Osteochondral Tissue-On-a-Chip: A Novel Model for Osteoarthritis Research.

The existing in vitro and in vivo models for studying osteoarthritis have significant limitations in replicating the complexity of joint tissues. This research aims to validate a Tissue-On-a-Chip system for osteoarthritis research. Osteochondral tissues obtained from knee replacement surgeries of patients with osteoarthritis were cultured in an Organ-On-a-Chip system. This system was designed to supply oxygen and glucose to the cartilage from the bone. The distribution of oxygen and glucose was evaluated by fluorescence using Image-iT Green Hypoxia and 2-NBDG, respectively. Cytotoxicity was measured using lactate dehydrogenase (LDH) levels in chip cultures compared to plate cultures (12 tissues per method). Glycosaminoglycans (GAGs), alkaline phosphatase (ALP), Coll2-1, and procollagen type II N-terminal propeptide (PIINP) were measured in the perfused medium of the Tissue-On-a-Chip over a period of 70 days. Fluorescence of Image-iT Green Hypoxia was observed only in the cartilage area, while 2-NBDG was distributed throughout the tissue. An increase in LDH levels was noted in the plate cultures on day 24 and in the Tissue-On-a-Chip cultures on day 63. Compared to the start of the culture, GAG content increased on day 52, while ALP showed variations. A notable increase in GAG, ALP, and Coll2-1 levels was observed on day 59. PIINP levels remained stable throughout the experiment. The validated osteochondral Tissue-On-a-Chip system can replicate the joint microenvironment, with hypoxic conditions in cartilage and normoxic conditions in bone. Tissue survival and component stability were maintained for approximately two months. This platform is a useful tool for evaluating new drugs and represents a viable alternative to animal models.

Limited dorsal myeloschisis without extradural stalk continuity to coexisting congenital dermal sinus.

Limited dorsal myeloschisis (LDM) is characterized by a fibroneural tethering stalk linking the skin lesion to the underlying spinal cord. LDM without an extradural stalk is rare. A full-term boy was noted at birth to have a dimple in the upper back (dorsal skin of the lower thoracic region). Computed tomographic scan showed spina bifida at the T9-12 vertebral level and osteochondral tissue at the T10 level. Magnetic resonance imaging (MRI) demonstrated a tiny dorsal lipoma at the T8 vertebral level, but the intradural tethering tract was not apparent. At 18 days of age, the congenital dermal sinus (CDS) tract started from the dimple and terminated at the osteochondral tissue, without continuity of the dura mater, and the osteochondral tissues were resected. At age 2 years 8 months, he developed spastic paresis of the right foot. On MRI, the tethering tract from the dorsal lipoma became apparent. During the second surgery at age 2 years 11 months, the intradural stalk started from the dorsal lipoma and joined the inner surface of the dura mater was untethering from the cord. Postoperatively, right spastic paresis was improved. Histological examination of the intradural stalk revealed the distribution of S100-immunopositive peripheral nerve fibers, which is one of the histopathological hallmarks of LDM. We speculated that the extradural stalk with coexisting CDS originally linked from the skin lesion subsequently regressed and was replaced by fibroadipose tissue with osteochondral tissue migration. Intradural exploration should always be seriously considered in these disorders of persisting neurocutaneous connection.

Advances in Translational 3D Printing for Cartilage, Bone, and Osteochondral Tissue Engineering.

The regeneration of 3D tissue constructs with clinically relevant sizes, structures, and hierarchical organizations for translational tissue engineering remains challenging. 3D printing, an additive manufacturing technique, has revolutionized the field of tissue engineering by fabricating biomimetic tissue constructs with precisely controlled composition, spatial distribution, and architecture that can replicate both biological and functional native tissues. Therefore, 3D printing is gaining increasing attention as a viable option to advance personalized therapy for various diseases by regenerating the desired tissues. This review outlines the recently developed 3D printing techniques for clinical translation and specifically summarizes the applications of these approaches for the regeneration of cartilage, bone, and osteochondral tissues. The current challenges and future perspectives of 3D printing technology are also discussed.

