Interfacial Tissue Regeneration with Bone.

PURPOSE OF REVIEW: Interfacial tissue exists throughout the body at cartilage-to-bone (osteochondral interface) and tendon-to-bone (enthesis) interfaces. Healing of interfacial tissues is a current challenge in regenerative approaches because the interface plays a critical role in stabilizing and distributing the mechanical stress between soft tissues (e.g., cartilage and tendon) and bone. The purpose of this review is to identify new directions in the field of interfacial tissue development and physiology that can guide future regenerative strategies for improving post-injury healing. RECENT FINDINGS: Cues from interfacial tissue development may guide regeneration including biological cues such as cell phenotype and growth factor signaling; structural cues such as extracellular matrix (ECM) deposition, ECM, and cell alignment; and mechanical cues such as compression, tension, shear, and the stiffness of the cellular microenvironment. In this review, we explore new discoveries in the field of interfacial biology related to ECM remodeling, cellular metabolism, and fate. Based on emergent findings across multiple disciplines, we lay out a framework for future innovations in the design of engineered strategies for interface regeneration. Many of the key mechanisms essential for interfacial tissue development and adaptation have high potential for improving outcomes in the clinic.

Understanding the Mechanics of the Temporomandibular Joint Osteochondral Interface from Micro- and Nanoscopic Perspectives.

The condylar cartilage of the temporomandibular joint (TMJ) is connected to the subchondral bone by an osteochondral interface that transmits loads without causing fatigue damage. However, the microstructure, composition, and mechanical properties of this interface remain elusive. In this study, we found that structurally, a spatial gradient assembly of hydroxyapatite (HAP) particles exists in the osteochondral interface, with increasing volume of apatite crystals with depth and a tendency to form denser and stacked structures. Combined with nanoindentation, this complex assembly of nanoscale structures and components enhanced energy dissipation at the osteochondral interface, achieving a smooth stress transition between soft and hard tissues. This study comprehensively demonstrates the elemental composition and complex nanogradient spatial assembly of the osteochondral interface at the ultramicroscopic scale, providing a basis for exploring the construction of complex mechanical models of the interfacial region.

The deterioration of calcified cartilage integrity reflects the severity of osteoarthritis-A structural, molecular, and biochemical analysis.

The calcified cartilage zone (CCZ) is a thin interlayer between the hyaline articular cartilage and the subchondral bone and plays an important role in maintaining the joint homeostasis by providing biological and mechanical support from unmineralized cartilage to the underlying mineralized subchondral bone. The hallmark of CCZ characteristics in osteoarthritis (OA) is less well known. The aim of our study is to evaluate the structural, molecular, and biochemical composition of CCZ in tissues affected by primary knee OA and its relationship with disease severity. We collected osteochondral tissue samples stratified according to disease severity, from 16 knee OA patients who underwent knee replacement surgery. We also used meniscectomy-induced rat samples to confirm the pathophysiologic changes of human samples. We defined the characteristics of the calcified cartilage layer using a combination of morphological, biochemical, proteomic analyses on laser micro-dissected tissue. Our results demonstrated that the Calcium/Phosphate ratio is unchanged during the OA progression, but the calcium-binding protein and cadherin binding protein, as well as carbohydrate metabolism-related proteins, undergo significant changes. These changes were further accompanied by thinning of the CCZ, loss of collagen and proteoglycan content, the occurrence of the endochondral ossification, neovasculature, loss of the elastic module, loss of the collagen direction, and increase of the tortuosity indicating an altered structural and mechanical properties of the CCZ in OA. In conclusion, our results suggest that the calcified cartilage changes can reflect the disease progression.

A technique for preparing undecalcified osteochondral fresh frozen sections for elemental mapping and understanding disease etiology.

The anatomy of the osteochondral junction is complex because several tissue components exist as a unit, including uncalcified cartilage (with superficial, middle, and deep layers), calcified cartilage, and subchondral bone. Furthermore, it is difficult to study because this region is made up of a variety of cell types and extracellular matrix compositions. Using X-ray fluorescence microscopy, we present a protocol for simultaneous elemental detection on fresh frozen samples. We transferred the osteochondral sample using a tape-assisted system and successfully tested it in synchrotron X-ray fluorescence. This protocol elucidates the distinct distribution of elements at the human knee's osteochondral junction, making it a useful tool for analyzing the co-distribution of various elements in both healthy and diseased states.

