage and subchondral bone in vivo using MSCs induced by a spatially controlled gene delivery system in bilayered integrated scaffolds. Biomaterials 32, 4793, 2011. 111. Yang, Q., Peng, J., Lu, S.B., Guo, Q.Y., Zhao, B., Zhang, L., Wang, A.Y., Xu, W.J., Xia, Q., Ma, X.L., Hu, Y.C., and Xu, B.S. Evaluation of an extracellular matrix-derived acellular biphasic scaffold/cell construct in the repair of a large articular high-load-bearing osteochondral defect in a canine model. Chin Med J (Engl) 124, 3930, 2011. 112. Miot, S., Brehm, W., Dickinson, S., Sims, T., Wixmerten, A., Longinotti, C., Hollander, A.P., Mainil-Varlet, P., and Martin, I. Influence of in vitro maturation of engineered cartilage on the outcome of osteochondral repair in a goat model. Eur Cell Mater 23, 222, 2012. 113. Pei, M., He, F., Boyce, B.M., and Kish, V.L. Repair of full-thickness femoral condyle cartilage defects using allogeneic synovial cell-engineered tissue constructs. Osteoarthritis Cartilage 17, 714, 2009. 114. Reyes, R., Delgado, A., Sanchez, E., Fernandez, A., Hernandez, A., and Evora, C. Repair of an osteochondral defect by sustained delivery of BMP-2 or TGFbeta1 from a bilayered alginate-PLGA scaffold. J Tissue Eng Regen Med 2012. DOI: 10.1002/term.1549. [Epub ahead of print.] 115. Deng, T., Lv, J., Pang, J., Liu, B., and Ke, J. Construction of tissue-engineered osteochondral composites and repair of large joint defects in rabbit. J Tissue Eng Regen Med 2012. [Epub ahead of print]; DOI: 10.1002/term.1556. 116. Re’em, T., Witte, F., Willbold, E., Ruvinov, E., and Cohen, S. Simultaneous regeneration of articular cartilage and subchondral bone induced by spatially presented TGF-beta and BMP-4 in a bilayer affinity binding system. Acta Biomater 8, 3283, 2012. 117. Getgood, A.M., Kew, S.J., Brooks, R., Aberman, H., Simon, T., Lynn, A.K., and Rushton, N. Evaluation of earlystage osteochondral defect repair using a biphasic scaffold based on a collagen-glycosaminoglycan biopolymer in a caprine model. Knee 19, 422, 2012. 118. Marmotti, A., Bruzzone, M., Bonasia, D.E., Castoldi, F., Rossi, R., Piras, L., Maiello, A., Realmuto, C., and Peretti, G.M. One-step osteochondral repair with cartilage fragments in a composite scaffold. Knee Surg Sports Traumatol Arthrosc 20, 2590, 2012. 119. Kon, E., Filardo, G., Robinson, D., Eisman, J.A., Levy, A., Zaslav, K., Shani, J., and Altschuler, N. Osteochondral regeneration using a novel aragonite-hyaluronate biphasic scaffold in a goat model. Knee Surg Sports Traumatol Arthrosc 2013. DOI: 10.1007/s00167-013-2467-2. 120. Duan, P., Pan, Z., Cao, L., He, Y., Wang, H., Qu, Z., Dong, J., and Ding, J. The effects of pore size in bilayered poly(lactide-co-glycolide) scaffolds on restoring osteochondral defects in rabbits. J Biomed Mater Res A 2013. [Epub ahead of print]; DOI: 10.1002/jbm.a.34683. 121. Zhang, S., Chen, L., Jiang, Y., Cai, Y., Xu, G., Tong, T., Zhang, W., Wang, L., Ji, J., Shi, P., and Ouyang, H.W. Bilayer collagen/microporous electrospun nanofiber scaffold improves the osteochondral regeneration. Acta Biomater 9, 7236, 2013. 122. Knutsen, G., Drogset, J.O., Engebretsen, L., Grontvedt, T., Isaksen, V., Ludvigsen, T.C., Roberts, S., Solheim, E., Strand, T., and Johansen, O. A randomized trial comparing autologous chondrocyte implantation with micro-

2076

123.