Piezoelectric Electrospun Fibrous Scaffolds for Bone, Articular Cartilage and Osteochondral Tissue Engineering.

Osteochondral tissue (OCT) related diseases, particularly osteoarthritis, number among the most prevalent in the adult population worldwide. However, no satisfactory clinical treatments have been developed to date to resolve this unmet medical issue. Osteochondral tissue engineering (OCTE) strategies involving the fabrication of OCT-mimicking scaffold structures capable of replacing damaged tissue and promoting its regeneration are currently under development. While the piezoelectric properties of the OCT have been extensively reported in different studies, they keep being neglected in the design of novel OCT scaffolds, which focus primarily on the tissue's structural and mechanical properties. Given the promising potential of piezoelectric electrospun scaffolds capable of both recapitulating the piezoelectric nature of the tissue's fibrous ECM and of providing a platform for electrical and mechanical stimulation to promote the regeneration of damaged OCT, the present review aims to examine the current state of the art of these electroactive smart scaffolds in OCTE strategies. A summary of the piezoelectric properties of the different regions of the OCT and an overview of the main piezoelectric biomaterials applied in OCTE applications are presented. Some recent examples of piezoelectric electrospun scaffolds developed for potentially replacing damaged OCT as well as for the bone or articular cartilage segments of this interfacial tissue are summarized. Finally, the current challenges and future perspectives concerning the use of piezoelectric electrospun scaffolds in OCT regeneration are discussed.

Bilayer Scaffolds for Interface Tissue Engineering and Regenerative Medicine: A Systematic Reviews.

PURPOSE: This systematic review focus on the application of bilayer scaffolds as an engaging structure for the engineering of multilayered tissues, including vascular and osteochondral tissues, skin, nerve, and urinary bladder. This article provides a concise literature review of different types of bilayer scaffolds to understand their efficacy in targeted tissue engineering. METHODS: To this aim, electronic search in the English language was performed in PMC, NBCI, and PubMed from April 2008 to December 2019 based on the PRISMA guidelines. Animal studies, including the "bilayer scaffold" and at least one of the following items were examined: osteochondral tissue, bone, skin, neural tissue, urinary bladder, vascular system. The articles which didn't include "tissue engineering" and just in vitro studies were excluded. RESULTS: Totally, 600 articles were evaluated; related articles were 145, and 35 full-text English articles met all the criteria. Fifteen articles in soft tissue engineering and twenty items in hard tissue engineering were the results of this exploration. Based on selected papers, it was revealed that the bilayer scaffolds were used in the regeneration of the multilayered tissues. The highest multilayered tissue regeneration has been achieved when bilayer scaffolds were used with mesenchymal stem cells and differentiation medium before implanting. Among the studies being reported in this review, bone marrow mesenchymal stem cells are the most studied mesenchymal stem cells. Among different kinds of multilayer tissue, the bilayer scaffold has been most used in osteochondral tissue engineering in which collagen and PLGA have been the most frequently used biomaterials. After osteochondral tissue engineering, bilayer scaffolds were widely used in skin tissue engineering. CONCLUSION: The current review aimed to manifest the researcher and surgeons to use a more sophisticated bilayer scaffold in combinations of appropriate stem cells, and different can improve multilayer tissue regeneration. This systematic review can pave a way to design a suitable bilayer scaffold for a specific target tissue and conjunction with proper stem cells.

Osteochondral tissue coculture: An in vitro and in silico approach.