Micro/Nano Scaffolds for Osteochondral Tissue Engineering.

To develop an osteochondral tissue regeneration strategy it is extremely important to take into account the multiscale organization of the natural extracellular matrix. The structure and gradients of organic and inorganic components present in the cartilage and bone tissues must be considered together. Another critical aspect is an efficient interface between both tissues. So far, most of the approaches were focused on the development of multilayer or stratified scaffolds which resemble the structural composition of bone and cartilage, not considering in detail a transitional interface layer. Typically, those scaffolds have been produced by the combined use of two or more processing techniques (microtechnologies and nanotechnologies) and materials (organic and inorganic). A significant number of works was focused on either cartilage or bone, but there is a growing interest in the development of the osteochondral interface and in tissue engineering models of composite constructs that can mimic the cartilage/bone tissues. The few works that give attention to the interface between cartilage and bone, as well as to the biochemical gradients observed at the osteochondral unit, are also herein described.

Osteochondral Interface: Regenerative Engineering and Challenges.

Osteochondral (OC) defects are debilitating for patients and represent a significant clinical problem for orthopedic surgeons as well as regenerative engineers due to their potential complications, which are likely to lead to osteoarthritis and related diseases. If they remain untreated or are treated suboptimally, OC lesions are known to impact the articular cartilage and the transition from cartilage to bone, that is, the cartilage-bone interface. An important component of the OC interface, that is, a selectively permeable membrane, the tidemark, still remains unaddressed in more than 90% of the published research in the past decade. This review focuses on the structure, composition, and function of the OC interface, regenerative engineering attempts with different scaffolding strategies and challenges ahead of us in recapitulating the native OC interface. There are different schools of thought regarding the structure of the native OC interface: stratified and graded. The former assumes the cartilage-to-bone interface to be hierarchically divided into distinct yet continuous zones of uncalcified cartilage-calcified cartilage-subchondral bone. The latter assumes the interface is continuously graded, that is, formed by an infinite number of layers. The cellular composition of the interface, either in respective layers or continuously changing in a graded manner, is chondrocytes, hypertrophic chondrocytes, and osteoblasts as moved from cartilage to bone. Functionally, the interface is assumed to play a role in enabling a smooth transition of loads exerted on the cartilage surface to the bone underneath. Regenerative engineering involves, first, a characterization of the native OC interface in terms of the composition, structure, and function, and, then, proposes the appropriate biomaterials, cells, and biomolecules either alone or in combination to eventually form a structure that mimics and functionally behaves similar to the native interface. The major challenge regarding regeneration of the OC interface appears to lie, in addition to others, in the formation of tidemark, which is a thin membrane separating the OC interface into two distinct zones: the avascular OC interface and the vascular OC interface. There is a significant amount of literature on regenerative approaches to the OC interface; however, only a small portion of them consider the importance of tidemark. Therefore, this review aims at highlighting the significance of the structural organization of the components of the OC interface and increasing the awareness of the orthopedics community regarding the importance of tidemark formation after clinical interventions or regenerative engineering attempts.

Development of a method to investigate strain distribution across the cartilage-bone interface in guinea pig model of spontaneous osteoarthritis using lab-based contrast enhanced X-ray-computed tomography and digital volume correlation.