124.

125.

126.

127.

128.

129.

130.

131.

132.

LOPA AND MADRY

fracture. Findings at five years. J Bone Joint Surg Am 89, 2105, 2007. Kon, E., Mutini, A., Arcangeli, E., Delcogliano, M., Filardo, G., Nicoli Aldini, N., Pressato, D., Quarto, R., Zaffagnini, S., and Marcacci, M. Novel nanostructured scaffold for osteochondral regeneration: pilot study in horses. J Tissue Eng Regen Med 4, 300, 2010. Kon, E., Delcogliano, M., Filardo, G., Fini, M., Giavaresi, G., Francioli, S., Martin, I., Pressato, D., Arcangeli, E., Quarto, R., Sandri, M., and Marcacci, M. Orderly osteochondral regeneration in a sheep model using a novel nano-composite multilayered biomaterial. J Orthop Res 28, 116, 2010. Kon, E., Filardo, G., Delcogliano, M., Fini, M., Salamanna, F., Giavaresi, G., Martin, I., and Marcacci, M. Platelet autologous growth factors decrease the osteochondral regeneration capability of a collagen-hydroxyapatite scaffold in a sheep model. BMC Musculoskelet Disord 11, 220, 2010. Cao, Z., Hou, S., Sun, D., Wang, X., and Tang, J. Osteochondral regeneration by a bilayered construct in a cell-free or cell-based approach. Biotechnol Lett 34, 1151, 2012. Tampieri, A., Celotti, G., Landi, E., Sandri, M., Roveri, N., and Falini, G. Biologically inspired synthesis of bonelike composite: self-assembled collagen fibers/hydroxyapatite nanocrystals. J Biomed Mater Res A 67, 618, 2003. Tampieri, A., Sandri, M., Landi, E., Pressato, D., Francioli, S., Quarto, R., and Martin, I. Design of graded biomimetic osteochondral composite scaffolds. Biomaterials 29, 3539, 2008. Dormer, N.H., Singh, M., Zhao, L., Mohan, N., Berkland, C.J., and Detamore, M.S. Osteochondral interface regeneration of the rabbit knee with macroscopic gradients of bioactive signals. J Biomed Mater Res A 100, 162, 2012. Dormer, N.H., Busaidy, K., Berkland, C.J., and Detamore, M.S. Osteochondral interface regeneration of rabbit mandibular condyle with bioactive signal gradients. J Oral Maxillofac Surg 69, e50, 2011. Seo, J.P., Tanabe, T., Tsuzuki, N., Haneda, S., Yamada, K., Furuoka, H., Tabata, Y., and Sasaki, N. Effects of bilayer gelatin/beta-tricalcium phosphate sponges loaded with mesenchymal stem cells, chondrocytes, bone morphogenetic protein-2, and platelet rich plasma on osteochondral defects of the talus in horses. Res Vet Sci 95, 1210, 2013. Chen, K., Teh, T.K., Ravi, S., Toh, S.L., and Goh, J.C. Osteochondral interface generation by rabbit bone marrow stromal cells and osteoblasts coculture. Tissue Eng Part A 18, 1902, 2012.

