Silk Fiber-Reinforced Hyaluronic Acid-Based Hydrogel for Cartilage Tissue Engineering

A continuing challenge in cartilage tissue engineering for cartilage regeneration is the creation of a suitable synthetic microenvironment for chondrocytes and tissue regeneration. The aim of this study was to develop a highly tunable hybrid scaffold based on a silk fibroin matrix (SM) and a hyaluronic acid (HA) hydrogel. Human articular chondrocytes were embedded in a porous 3-dimensional SM, before infiltration with tyramine modified HA hydrogel. Scaffolds were cultured in chondropermissive medium with and without TGF-β1. Cell viability and cell distribution were assessed using CellTiter-Blue assay and Live/Dead staining. Chondrogenic marker expression was detected using qPCR. Biosynthesis of matrix compounds was analyzed by dimethylmethylene blue assay and immuno-histology. Differences in biomaterial stiffness and stress relaxation were characterized using a one-step unconfined compression test. Cell morphology was investigated by scanning electron microscopy. Hybrid scaffold revealed superior chondro-inductive and biomechanical properties compared to sole SM. The presence of HA and TGF-β1 increased chondrogenic marker gene expression and matrix deposition. Hybrid scaffolds offer cytocompatible and highly tunable properties as cell-carrier systems, as well as favorable biomechanical properties.     

Enhanced function of chondrocytes in a chitosan-based hydrogel to regenerate cartilage tissues by accelerating degradability of the hydrogel via a hydrolysable crosslinker

Chitosan-based hydrogels as scaffolds for culturing chondrocytes were prepared using linkers with and without hydrolysable poly(dl -lactide) (PLA) segments. The evaluation of the cultured chondrocytes in them indicated that the accelerated degradation of the hydrogel via hydrolysis of the PLA slightly promoted production of the sulfated glycosaminoglycan and drastically improved that of collagen from the encapsulated chondrocytes, which are the chondrospecific extracellular matrix components. Furthermore, the accelerated degradability significantly upregulated the gene expression for Collagen II production and downregulated that for Collagen I production of the encapsulated chondrocytes. Because major component of the articular cartilage tissue is Collagen II-rich hyaline cartilage, these results suggest the degradation of the scaffolds is an important parameter in cartilage tissue regeneration and the accelerated degradability may have benefits on promotion of cartilage tissue regeneration especially from the viewpoint of hyaline cartilage-like collagen production. © 2019 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2019 , 137, 48893.     

Electrospun nanofibrous alginate sulfate scaffolds promote mesenchymal stem cells differentiation to chondrocytes

Cartilage tissue engineering is one of the interesting approaches used for repairing cartilage injuries. This study reports the fabrication of polyvinyl alcohol/alginate sulfate (PVA/ALG-S) nanofibrous mats as a functional support for chondrogenic differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs). The PVA/ALG-S nanofibers were obtained through electrospinning of PVA solutions containing 10, 20, and 30 wt% of ALG-S. The appearance of a band at 833 cm⁻¹ assigned to the symmetrical C O S vibration associated to a C O SO₃ group confirmed the presence of ALG-S in nanofibrous mat. The SEM images illustrated the bead-free and smooth morphology of PVA/ALG-S nanofibers with a mean diameter of 185 ± 0.06 nm. The MTT assay of the hBM-MSCs seeded on scaffolds indicated the appropriate cytocompatibility of nanofibrous PVA/ALG-S scaffolds. Furthermore, the appropriate attachment and spreading of the hBM-MSCs based on SEM images, and their differentiation to the chondrocyte-like cells accompanied by a decrease in cell growth on MTT analysis and more color absorption in alician blue staining indicated the effective role of alginate sulfate on cell differentiation. Finally, the expression of Type II collagen by RT-PCR and immunocytochemistry analyses revealed the chondrogenic differentiation of hBM-MSCs on alginate sulfate nanofibers.     