OBJECTIVE: Strain changes at the cartilage-bone interface play a crucial role in osteoarthritis (OA) development. Contrast-Enhanced X-ray Computed Tomography (CECT) and Digital Volume Correlation (DVC) can measure 3D strain changes at the osteochondral interface. Using lab-based CT systems it is often difficult to visualise soft tissues such as articular cartilage without staining to enhance contrast. Contrast-Enhancing Staining Agents (CESAs), such as Phosphotungstic Acid (PTA) in 70% ethanol, can cause tissue shrinkage and alter tissue mechanics. The aims of this study were, firstly, to assess changes to the mechanical properties of osteochondral tissue after staining with a PTA/PBS solution, and secondly, to visualise articular cartilage during loading and with CECT imaging in order to compare strain across the interface in both healthy and OA joints using DVC. DESIGN: Nanoindentation was used to assess changes to mechanical properties in articular cartilage and subchondral bone before and after staining. Hindlimbs from Dunkin-Hartley guinea pigs were stained with 1% PTA/PBS at room temperature for 6 days. Two consecutive CECT datasets were acquired for DVC error analysis. In-situ compression with a load corresponding to 2x body weight was applied, the specimen was re-imaged, and DVC was performed between the pre- and post-load tomograms. RESULTS: Nanoindentation before and after PTA/PBS staining showed similar cartilage stiffness (p < 0.05), however, staining significantly decreased the stiffness of subchondral bone (∼9-fold; p = 0.0012). In severe OA specimens, third principal/compressive (εp3) strain was 141.7% higher and shear strain (γ) was 98.2% higher in tibial articular cartilage compared to non-OA (2 - month) specimens. A 23.1% increase in third principal stain strain and a 54.5% significant increase in the shear (γ) strain (p = 0.0027) was transferred into the mineralised regions of calcified cartilage and subchondral bone in severe OA specimens. CONCLUSIONS: These results indicate the suitability of PTA in PBS as a contrast agent for the visualisation of cartilage during CECT imaging and allowed DVC computation of strain across the cartilage-bone interface. However, further research is needed to address the reduction in stiffness of subchondral bone after incubation in PBS.

3D bioprinting of tyramine modified hydrogels under visible light for osteochondral interface.

Recent advancements in tissue engineering have demonstrated a great potential for the fabrication of three-dimensional (3D) tissue structures such as cartilage and bone. However, achieving structural integrity between different tissues and fabricating tissue interfaces are still great challenges. In this study, anin situcrosslinked hybrid, multi-material 3D bioprinting approach was used for the fabrication of hydrogel structures based on an aspiration-extrusion microcapillary method. Different cell-laden hydrogels were aspirated in the same microcapillary glass and deposited in the desired geometrical and volumetric arrangement directly from a computer model. Alginate and carboxymethyl cellulose were modified with tyramine to enhance cell bioactivity and mechanical properties of human bone marrow mesenchymal stem cells-laden bioinks. Hydrogels were prepared for extrusion by gelling in microcapillary glass utilizing anin situcrosslink approach with ruthenium (Ru) and sodium persulfate photo-initiating mechanisms under visible light. The developed bioinks were then bioprinted in precise gradient composition for cartilage-bone tissue interface using microcapillary bioprinting technique. The biofabricated constructs were co-cultured in chondrogenic/osteogenic culture media for three weeks. After cell viability and morphology evaluations of the bioprinted structures, biochemical and histological analyses, and a gene expression analysis for the bioprinted structure were carried out. Analysis of cartilage and bone formation based on cell alignment and histological evaluation indicated that mechanical cues in conjunction with chemical cues successfully induced MSC differentiation into chondrogenic and osteogenic tissues with a controlled interface.

Early Degenerative Changes in a Spontaneous Osteoarthritis Model Assessed by Nanoindentation.

Understanding early mechanical changes in articular cartilage (AC) and subchondral bone (SB) is crucial for improved treatment of osteoarthritis (OA). The aim of this study was to develop a method for nanoindentation of fresh, unfixed osteochondral tissue to assess the early changes in the mechanical properties of AC and SB. Nanoindentation was performed throughout the depth of AC and SB in the proximal tibia of Dunkin Hartley guinea pigs at 2 months, 3 months, and 2 years of age. The contralateral tibias were either histologically graded for OA or analyzed using immunohistochemistry. The results showed an increase in the reduced modulus (Er) in the deep zone of AC during early-stage OA (6.0 ± 1.75 MPa) compared to values at 2 months (4.04 ± 1.25 MPa) (*** p < 0.001). In severe OA (2-year) specimens, there was a significant reduction in Er throughout the superficial and middle AC zones, which correlated to increased ADAMTS 4 and 5 staining, and proteoglycan loss in these regions. In the subchondral bone, a 35.0% reduction in stiffness was observed between 2-month and 3-month specimens (*** p < 0.001). The severe OA age group had significantly increased SB stiffness of 36.2% and 109.6% compared to 2-month and 3-month-old specimens respectively (*** p < 0.001). In conclusion, this study provides useful information about the changes in the mechanical properties of both AC and SB during both early- and late-stage OA and indicates that an initial reduction in stiffness of the SB and an increase in stiffness in the deep zone of AC may precede early-stage cartilage degeneration.

Computational investigation of interface printing patterns within 3D printed multilayered scaffolds for osteochondral tissue engineering.