Osteochondral tissue engineering aims to regenerate functional tissue-mimicking physiological properties of injured cartilage and its subchondral bone. Given the distinct structural and biochemical difference between bone and cartilage, bilayered scaffolds, and bioreactors are commonly employed. We present an osteochondral culture system which cocultured ATDC5 and MC3T3-E1 cells on an additive manufactured bilayered scaffold in a dual-chamber perfusion bioreactor. Also, finite element models (FEM) based on the microcomputed tomography image of the manufactured scaffold as well as on the computer-aided design (CAD) were constructed; the microenvironment inside the two FEM was studied and compared. In vitro results showed that the coculture system supported osteochondral tissue growth in terms of cell viability, proliferation, distribution, and attachment. In silico results showed that the CAD and the actual manufactured scaffold had significant differences in the flow velocity, differentiation media mixing in the bioreactor and fluid-induced shear stress experienced by the cells. This system was shown to have the desired microenvironment for osteochondral tissue engineering and it can potentially be used as an inexpensive tool for testing newly developed pharmaceutical products for osteochondral defects.

Continuous Passive Motion Promotes and Maintains Chondrogenesis in Autologous Endothelial Progenitor Cell-Loaded Porous PLGA Scaffolds during Osteochondral Defect Repair in a Rabbit Model.

Continuous passive motion (CPM) is widely used after total knee replacement. In this study, we investigated the effect of CPM combined with cell-based construct-transplantation in osteochondral tissue engineering. We created osteochondral defects (3 mm in diameter and 3 mm in depth) in the medial femoral condyle of 36 knees and randomized them into three groups: ED (empty defect), EPC/PLGA (endothelial progenitor cells (EPCs) seeded in the poly lactic-co-glycolic acid (PLGA) scaffold), or EPC/PLGA/CPM (EPC/PLGA scaffold complemented with CPM starting one day after transplantation). We investigated the effects of CPM and the EPC/PLGA constructs on tissue restoration in weight-bearing sites by histological observation and micro-computed tomography (micro-CT) evaluation 4 and 12 weeks after implantation. After CPM, the EPC/PLGA construct exhibited early osteochondral regeneration and prevention of subchondral bone overgrowth and cartilage degeneration. CPM did not alter the microenvironment created by the construct; it up-regulated the expression of the extracellular matrix components (glycosaminoglycan and collagen), down-regulated bone formation, and induced the biosynthesis of lubricin, which appeared in the EPC/PLGA/CPM group after 12 weeks. CPM can provide promoting signals during osteochondral tissue engineering and achieve a synergistic effect when combined with EPC/PLGA transplantation, so it should be considered a non-invasive treatment to be adopted in clinical practices.

A Cellularized Biphasic Implant Based on a Bioactive Silk Fibroin Promotes Integration and Tissue Organization during Osteochondral Defect Repair in a Porcine Model.

In cartilage tissue engineering, biphasic scaffolds (BSs) have been designed not only to influence the recapitulation of the osteochondral architecture but also to take advantage of the healing ability of bone, promoting the implant's integration with the surrounding tissue and then bone restoration and cartilage regeneration. This study reports the development and characterization of a BS based on the assembly of a cartilage phase constituted by fibroin biofunctionalyzed with a bovine cartilage matrix, cellularized with differentiated autologous pre-chondrocytes and well attached to a bone phase (decellularized bovine bone) to promote cartilage regeneration in a model of joint damage in pigs. BSs were assembled by fibroin crystallization with methanol, and the mechanical features and histological architectures were evaluated. The scaffolds were cellularized and matured for 12 days, then implanted into an osteochondral defect in a porcine model (n = 4). Three treatments were applied per knee: Group I, monophasic cellular scaffold (single chondral phase); group II (BS), cellularized only in the chondral phase; and in order to study the influence of the cellularization of the bone phase, Group III was cellularized in chondral phases and a bone phase, with autologous osteoblasts being included. After 8 weeks of surgery, the integration and regeneration tissues were analyzed via a histology and immunohistochemistry evaluation. The mechanical assessment showed that the acellular BSs reached a Young's modulus of 805.01 kPa, similar to native cartilage. In vitro biological studies revealed the chondroinductive ability of the BSs, evidenced by an increase in sulfated glycosaminoglycans and type II collagen, both secreted by the chondrocytes cultured on the scaffold during 28 days. No evidence of adverse or inflammatory reactions was observed in the in vivo trial; however, in Group I, the defects were not reconstructed. In Groups II and III, a good integration of the implant with the surrounding tissue was observed. Defects in group II were fulfilled via hyaline cartilage and normal bone. Group III defects showed fibrous repair tissue. In conclusion, our findings demonstrated the efficacy of a biphasic and bioactive scaffold based on silk fibroin and cellularized only in the chondral phase, which entwined chondroinductive features and a biomechanical capability with an appropriate integration with the surrounding tissue, representing a promising alternative for osteochondral tissue-engineering applications.