Enhanced chondrogenic differentiation of human bone marrow mesenchymal stem cells on PCL/PLGA electrospun with different alignments and compositions

The simultaneous effect of electrospun scaffold alignment and polymer composition on chondrogenic differentiation of human bone marrow mesenchymal stem cells (hBMMSC) is investigated. Aligned and randomly oriented polycaprolactone/poly(lactic-co-glycolic acid) (PLGA) hybrid electrospun scaffolds with two different ratios are fabricated by electrospinning. It is found that aligned nanofibrous scaffolds support higher chondrogenic differentiation of hBMMSCs compared to random ones. The aligned scaffolds show a higher expression level of chondrogenic markers such as type II collagen and aggrecan. It is concluded that the aligned nanofibrous scaffold with higher PLGA ratio could significantly enhance hBMMSC proliferation and differentiation to chondrocytes.     

Incorporation of chitosan nanoparticles into silk fibroin-based porous scaffolds: Chondrogenic differentiation of stem cells

A silk fibroin-chondroitin sulfate-sodium alginate (SF-CHS-SA) porous scaffold containing chitosan nanoparticles (NPs) was investigated. The proliferation of adipose-derived stem cells (ASCs) was studied by SEM, fluorescent microscopy, alcian blue staining, dimethylmethylene blue assay, and real-time polymerization chain reaction. The results showed that incorporation of NPs into the scaffold improved compressive modulus (5.6 ± 0.15 MPa). The amount of glycosaminoglycan expression of the ASCs was reached to 8.9 ± 0.3 µg/mL. The gene expressions of aggrecan, collagen II, and SOX9 of the ASCs were significantly improved. This study revealed that the prepared scaffold can be used as a substrate for cartilage tissue engineering.     

Interpenetrating alginate on gelatin–poly(2-hydroxyethyl methacrylate) as a functional polymeric matrix for cartilage tissue engineering

In this work, we report the fabrication of an interpenetrating alginate network on a gelatin and (2-hydroxyethyl methacrylate) (GAH). The anisotropic architecture of scaffold promotes the proliferation of chondrocytes. The anisotropy provided by the alginate influences the cellular response because high collagen and glycosaminoglycan (GAG) contents were found in the cells cultured on the GAH scaffold compared to the scaffold (GH) without alginate. The in vivo experiments demonstrate that the scaffold and the disposition of matrix components, particularly collagen, could promote neovascularization with the ability to recruit cells from the surrounding tissue.     

Alginate Based Scaffolds for Cartilage Tissue Engineering: A Review

Abstract

Many people, especially old and middle-aged, suffer from pain and disabilities caused by cartilage degradation. There are many surgical methods for cartilage treatment, however, none of them have shown acceptable results in long-term. Tissue engineering would be an acceptable approach for cartilage treatment. This includes cells, a carrier such as a matrix scaffold and signaling molecule. An ideal scaffold for cartilage tissue engineering should meet some requirements includes biocompatibility, biodegradability, and sufficient mechanical characteristic. While there are many suitable scaffolds made by natural and synthesis polymers, alginate- a natural polymer- has received good attention. Alginate offers many advantages for cartilage treatment; it has great potential in having tunable mechanical properties and easy manufacturing process. In the present paper, focusing on alginate as main scaffold constructive component, different studies on alginate based scaffolds in the form of physically, chemically and biologically crosslinked hydrogel, sponge, fiber, micro/nano particles and 3?D printed for articular cartilage tissue engineering are discussed and reviewed.     

Surface treatment with amino acids of porous collagen based scaffolds to improve cell adhesion and proliferation

The purpose of this study was to improve the biocompatibility of glutaraldehyde (GA) cross‐linked chitosan coated collagen scaffold for cartilage tissue regeneration. In order to prevent the potential toxicity of GA, we treated the designed scaffold with either glutamic acid or glycine. Amino acid treated scaffolds were characterized by scanning electron microscopy (SEM) techniques. Afterward, chondrocyte interaction with the composite scaffold was investigated assessing cell adhesion and proliferation using Hoechst staining and MTT cell proliferation assay, respectively. The SEM analyses of the scaffolds’ surface and cross‐section confirmed the adhesion of amino acids on the surface of the scaffolds. We also observed that scaffolds’ porosity was reduced due to the coverage of the pores by chitosan and amino acids, leading to low porosity. The use of amino acid improved the chondrocyte adhesion and proliferation inside the scaffolds’ pores when cells were cultured onto the chitosan‐coated collagen scaffolds. Overall, our in vitro results suggest the use of amino acid to improve the biocompatibility of natural polymer composite scaffold being crosslinked with glutaraldehyde. Such scaffold has improved mechanical properties; biocompatibility thus may be useful for tissue regeneration such as cartilage.