Osteoarthritis is a highly prevalent rheumatic musculoskeletal disorder that commonly affects many joints. Repetitive joint overloading perpetuates the damage to the affected cartilage, which undermines the structural integrity of the osteochondral unit. Various tissue engineering strategies have been employed to design multiphasic osteochondral scaffolds that recapitulate layer-specific biomechanical properties, but the inability to fully satisfy mechanical demands within the joint has limited their success. Through computational modeling and extrusion-based bioprinting, we attempted to fabricate a biphasic osteochondral scaffold with improved shear properties and a mechanically strong interface. A 3D stationary solid mechanics model was developed to simulate the effect of lateral shear force on various thermoplastic polymer/hydrogel scaffolds with a patterned interface. Additionally, interfacial shear tests were performed on bioprinted polycaprolactone (PCL)/hydrogel interface scaffolds. The first simulation showed that the PCL/gelatin methacrylate (GelMA) and PCL/polyethylene glycol diacrylate (PEGDA) scaffolds interlocking hydrogel and PCL at interface in a 1:1 ratio possessed the largest average tensile (PCL/GelMA: 80.52 kPa; PCL/PEGDA: 79.75 kPa) and compressive stress (PCL/GelMA: 74.71 kPa; PCL/PEGDA: 73.83 kPa). Although there were significant differences in shear strength between PCL/GelMA and PCL/PEGDA scaffolds, no significant difference was observed among the treatment groups within both scaffold types. Lastly, the hypothetical simulations of potential biphasic 3D printed scaffolds showed that for every order of magnitude decrease in Young's modulus (E) of the soft bioink, all the scaffolds underwent an exponential increase in average displacement at the cartilage and interface layers. The following work provides valuable insights into the biomechanics of 3D printed osteochondral scaffolds, which will help inform future scaffold designs for enhanced regenerative outcomes.

Progress in Osteochondral Regeneration with Engineering Strategies.

Osteoarthritis, the main cause of disability worldwide, involves not only cartilage injury but also subchondral bone injury, which brings challenges to clinical repair. Tissue engineering strategies provide a promising solution to this degenerative disease. Articular cartilage connects to subchondral bone through the osteochondral interfacial tissue, which has a complex anatomical architecture, distinct cell distribution and unique biomechanical properties. Forming a continuous and stable osteochondral interface between cartilage tissue and subchondral bone is challenging. Thus, successful osteochondral regeneration with engineering strategies requires intricately coordinated interplay between cells, materials, biological factors, and physical/chemical factors. This review provides an overview of the anatomical composition, microstructure, and biomechanical properties of the osteochondral interface. Additionally, the latest research on the progress related to osteochondral regeneration is reviewed, especially discussing the fabrication of biomimetic scaffolds and the regulation of biological factors for osteochondral defects.

Gravity-based patterning of osteogenic factors to preserve bone structure after osteochondral injury in a large animal model.

Chondral and osteochondral repair strategies are limited by adverse bony changes that occur after injury. Bone resorption can cause entire scaffolds, engineered tissues, or even endogenous repair tissues to subside below the cartilage surface. To address this translational issue, we fabricated thick-shelled poly(D,L-lactide-co-glycolide) microcapsules containing the pro-osteogenic agents triiodothyronine andß-glycerophosphate, and delivered these microcapsules in a large animal model of osteochondral injury to preserve bone structure. We demonstrate that the developed microcapsules rupturedin vitrounder increasing mechanical loads, and readily sink within a liquid solution, enabling gravity-based patterning along the osteochondral surface. In a large animal, these mechanically-activated microcapsules (MAMCs) were assessed through two different delivery strategies. Intra-articular injection of control MAMCs enabled fluorescent quantification of MAMC rupture and cargo release in a synovial joint setting over timein vivo. This joint-wide injection also confirmed that the MAMCs do not elicit an inflammatory response. In the contralateral hindlimbs, chondral defects were created, MAMCs were patternedin situ, and nanofracture (Nfx), a clinically utilized method to promote cartilage repair, was performed. The Nfx holes enabled marrow-derived stromal cells to enter the defect area and served as repeatable bone injury sites to monitor over time. Animals were evaluated one and two weeks after injection and surgery. Analysis of injected MAMCs showed that bioactive cargo was released in a controlled fashion over two weeks. A bone fluorochrome label injected at the time of surgery displayed maintenance of mineral labeling in the therapeutic group, but resorption in both control groups. Alkaline phosphatase (AP) staining at the osteochondral interface revealed higher AP activity in defects treated with therapeutic MAMCs. Overall, this study develops a gravity-based approach to pattern bioactive factors along the osteochondral interface, and applies this novel biofabrication strategy to preserve bone structure after osteochondral injury.