Bioreactors and Microfluidics for Osteochondral Interface Maturation.

The cell culture techniques are in the base of any biology-based science. The standard techniques are commonly static platforms as Petri dishes, tissue culture well plates, T-flasks, or well plates designed for spheroids formation. These systems faced a paradigm change from 2D to 3D over the current decade driven by the tissue engineering (TE) field. However, 3D static culture approaches usually suffer from several issues as poor homogenization of the formed tissues and development of a necrotic center which limits the size of in vitro tissues to hundreds of micrometers. Furthermore, for complex tissues as osteochondral (OC), more than recovering a 3D environment, an interface needs to be replicated. Although 3D cell culture is already the reality adopted by a newborn market, a technological revolution on cell culture devices needs a further step from static to dynamic already considering 3D interfaces with dramatic importance for broad fields such as biomedical, TE, and drug development. In this book chapter, we revised the existing approaches for dynamic 3D cell culture, focusing on bioreactors and microfluidic systems, and the future directions and challenges to be faced were discussed. Basic principles, advantages, and challenges of each technology were described. The reported systems for OC 3D TE were focused herein.

Natural Origin Materials for Osteochondral Tissue Engineering.

Materials selection is a critical aspect for the production of scaffolds for osteochondral tissue engineering. Synthetic materials are the result of man-made operations and have been investigated for a variety of tissue engineering applications. Instead, the products of physiological processes and the metabolic activity of living organisms are identified as natural materials. Over the recent decades, a number of natural materials, namely, biopolymers and bioceramics, have been proposed as the main constituent of osteochondral scaffolds, but also as cell carriers and signaling molecules. Overall, natural materials have been investigated both in the bone and in the cartilage compartment, sometimes alone, but often in combination with other biopolymers or synthetic materials. Biopolymers and bioceramics possess unique advantages over their synthetic counterparts due similarity with natural extracellular matrix, the presence of cell recognition sites and tunable chemistry. However, the characteristics of natural origin materials can vary considerably depending on the specific source and extraction process. A deeper understanding of the relationship between material variability and biological activity and the definition of standardized manufacturing procedures will be crucial for the future of natural materials in tissue engineering.

Large Animal Models for Osteochondral Regeneration.

Namely, in the last two decades, large animal models - small ruminants (sheep and goats), pigs, dogs and horses - have been used to study the physiopathology and to develop new therapeutic procedures to treat human clinical osteoarthritis. For that purpose, cartilage and/or osteochondral defects are generally performed in the stifle joint of selected large animal models at the condylar and trochlear femoral areas where spontaneous regeneration should be excluded. Experimental animal care and protection legislation and guideline documents of the US Food and Drug Administration, the American Society for Testing and Materials and the International Cartilage Repair Society should be followed, and also the specificities of the animal species used for these studies must be taken into account, such as the cartilage thickness of the selected defect localization, the defined cartilage critical size defect and the joint anatomy in view of the post-operative techniques to be performed to evaluate the chondral/osteochondral repair. In particular, in the articular cartilage regeneration and repair studies with animal models, the subchondral bone plate should always be taken into consideration. Pilot studies for chondral and osteochondral bone tissue engineering could apply short observational periods for evaluation of the cartilage regeneration up to 12 weeks post-operatively, but generally a 6- to 12-month follow-up period is used for these types of studies.

Advances for Treatment of Knee OC Defects.

