An additive manufacturing-based PCL-alginate-chondrocyte bioprinted scaffold for cartilage tissue engineering

Cartilage tissue is under extensive investigation in tissue engineering and regenerative medicine studies because of its limited regenerative potential. Currently, many scaffolds are undergoing scientific and clinical research. A key for appropriate scaffolding is the assurance of a temporary cellular environment that allows the cells to function as in native tissue. These scaffolds should meet the relevant requirements, including appropriate architecture and physicochemical and biological properties. This is necessary for proper cell growth, which is associated with the adequate regeneration of cartilage. This paper presents a review of the development of scaffolds from synthetic polymers and hybrid materials employed for the engineering of cartilage tissue and regenerative medicine. Initially, general information on articular cartilage and an overview of the clinical strategies for the treatment of cartilage defects are presented. Then, the requirements for scaffolds in regenerative medicine, materials intended for membranes, and methods for obtaining them are briefly described. We also describe the hybrid materials that combine the advantages of both synthetic and natural polymers, which provide better properties for the scaffold. The last part of the article is focused on scaffolds in cartilage tissue engineering that have been confirmed by undergoing preclinical and clinical tests.

Tissue grafting is mostly used for repair and replacement of severely damaged tissues, the key challenges are compatibility, availability of the grafts, complex surgical process and post-operative complications. Hence, additive technologies such as three-dimensional (3D) bioprinting have emerged as promising alternative for tissue engineering in order to ensure safety, compatibility, and rapid healing. The aim of this chapter is to give an elaborate account of 3D printed scaffolds for bone, cartilage, cardio-vascular and nerve tissue engineering. Various components such as polycaprolactone, poly (lactic-co-glycolic acid), and β-tricalcium phosphate, bioglass 45S5, and nano-hydroxyapatite are combined with collagen and its derivatives to achieve specific pore size in the scaffolds for effective restoration of the defects of soft or hard tissues. Likewise, proanthocyanidin, oxidized hyaluronic acid, methacrylated gelatin, are used in collagen based 3D printed scaffolds for cartilage tissue engineering. Bioink with collagen as active component is also used for developing cardio-vascular implants with recellularizing properties. Collagen in combination with silk fibroin, chitosan, heparin sulphate and others are ideal for fabrication of elastic nerve guidance conduits. In view of the background, collagen-supplemented hydrogels can revolutionize future biomedical approaches for the development of complex scaffolds for tissue engineering.

Selective laser sintered poly-ε-caprolactone scaffold hybridized with collagen hydrogel for cartilage tissue engineering

Hybrid scaffolds for cartilage tissue engineering provide the potential for high stiffness properties in tension and compression while exhibiting the viscoelastic response found in hydrogels and native cartilage tissue. We investigate the impact of a hybrid scaffold fabricated from a hyaluronic acid (HA)-based hydrogel combined with porous poly(ε-caprolactone) (PCL) material formed by a particulate leaching method to study dedifferentiated chondrocyte response. The material properties of the hybrid scaffold showed mean Young's moduli in tension which were similar to human articular cartilage but not statistically different between the hybrid and porous PCL scaffolds at 2.02 and 2.07 MPa, respectively. For both the hybrid and porous PCL control scaffolds in compression at low loading frequencies (<1 Hz) and 10% strain peak amplitude the Young's moduli are not statistically distinct. However, at frequencies in the range of normal human gait from 1 to2 Hz, hybrid scaffolds exhibit significantly (p < 0.01) increased loss moduli indicating additional contribution of the viscous phase to stiffness. Dedifferentiated chondrocytes seeded onto the scaffolds exhibited a rounded morphology in hybrid scaffolds however ECM protein expression levels of collagen type I, collagen type II, and aggrecan are not different from the PCL control scaffolds. These results provide a model platform to investigate cell response to mechanical and chemical cues in a hybrid scaffold system with mechanical properties similar to human cartilage that does not contribute to differentiation in order to identify the appropriate design and development parameters to promote formation of extracellular matrix and investigate chondrocyte scaffold interactions.