Bio-inspired zonal-structured matrices for bone-cartilage interface engineering.

Design and development of scaffold structures for osteochondral (OC) interface regeneration is a significant engineering challenge. Recent efforts are aimed at recapitulating the unique compositional and hierarchical structure of an OC interface. Conventional scaffold fabrication techniques often have limited design control and reproducibility, and the development of OC scaffolds with zonal hierarchy and structural integrity between zones is especially challenging. In this study, a series of multi-zonal and gradient structures were designed and fabricated using three-dimensional bioprinting. We developed OC scaffolds with bi-phasic and tri-phasic configurations to support the zonal structure of OC tissue, and gradient scaffold configurations to enable smooth transitions between the zones to more closely mimic a bone-cartilage interface. A biodegradable polymer, polylactic acid, was used for the fabrication of zonal/gradient scaffolds to provide mechanical strength and support OC function. The formation of the multi-zonal and gradient scaffolds was confirmed through scanning electron microscopy imaging and micro-computed tomography scanning. Precisely controlled hierarchy with tunable porosity along the scaffold length established the formation of the bio-inspired scaffolds with different zones/gradient structure. In addition, we also developed a novel bioprinting method to selectively introduce cells into desired scaffold zones of the zonal/gradient scaffolds via concurrent printing of a cell-laden hydrogel within the porous template. Live/dead staining of the cell-laden hydrogel introduced in the cartilage zone showed uniform cell distribution with high cell viability. Overall, our study developed bio-inspired scaffold structures with structural hierarchy and mechanical integrity for bone-cartilage interface engineering.

Bio-inspired zonal-structured matrices for bone-cartilage interface engineering.

Design and development of scaffold structures for osteochondral (OC) interface regeneration is a significant engineering challenge. Recent efforts are aimed at recapitulating the unique compositional and hierarchical structure of an OC interface. Conventional scaffold fabrication techniques often have limited design control and reproducibility, and the development of OC scaffolds with zonal hierarchy and structural integrity between zones is especially challenging. In this study, a series of multi-zonal and gradient structures were designed and fabricated using three-dimensional (3D) bioprinting. We developed OC scaffolds with bi-phasic and tri-phasic configurations to support the zonal structure of OC tissue, and gradient scaffold configurations to enable smooth transitions between the zones to more closely mimic a bone-cartilage interface. A biodegradable polymer, polylactic acid (PLA), was used for the fabrication of zonal/gradient scaffolds to provide mechanical strength and support OC function. The formation of the multi-zonal and gradient scaffolds was confirmed through SEM imaging and micro-CT scanning. Precisely controlled hierarchy with tunable porosity along the scaffold length established the formation of the bio-inspired scaffolds with different zones/gradient structure. In addition, we also developed a novel bioprinting method to selectively introduce cells into desired scaffold zones of the zonal/gradient scaffolds via concurrent printing of a cell-laden hydrogel within the porous template. Live/dead staining of the cell-laden hydrogel introduced in the cartilage zone showed uniform cell distribution with high cell viability. Overall, our study developed bio-inspired scaffold structures with structural hierarchy and mechanical integrity for bone-cartilage interface engineering.

Porous Scaffold-Hydrogel Composites Spatially Regulate 3D Cellular Mechanosensing.