133. Crowley, C., Wong, J.M., Fisher, D.M., and Khan, W.S. A systematic review on preclinical and clinical studies on the use of scaffolds for bone repair in skeletal defects. Curr Stem Cell Res Ther 8, 243, 2013. 134. Carmont, M.R., Carey-Smith, R., Saithna, A., Dhillon, M., Thompson, P., and Spalding, T. Delayed incorporation of a TruFit plug: perseverance is recommended. Arthroscopy 25, 810, 2009. 135. Bekkers, J.E., Bartels, L.W., Vincken, K.L., Dhert, W.J., Creemers, L.B., and Saris, D.B. Articular Cartilage Evaluation After TruFit Plug Implantation Analyzed by Delayed Gadolinium-Enhanced MRI of Cartilage (dGEMRIC). Am J Sports Med 41, 1290, 2013. 136. Filardo, G., Kon, E., Di Martino, A., Busacca, M., Altadonna, G., and Marcacci, M. Treatment of knee osteochondritis dissecans with a cell-free biomimetic osteochondral scaffold: clinical and imaging evaluation at 2-year follow-up. Am J Sports Med 41, 1786, 2013. 137. Zhang, W., Wang, G., Liu, Y., Zhao, X., Zou, D., Zhu, C., Jin, Y., Huang, Q., Sun, J., Liu, X., Jiang, X., and Zreiqat, H. The synergistic effect of hierarchical micro/nano-topography and bioactive ions for enhanced osseointegration. Biomaterials 34, 3184, 2013. 138. Roohani-Esfahani, S.I., Nouri-Khorasani, S., Lu, Z., Appleyard, R., and Zreiqat, H. The influence hydroxyapatite nanoparticle shape and size on the properties of biphasic calcium phosphate scaffolds coated with hydroxyapatitePCL composites. Biomaterials 31, 5498, 2010. 139. Fedorovich, N.E., Schuurman, W., Wijnberg, H.M., Prins, H.J., van Weeren, P.R., Malda, J., Alblas, J., and Dhert, W.J. Biofabrication of osteochondral tissue equivalents by printing topologically defined, cell-laden hydrogel scaffolds. Tissue Eng Part C Methods 18, 33, 2012. 140. Cui, X., Breitenkamp, K., Finn, M.G., Lotz, M., and D’Lima, D.D. Direct human cartilage repair using threedimensional bioprinting technology. Tissue Eng Part A 18, 1304, 2012.

Address correspondence to: Henning Madry, MD Center of Experimental Orthopaedics Saarland University Homburg/Saar D-66421 Germany E-mail: [email protected] Received: June 13, 2013 Accepted: October 16, 2013 Online Publication Date: January 27, 2014

Development of novel three-dimensional printed scaffolds for osteochondral regeneration.

Cell-laden biphasic scaffolds with anisotropic structure for the regeneration of osteochondral tissue.

Strategies for osteochondral repair: Focus on scaffolds.

Surface energy and stiffness discrete gradients in additive manufactured scaffolds for osteochondral regeneration.

ECM scaffolds for cartilage regeneration.

Decellularized Tooth Bud Scaffolds for Tooth Regeneration.

Clinical results of multilayered biomaterials for osteochondral regeneration.

3D printing of novel osteochondral scaffolds with graded microstructure.

Magnesium-containing nanostructured hybrid scaffolds for enhanced dentin regeneration.

Electrospun soy protein nanofiber scaffolds for tissue regeneration.

Tuning dual-drug release from composite scaffolds for bone regeneration.

Highly Porous Gelatin Reinforced 3D Scaffolds for Articular Cartilage Regeneration.

gelatin hybrid nanofibrous scaffolds encapsulating EGF for skin regeneration.

Triphasic scaffolds for the regeneration of the bone-ligament interface.

Three-dimensional scaffolds of carbonized polyacrylonitrile for bone tissue regeneration.

Design strategies of biodegradable scaffolds for tissue regeneration.

3D Printing of Scaffolds for Tissue Regeneration Applications.

Bone Tissue Engineering: Scaffolds with Osteoinductivity for Bone Regeneration.

Functionalized scaffolds to enhance tissue regeneration.

Scaffolds in vascular regeneration: current status.

Integrating biologically inspired nanomaterials and table-top stereolithography for 3D printed biomimetic osteochondral scaffolds.

Osteochondral tissue regeneration through polymeric delivery of DNA encoding for the SOX trio and RUNX2.

Evaluation of oriented electrospun fibers for periosteal flap regeneration in biomimetic triphasic osteochondral implant.

Osteochondral Allograft Transplantation for Treatment of Focal Patellar Osteochondral Lesion.