A cutting-edge field of study to create biological substitutes to maintain, restore, or enhance cartilage function is articular cartilage (AC) tissue engineering. Employing this innovative approach, polymer composites that mimic the structure and function of real cartilage tissues are created by carefully mixing biomaterials of scaffolds, cells, and biochemical components. Naturally polymers have recently gained attention as biomaterials to fabricate scaffolds, for instance, chitosan. Despite its shortcomings such as poor mechanical strength and low stiffness, chitosan-based hydrogel scaffolds have been cross-linked with other synthetic polymers, namely hybrid scaffolds, for future perspective of AC tissue engineering. To mimic AC compartment design, additive manufacturing closely resembles native tissue, and its flexibility is possibly tailored. HIGHLIGHTS Chitosan emerges as a valuable scaffold due to its structural similarity to glycine aminoglycan of extracellular matrix (ECM). Overcoming obstacles in scaffold design, innovative approaches like gradients and composite multilayer scaffolds resembling cartilage tissue are developed. The integration of elements like minerals, growth factors, and cells, along with advancements in mechanobiology and bioreactor design, is set to play a crucial role in cartilage tissue engineering. The advent of three-dimensional (3D) printing, especially with chitosan and synthetic polymer hydrogel inks, holds promise for creating stable structures. GRAPHICAL ABSTRACT

In order to contribute to the development of minimally invasive surgery techniques for autologous chondrocyte implantation, a novel self-assembling biomaterial consisting of thermoresponsive poly(N-isopropylacrylamide)-grafted hyaluronan (PNIPAAm-g-HA) has been synthesized as an injectable scaffold for cartilage tissue engineering. The aim of this study was to investigate the efficacy and cytocompatibility of PNIPAAm-g-HA to normal chondrocytes by using reverse transcription-polymerase chain reaction (RT-PCR) analysis and histochemical staining in preliminary in vitro and in vitro experiments. Hematoxylin and eosin staining showed homogeneous distribution of cells in the PNIPAAm-g-HA hydrogel in 3-dimensional in vitro cultivation. Alcian blue staining also indicated that abundant extracellular matrix formation, including acidic glycosaminoglycans, occurred in tissue-engineered cartilage over time in vitro. Cartilage-related gene expression patterns, which were tested in rabbit normal chondrocytes embedded in the hydrogel, were almost maintained for 4 weeks. Transforming growth factor-β1 (TGF-β1) stimulation enhanced the expression of SRY-related HMG box-containing gene 9 (Sox9) and type X collagen genes suggesting promotion of chondrogenic differentiation. Histochemical evaluation showed neocartilage formation following subcutaneous implantation of the chondrocyte-gel mixture in nude mice. Furthermore, TGF-β1 stimulation promoted production and maturation of the extracellular matrix of the in situ tissue engineered hyaline cartilage. These data suggested that PNIPAAm-g-HA could be a promising biomaterial, i.e., a self-assembling and injectable scaffold for cartilage tissue engineering.

In this study, three-dimensional (3D) porous scaffolds were developed for the repair of articular cartilage defects. Novel collagen/polylactide (PLA), chitosan/PLA, and collagen/chitosan/PLA hybrid scaffolds were fabricated by combining freeze-dried natural components and synthetic PLA mesh, where the 3D PLA mesh gives mechanical strength, and the natural polymers, collagen and/or chitosan, mimic the natural cartilage tissue environment of chondrocytes. In total, eight scaffold types were studied: four hybrid structures containing collagen and/or chitosan with PLA, and four parallel plain scaffolds with only collagen and/or chitosan. The potential of these types of scaffolds for cartilage tissue engineering applications were determined by the analysis of the microstructure, water uptake, mechanical strength, and the viability and attachment of adult bovine chondrocytes to the scaffolds. The manufacturing method used was found to be applicable for the manufacturing of hybrid scaffolds with highly porous 3D structures. All the hybrid scaffolds showed a highly porous structure with open pores throughout the scaffold. Collagen was found to bind water inside the structure in all collagen-containing scaffolds better than the chitosan-containing scaffolds, and the plain collagen scaffolds had the highest water absorption. The stiffness of the scaffold was improved by the hybrid structure compared to plain scaffolds. The cell viability and attachment was good in all scaffolds, however, the collagen hybrid scaffolds showed the best penetration of cells into the scaffold. Our results show that from the studied scaffolds the collagen/PLA hybrids are the most promising scaffolds from this group for cartilage tissue engineering.