Cells encapsulated in 3D hydrogels exhibit differences in cellular mechanosensing based on their ability to remodel their surrounding hydrogel environment. Although cells in tissue interfaces feature a range of mechanosensitive states, it is challenging to recreate this in 3D biomaterials. Human mesenchymal stem cells (MSCs) encapsulated in methacrylated gelatin (GelMe) hydrogels remodel their local hydrogel environment in a time-dependent manner, with a significant increase in cell volume and nuclear Yes-associated protein (YAP) localization between 3 and 5 days in culture. A finite element analysis model of compression showed spatial differences in hydrogel stress of compressed GelMe hydrogels, and MSC-laden GelMe hydrogels were compressed (0-50%) for 3 days to evaluate the role of spatial differences in hydrogel stress on 3D cellular mechanosensing. MSCs in the edge (high stress) were significantly larger, less round, and had increased nuclear YAP in comparison to MSCs in the center (low stress) of 25% compressed GelMe hydrogels. At 50% compression, GelMe hydrogels were under high stress throughout, and this resulted in a consistent increase in MSC volume and nuclear YAP across the entire hydrogel. To recreate heterogeneous mechanical signals present in tissue interfaces, porous polycaprolactone (PCL) scaffolds were perfused with an MSC-laden GelMe hydrogel solution. MSCs in different pore diameter (~280-430 µm) constructs showed an increased range in morphology and nuclear YAP with increasing pore size. Hydrogel stress influences MSC mechanosensing, and porous scaffold-hydrogel composites that expose MSCs to diverse mechanical signals are a unique biomaterial for studying and designing tissue interfaces.

Regulation of chondrocyte hypertrophy in an osteochondral interface mimicking gel matrix.

Calcified cartilage extracellular matrix (ECM) is a critical interface at the osteochondral junction which plays an important role in maintaining the structural continuity between articular cartilage and subchondral bone. This mineralized network is primarily composed of glycosaminoglycans (GAGs) and collagen type II (col II) and hosts hypertrophic chondrocytes. This work aimed to investigate the effect of gel composition and collagen II content on the behavior and hypertrophic differentiation of ATDC5 cells for regeneration of calcified cartilage tissue. For this purpose, chitosan/collagen type II/nanohydroxyapatite (chi/col II/nHA) composite hydrogels were prepared to mimic the calcified cartilage ECM. ATDC5 cells were encapsulated within the composite gels and the viability, ECM production and hypertrophic gene expression were assessed during culture. All composites were favorable for ATDC5 viability and proliferation, whereas specific ECM production and hypertrophic differentiation were dependent on gel composition. Chitosan: collagen II ratio had an impact on ATDC5 cell fate. Hypertrophic differentiation was best pronounced in chi/col II/nHA 70:30 composition. The results obtained from this study offers a scaffold-based approach for calcified cartilage regeneration and provide an insight for biomimetic design and preparation of more complicated gradient osteochondral units.

Cartilage/bone interface fabricated under perfusion: Spatially organized commitment of adipose-derived stem cells without medium supplementation.

Tissue engineering of an osteochondral interface demands for a gradual transition of chondrocyte- to osteoblast-prevailing tissue. If stem cells are used as a single cell source, an appropriate cue to trigger the desired differentiation is the use of composite materials with different amounts of calcium phosphate. Electrospun meshes of poly-lactic-co-glycolic acid and amorphous calcium phosphate nanoparticles (PLGA/aCaP) in weight ratios of 100:0; 90:10, 80:20, and 70:30 were seeded with human adipose-derived stem cells (ASCs) and cultured in DMEM without chemical supplementation. After 2 weeks of static cultivation, they were either further cultivated statically for another 2 weeks (group 1), or placed in a Bose® bioreactor with a flow rate per area of 0.16 mL cm-2 min-1 (group 2). Markers for stem cell criteria, chondrogenesis, osteogenesis, adipogenesis and angiogenesis were analyzed by quantitative real-time PCR. Cell distribution, Sox9 protein expression and proteoglycans were assessed by histology. In group 2 (perfusion culture), chondrogenic Sox9 was upregulated toward the cartilage-mimicking side compared to pure PLGA. On the bone-mimicking side, Sox9 experienced a downregulation, which was confirmed on the protein level. Vice versa, expression of osteocalcin was upregulated on the bone-mimicking side, while it was unchanged on the cartilage-mimicking side. In group 1 (static culture), CD31 was upregulated in the presence of aCaP compared to pure PLGA, whereas Sox9 and osteocalcin expression were not affected. aCaP nanoparticles incorporated in electrospun PLGA drive the differentiation behavior of human ASCs in a dose-dependent manner. Discrete gradients of aCaP may act as promising osteochondral interfaces. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1833-1843, 2019.

Icariin conjugated hyaluronic acid/collagen hydrogel for osteochondral interface restoration.