Osteochondral (OC) defects are prevalent among young adults and are notorious for being unable to heal. Although they are traumatic in nature, they often develop silently. Detection of many OC defects is challenging, despite the criticality of early care. Current repair approaches face limitations and cannot provide regenerative or long-standing solution. Clinicians and researchers are working together in order to develop approaches that can regenerate the damaged tissues and protect the joint from developing osteoarthritis. The current concepts of tissue engineering and regenerative medicine, which have brought many promising applications to OC management, are overviewed herein. We will also review the types of stem cells that aim to provide sustainable cell sources overcoming the limitation of autologous chondrocyte-based applications. The various scaffolding materials that can be used as extracellular matrix mimetic and having functional properties similar to the OC unit are also discussed.

Osteochondral Tissue Engineering: Translational Research and Turning Research into Products.

Osteochondral (OC) defect repair is a significant clinical challenge. Osteoarthritis results in articular cartilage/subchondral bone tissue degeneration and tissue loss, which in the long run results in cartilage/ostecochondral defect formation. OC defects are commonly approached with autografts and allografts, and both these options have found limitations. Alternatively, tissue engineered strategies with biodegradable scaffolds with and without cells and growth factors have been developed. In order to approach regeneration of complex tissues such as osteochondral, advanced tissue engineered grafts including biphasic, triphasic, and gradient configurations are considered. The graft design is motivated to promote cartilage and bone layer formation with an interdigitating transitional zone (i.e., bone-cartilage interface). Some of the engineered OC grafts with autologous cells have shown promise for OC defect repair and a few of them have advanced into clinical trials. This chapter presents synthetic osteochondral designs and the progress that has been made in terms of the clinical translation.

Biphasic Scaffolds from Marine Collagens for Regeneration of Osteochondral Defects.

BACKGROUND: Collagens of marine origin are applied increasingly as alternatives to mammalian collagens in tissue engineering. The aim of the present study was to develop a biphasic scaffold from exclusively marine collagens supporting both osteogenic and chondrogenic differentiation and to find a suitable setup for in vitro chondrogenic and osteogenic differentiation of human mesenchymal stroma cells (hMSC). METHODS: Biphasic scaffolds from biomimetically mineralized salmon collagen and fibrillized jellyfish collagen were fabricated by joint freeze-drying and crosslinking. Different experiments were performed to analyze the influence of cell density and TGF-ß on osteogenic differentiation of the cells in the scaffolds. Gene expression analysis and analysis of cartilage extracellular matrix components were performed and activity of alkaline phosphatase was determined. Furthermore, histological sections of differentiated cells in the biphasic scaffolds were analyzed. RESULTS: Stable biphasic scaffolds from two different marine collagens were prepared. An in vitro setup for osteochondral differentiation was developed involving (1) different seeding densities in the phases; (2) additional application of alginate hydrogel in the chondral part; (3) pre-differentiation and sequential seeding of the scaffolds and (4) osteochondral medium. Spatially separated osteogenic and chondrogenic differentiation of hMSC was achieved in this setup, while osteochondral medium in combination with the biphasic scaffolds alone was not sufficient to reach this ambition. CONCLUSIONS: Biphasic, but monolithic scaffolds from exclusively marine collagens are suitable for the development of osteochondral constructs.

The osteochondral interface as a gradient tissue: from development to the fabrication of gradient scaffolds for regenerative medicine.

The osteochondral (OC) interface is not only the interface between two tissues, but also the evolution of hard and stiff bone tissue to the softer and viscoelastic articular cartilage covering the joint surface. To generate a smooth transition between two tissues with such differences in many of their characteristics, several gradients are recognizable when moving from the bone side to the joint surface. It is, therefore, necessary to implement such gradients in the design of scaffolds to regenerate the OC interface, so to mimic the anatomical, biological, and physicochemical properties of bone and cartilage as closely as possible. In the past years, several scaffolds were developed for OC regeneration: biphasic, triphasic, and multilayered scaffolds were used to mimic the compartmental nature of this tissue. The structure of these scaffolds presented gradients in mechanical, physicochemical, or biological properties. The use of gradient scaffolds with already differentiated or progenitor cells has been recently proposed. Some of these approaches have also been translated in clinical trials, yet without the expected satisfactory results, thus suggesting that further efforts in the development of constructs, which can lead to a functional regeneration of the OC interface by presenting gradients more closely resembling its native environment, will be needed in the near future. The aim of this review is to analyze the gradients present in the OC interface from the early stage of embryonic life up to the adult organism, and give an overview of the studies, which involved gradient scaffolds for its regeneration.