Articular cartilage has a very limited regeneration capacity. Therefore, injury or degeneration of articular cartilage results in an inferior mechanical stability, load-bearing capacity, and lubrication capability. Here, we developed a biomimetic scaffold consisting of macroporous polyvinyl alcohol (PVA) sponges as a platform material for the incorporation of cell-embedded photocrosslinkable poly(ethylene glycol) diacrylate (PEGDA), PEGDA-methacrylated chondroitin sulfate (PEGDA-MeCS; PCS), or PEGDA-methacrylated hyaluronic acid (PEGDA-MeHA; PHA) within its pores to improve in vitro chondrocyte functions and subsequent in vivo ectopic cartilage tissue formation. Our findings demonstrated that chondrocytes encapsulated in PCS or PHA and loaded into macroporous PVA hybrid scaffolds maintained their physiological phenotypes during in vitro culture, as shown by the upregulation of various chondrogenic genes. Further, the cell-secreted extracellular matrix (ECM) improved the mechanical properties of the PVA-PCS and PVA-PHA hybrid scaffolds by 83.30% and 73.76%, respectively, compared to their acellular counterparts. After subcutaneous transplantation in vivo, chondrocytes on both PVA-PCS and PVA-PHA hybrid scaffolds significantly promoted ectopic cartilage tissue formation, which was confirmed by detecting cells positively stained with Safranin-O and for type II collagen. Consequently, the mechanical properties of the hybrid scaffolds were biomimetically reinforced by 80.53% and 210.74%, respectively, compared to their acellular counterparts. By enabling the recapitulation of biomimetically relevant structural and functional properties of articular cartilage and the regulation of in vivo mechanical reinforcement mediated by cell–matrix interactions, this biomimetic material offers an opportunity to control the desired mechanical properties of cell-laden scaffolds for cartilage tissue regeneration.

Biomaterials for Regenerative Medicine

The limited supply of cartilage tissue with appropriate sizes and shapes needed for reconstruction and repair has stimulated research in the area of hydrogels as scaffolds for cartilage tissue engineering. In this study we demonstrate that poly(ethylene glycol) (PEG)-based semi-interpenetrating (sIPN) network hydrogels, made with a crosslinkable poly(ethylene glycol)-dimethacrylate (PEGDM) component and a non-crosslinkable interpenetration poly(ethylene oxide) (PEO) component, and seeded with chondrocytes support cartilage construct growth having nominal thicknesses of 6 mm and relatively uniform safranin-O stained matrix when cultured statically, unlike constructs grown with prefabricated macroporous scaffolds. Even though changing the molecular weight of the PEO from 100 to 20 kDa reduces the viscosity of the precursor polymer solution, we have demonstrated that it does not appear to affect the histological or biochemical characteristics of cartilaginous constructs. Extracellular matrix (ECM) accumulation and the spatial uniformity of the ECM deposited by the embedded chondrocytes decreased, and hydrogel compressive properties increased, as the ratio of the PEGDM:PEO in the hydrogel formulation increased (from 30:70 to 100:0 PEGDM:PEO). Total collagen and glycosaminoglycan contents per dry weight were highest using the 30:70 PEGDM:PEO formulation (24.4+/-3.5% and 7.1+/-0.9%, respectively). The highest equilibrium compressive modulus was obtained using the 100:0 PEGDM:PEO formulation (0.32+/-0.07 MPa), which is similar to the compressive modulus of native articular cartilage. These results suggest that the versatility of PEG-based sIPN hydrogels makes them an attractive scaffold for tissue engineering of cartilage.

Tissue engineering provides a new strategy for repairing damaged cartilage. Surface and mechanical properties of scaffolds play important roles in inducing cell growth. The aim of this study was to fabricate and characterize PLGA and gelatin/hyaluronic acid-treated PLGA (PLGA-GH) sponge scaffolds for articular cartilage tissue engineering. The PLGA-GH scaffolds were cross-linked with gelatin and hyaluronic acid. Primary chondrocytes isolated from porcine articular cartilages were used to assess cell compatibility. The characteristic PLGA-GH scaffold was higher in water uptake ratio and degradation rate within 42 days than the PLGA scaffold. The mean compressive moduli of PLGA and PLGA-GH scaffolds were 1.72 ± 0.50 MPa and 1.86 ± 0.90 MPa, respectively. The cell attachment ratio, proliferation, and extracellular matrix secretion on PLGA-GH scaffolds are superior to those of PLGA scaffolds. In our study, PLGA-GH scaffolds exhibited improvements in cell biocompatibility, cell proliferation, extracellular matrix synthesis, and appropriate mechanical and structural properties for potential engineering cartilage applications.