Over the past decades, numerous tissue-engineered constructs have been investigated for the osteochondral repair. However, it still remains a challenge to regenerate the functionalized calcified layer. In this study, the potential of icariin (Ica) conjugated hyaluronic acid/collagen (Ica-HA/Col) hydrogel to promote the osteochondral interface restoration was investigated. Compared with HA/Col hydrogel, Ica-HA/Col hydrogel simultaneously facilitated chondrogenesis and osteogenesis in vitro. The cells encapsulated in Ica-HA/Col hydrogel tended to aggregate into bigger clusters. The chondrogenic genes' expression level was remarkably up-regulated, and the matrix synthesis of sGAG and type II collagen was significantly enhanced. Similarly, the osteogenic genes, including RUNX2, ALP, and OCN were also up-regulated at early stage. Consequently, more calcium deposition was observed in the Ica-HA/Col hydrogel construct. Moreover, the gene expression and matrix synthesis of type X collagen, an important marker for the formation of calcified layer; were significantly higher in the Ica-HA/Col hydrogel. Furthermore, the in vivo study showed that Ica-HA/Col constructs facilitated the reconstruction of osteochondral interface in rabbit subchondral defects. In the Ica-HA/Col group, the neo-cartilage layer contained more type II collagen and the newly formed subchondral bone deposited more abundant type I collagen. Overall, the results indicated that Ica-HA/Col hydrogel might be a promising scaffold to reconstruct an osteochondral interface, therefore promoting restoring of osteochondral defect. STATEMENT OF SIGNIFICANCE: The osteochondral defect restoration not only involves the repair of damaged cartilage and the subchondral bone, but also the reconstruction of osteochondral interface (the functional calcified layer). The calcified layer regeneration is essential for integrative and functional osteochondral repair. Over the past decade, numerous tissue engineered constructs have been investigated for the osteochondral repair. However, it still remains a challenge to regenerate a functionalized calcified layer. The present study demonstrates that Ica-HA/Col hydrogel facilitates deposition of matrix related to calcified layer in mixed chondrogenic/osteogenic inductive media and restoration of osteochondral defect in vivo. Since, Ica-HA/Col hydrogel as is cheaper, easier and more efficient, it might be a desired scaffold for the osteochondral defects restoration.

Multilayered Scaffold with a Compact Interfacial Layer Enhances Osteochondral Defect Repair.

Repairing osteochondral defect (OCD) using advanced biomaterials that structurally, biologically, and mechanically fulfill the criteria for stratified tissue regeneration remains a significant challenge for researchers. Here, a multilayered scaffold (MLS) with hierarchical organization and heterogeneous composition is developed to mimic the stratified structure and complex components of natural osteochondral tissues. Specifically, the intermediate compact interfacial layer within the MLS is designed to resemble the osteochondral interface to realize the closely integrated layered structure. Subsequently, macroscopic observations, histological evaluation, and biomechanical and biochemical assessments are performed to evaluate the ability of the MLS of repairing OCD in a goat model. By 48 weeks postimplantation, superior hyalinelike cartilage and sound subchondral bone are observed in the MLS group. Furthermore, the biomimetic MLS significantly enhances the biomechanical and biochemical properties of the neo-osteochondral tissue. Taken together, these results confirm the potential of this optimized MLS as an advanced strategy for OCD repair.

Solute transport at the interface of cartilage and subchondral bone plate: Effect of micro-architecture.

Cross-talk of subchondral bone and articular cartilage could be an important aspect in the etiology of osteoarthritis. Previous research has provided some evidence of transport of small molecules (~370Da) through the calcified cartilage and subchondral bone plate in murine osteoarthritis models. The current study, for the first time, uses a neutral diffusing computed tomography (CT) contrast agent (iodixanol, ~1550Da) to study the permeability of the osteochondral interface in equine and human samples. Sequential CT monitoring of diffusion after injecting a finite amount of contrast agent solution onto the cartilage surface using a micro-CT showed penetration of the contrast molecules across the cartilage-bone interface. Moreover, diffusion through the cartilage-bone interface was affected by thickness and porosity of the subchondral bone as well as the cartilage thickness in both human and equine samples. Our results revealed that porosity of the subchondral plate contributed more strongly to the diffusion across osteochondral interface compared to other morphological parameters in healthy equine samples. However, thickness of the subchondral plate contributed more strongly to the diffusion in slightly osteoarthritic human samples.