The potential of encapsulating "raw materials" in 3D osteochondral gradient scaffolds.

Scaffolds with continuous gradients in material composition and bioactive signals enable a smooth transition of properties at the interface. Components like chondroitin sulfate (CS) and bioactive glass (BG) in 3D scaffolds may serve as "raw materials" for synthesis of new extracellular matrix (ECM), and may have the potential to completely or partially replace expensive growth factors. We hypothesized that scaffolds with gradients of ECM components would enable superior performance of engineered constructs. Raw material encapsulation altered the appearance, structure, porosity, and degradation of the scaffolds. They allowed the scaffolds to better retain their 3D structure during culture and provided a buffering effect to the cells in culture. Following seeding of rat mesenchymal stem cells, there were several instances where glycosaminoglycan (GAG), collagen, or calcium contents were higher with the scaffolds containing raw materials (CS or BG) than with those containing transforming growth factor (TGF)-ß3 or bone morphogenetic protein (BMP)-2. It was also noteworthy that a combination of both CS and TGF-ß3 increased the secretion of collagen type II. Moreover, cells seeded in scaffolds containing opposing gradients of CS/TGF-ß3 and BG/BMP-2 produced clear regional variations in the secretion of tissue-specific ECM. The study demonstrated raw materials have the potential to create a favorable microenvironment for cells; they can significantly enhance the synthesis of certain extracellular matrix (ECM) components when compared to expensive growth factors; either alone or in combination with growth factors they can enhance the secretion of tissue specific matrix proteins. Raw materials are promising candidates that can be used to either replace or be used in combination with growth factors. Success with raw materials in lieu of growth factors could have profound implications in terms of lower cost and faster regulatory approval for more rapid translation of regenerative medicine products to the clinic.

Gelatin-based hydrogels with tunable network structure and mechanical property for promoting osteogenic differentiation.

Osteoarthritis (OA) is a joint disease involving all joint components, including cartilage, calcified cartilage, and subchondral bone. The repair of osteochondral defects remains a significant challenge in orthopedics. Development of new strategies is essential for effective osteochondral injury repair. In this study, gelatin (Gel), polyethylene glycol diglycidyl ether (PEGDGE), hydroxyethyl cellulose (HEC) and chitosan (CS) were used to prepare semi-IPNs and IPNs hydrogels. Mechanical properties of Gel based hydrogels significantly improved with the semi-IPN and IPN structures. Tensile strength ranges from 238.7 KPa to 479.5 KPa, and its compressive strength ranges from 35.6 KPa to 112.7 KPa. Additionally, the stress relaxation rate increased with higher CS concentrations, ranging from 25 % to 35 %. The network structure of Gel-based hydrogels was a key factor in regulating stress relaxation. Viscoelasticity was adjusted by its network structures. Swelling and degradation behaviors of Gel based hydrogels were systematically investigated. Gel based hydrogels had good cytocompatibility. Both semi-IPN and IPN structures Gel based hydrogels could promote cell spreading and osteogenic differentiation. G10HEC1 and G10CS1 hydrogels show promise as candidates for osteochondral tissue regeneration, offering a new strategy for osteochondral tissue engineering.

Composite Spheroid-Laden Bilayer Hydrogel for Engineering Three-Dimensional Osteochondral Tissue.