Due to its poor self-repair ability, the cartilage injury has been a great challenge in clinical work. The conventional therapies for cartilage injury mostly form fibrocartilage with weaker strength and flexibility compared to natural cartilage. The development of tissue engineering technology provides a new approach for cartilage repair and regeneration. An appropriate scaffold is one of key factors in cartilage tissue engineering. The continuous nanofibers or submicron fibers obtained by electrospinning, a newly emerging technologies that has been increasingly emphasized for its simple manufacturing process and high efficiency, have been widely applied as scaffolds in tissue engineering, for its structure being similar with natural extracellular matrix. This chapter will summarize the current knowledge related to electrospun fibrous scaffolds in cartilage tissue engineering, especially focus on the materials, topography, modifications of electrospun fibrous scaffolds, and new electrospinning approaches in cartilage tissue engineering.

Owing to the limited repair capacity of articular cartilage, it is essential to develop tissue-engineered cartilage for patients suffering from joint disease and trauma. Herein, we prepared a novel hybrid scaffold composed of methacrylated chondroitin sulfate (CSMA), poly(ethylene glycol) methyl ether-ε-caprolactone-acryloyl chloride (MPEG-PCL-AC, PECA was used as abbreviation for MPEG-PCL-AC) and graphene oxide (GO) and evaluated its potential application in cartilage tissue engineering. To mimic the natural extracellular matrix (ECM) of cartilage, the scaffold had an adequate pore size, porosity, swelling ability, compression modulus and conductivity. Cartilage cells contacted with the scaffold remained viable and showed growth potential. Furthermore, CSMA/PECA/GO scaffold was biocompatible and had a favorable degradation rate. In the cartilage tissue repair of rabbit, Micro-CT and histology observation showed the group of CSMA/PECA/GO scaffold with cellular supplementation had better chondrocyte morphology, integration, continuous subchondral bone, and much thicker newly formed cartilage compared with scaffold group and control group. Our results show that the CSMA/PECA/GO hybrid porous scaffold can be applied in articular cartilage tissue engineering and may have great potential to in other types of tissue engineering applications.

Cartilage regeneration using a porous scaffold, a collagen sponge incorporating a hydroxyapatite/chondroitinsulfate composite

ABSTRACTThe development of porous biodegradable scaffolds is of great interest in tissue engineering. In this regard, exploration of novel biocompatible materials is needed. Silk fibroin‐chondroitin sulfate‐sodium alginate (SF‐CHS‐SA) porous hybrid scaffolds were successfully prepared via lyophilization method and crosslinked by 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide‐ethanol treatment. According to the scanning electron microscopy studies, mean pore diameters of the scaffolds were in the range of 60–187 μm. The porosity percentage of the scaffold with SF‐CHS‐SA ratio of 70 : 15 : 15 (w/w/w %) was 92.4 ± 3%. Attenuated total reflectance Fourier transform infrared spectroscopy, X‐ray diffraction, and differential scanning calorimetry results confirmed the transition from amorphous random coil to crystalline β‐sheet in treated SF‐CHS‐SA scaffold. Compressive modulus was significantly improved in hybrid scaffold with SF‐CHS‐SA ratio of 70 : 15 : 15 (3.35 ± 0.15 MPa). Cytotoxicity assay showed that the scaffolds have no toxic effects on chondrocytes. Attachment of chondrocytes was much more improved within the SF‐CHS‐SA hybrid scaffold. Real‐time polymerase chain reaction analyses showed a significant increase in gene expression of collagen type II, aggrecan, and SOX9 and decrease in gene expression of collagen type I for SF‐CHS‐SA compared with SF scaffold. This novel hybrid scaffold can be a good candidate to be utilized as an efficient scaffold for cartilage tissue engineering. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 41048.