A combination of hydrogels and stem cell spheroids has been used to engineer three-dimensional (3D) osteochondral tissue, but precise zonal control directing cell fate within the hydrogel remains a challenge. In this study, we developed a composite spheroid-laden bilayer hydrogel to imitate osteochondral tissue by spatially controlled differentiation of human adipose-derived stem cells. Meticulous optimization of the spheroid-size and mechanical strength of gelatin methacryloyl (GelMA) hydrogel enables the cells to homogeneously sprout within the hydrogel. Moreover, fibers immobilizing transforming growth factor beta-1 (TGF-ß1) or bone morphogenetic protein-2 (BMP-2) were incorporated within the spheroids, which induced chondrogenic or osteogenic differentiation of cells in general media, respectively. The spheroids-filled GelMA solution was crosslinked to create the bilayer hydrogel, which demonstrated a strong interfacial adhesion between the two layers. The cell sprouting enhanced the adhesion of each hydrogel, demonstrated by increase in tensile strength from 4.8 ± 0.4 to 6.9 ± 1.2 MPa after 14 days of culture. Importantly, the spatially confined delivery of BMP-2 within the spheroids increased mineral deposition and more than threefold enhanced osteogenic genes of cells in the bone layer while the cells induced by TGF-ß1 signals were apparently differentiated into chondrocytes within the cartilage layer. The results suggest that our composite spheroid-laden hydrogel could be used for the biofabrication of osteochondral tissue, which can be applied to engineer other complex tissues by delivery of appropriate biomolecules.

A comprehensive review on using injectable chitosan microgels for osteochondral tissue repair.

Restoring cartilage to healthy state is challenging due to low cell density and hence low regenerative capacity. The current platforms are not compatible with clinical translation and require dedicated handling of trained personnel. However, by engineering and implanting cell microaggregates in higher concentrations, efficient formation of new cartilage can be achieved, even in the absence of exogenous growth factors. Therefore, one-step surgeries are preferable for novel treatments and we need cell laden microgels allowing the formation of microaggregaets in vivo. Injectability is a key parameter for in situ forming the shape and minimally invasive clinical applications. Hydrogels as bioinks can restore damaged tissues to their primary shape. Chitosan is a polysaccharide derived from chitin with abundant usage in tissue engineering. This review highlights the use of chitosan as an injectable hydrogel for osteochondral defects. Several studies focused on encapsulating mesenchymal stem cells within chitosan hydrogels have been categorized and incorporating microfluidic devices has been identified in the forefront to form microgels. Additionally, the printability is another convenience of chitosan for using in 3D printing for cartilage tissue engineering which is described in this review.

Cell-free bilayer functionalized scaffold for osteochondral tissue engineering.

Osteochondral tissue engineering using layered scaffolds is a promising approach for treating osteochondral defects as an alternative to microfracture procedure, autologous chondrocyte implantation, and cartilage-bone grafting. The team previously investigated the chondrogenesis of mesenchymal stem cells (MSCs) on a polycaprolactone (PCL)/acetylated hyaluronic acid scaffold. The present study first focused on fabricating a novel osteoconductive scaffold utilizing bismuth-nanohydroxyapatite/reduced graphene oxide (Bi-nHAp/rGO) nanocomposite and electrospun PCL. The osteoconductive ability of the scaffold was investigated by evaluating the alkaline phosphatase (ALP) activity and the osteogenic genes expression in the adipose-derived MSCs. The expression of Runx2, collagen I, ALP, and osteocalcin as well as the result of ALP activity indicated the osteoconductive potential of the Bi-nHA-rGO/PCL scaffold. In the next step, a bilayer scaffold containing Bi-nHAp/rGO/PCL as an osteogenic layer and acetylated hyaluronic acid/PCL as a chondrogenic layer was prepared by the electrospinning technique and transplanted into osteochondral defects of rats. The chondrogenic and osteogenic markers corresponding to the surrounding tissues of the transplanted scaffold were surveyed 60 days later by real-time polymerase chain reaction (PCR) and immunohistochemistry methods. The results showed increased chondrogenic (Sox9 and collagen II) and osteogenic (osteocalcin and ALP) gene expression and augmented secretion of collagens II and X after transplantation. The results strongly support the efficacy of this constructed cell-free bilayer scaffold to induce osteochondral defect regeneration.