     

Chondrocyte Cultivation in Hyaluronan-Tyramine Cross-Linked Hydrogel

The application of chondrocyte-seeded hydrogels, such as tyramine-substituted hyaluronan, is a highly promising strategy for damaged cartilage treatments. The rheological characteristics of hyaluronan-tyramine derivative scaffold were determined together with the hydrogel swelling, the water uptake, and the morphological characterization. The chondrocytes seeded in hyaluronan-tyramine hydrogel were viable and expressed markers typical for differentiated chondrocytes with no signs of hypertrophy or extensive expression of matrix metalloproteinases. The lyophilized unseeded hyaluronan-tyramine scaffolds were also implantated into trochlea osteochondral defects in rabbits. In conclusion, the hyaluronan-tyramine derivative was proven to be advisable material as a scaffold for cartilage reconstitution.     

Chondrogenic stimulation in mesenchymal stem cells using scaffold-based sustained release of platelet-rich plasma

Developing minimal invasive strategies via injectable hydrogels for effective repairing of cartilage defects is highly desired. Injectable hydrogels, which can simultaneously embed cell and growth factors (GFs), serve as in situ formed scaffolds and could support an accelerated tissue regeneration process. The purpose of this study is to fabricate a composite injectable hydrogel, based on alginate (Alg)/polyvinyl alcohol (PVA) incorporating platelet rich plasma (PRP)-encapsulated Alg sulfate (AlgS) microbeads, as a localized sustained release system of GFs, for the articular cartilage regeneration. The results show that synthesized AlgS microbeads support the sustained release of PRP GFs during 14 days, where preserve the bioactivity of them more than the free PRP. Rabbit adipose-derived mesenchymal stem cells in contact with PRP-loaded AlgS beads show more proliferation (2.7 folds) and have obviously higher deposition of collagen type ΙΙ and GAGs than free PRP treated ones. In addition, cells encapsulated into the hydrogel including PRP sustained release system show upregulated expression of collagen type ΙΙ (61 folds), Aggrecan (294 folds) and SOX9 (71.5 folds), as cartilage-critical genes, compared to the direct treatment by PRP. To summarize, the developed hybrid Alg/PVA hydrogel embedding with PRP-encapsulated AlgS microbeads is suggested as a potential in situ formed scaffold for cartilage regeneration.     

Silk fibroin‐chondroitin sulfate‐alginate porous scaffolds: Structural properties and in vitro studies

The 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.     

Study of Different Delivery Modes of Chondroitin Sulfate Using Microspheres and Cryogel Scaffold for Application in Cartilage Tissue Engineering

This study focuses on the development of an efficient delivery modes designed for chondroitin sulfate (CS) for application in cartilage tissue engineering. Novel three-dimensional (3-D) scaffold fabricated from natural polymers such as chitosan and gelatin blended with chondroitin sulfate (CGC) were synthesized using cryogelation technology. Other methods to deliver CS were also tried, which included incorporation into microparticles for sustained release and embedding the CS loaded microparticles in CG (chitosan-gelatin) cryogel scaffold. Novel CGC scaffolds were characterized by rheology, scanning electron microscopy (SEM), and mechanical assay. Scaffolds exhibited compression modulus of 50 KPa confirming the utility of these scaffolds for cartilage tissue engineering. Primary goat chondrocytes were used for the in vitro testing of all the delivery modes. So this study shows that CS microparticles when given freely with matrix (chitosan–gelatin) or embedded into scaffold has potential to enhance chondrocyte proliferation together with improved matrix production than in control without microspheres.     

Integrated three‐dimensional fiber/hydrogel biphasic scaffolds for periodontal bone tissue engineering

Combining a tissue engineering scaffold made of a load‐bearing polymer with a hydrogel represents a powerful approach to enhancing the functionalities of the resulting biphasic construct, such as its mechanical properties or ability to support cellular colonization. This research activity was aimed at the development of biphasic scaffolds through the combination of an additively manufactured poly(?‐caprolactone) (PCL) fiber construct and a chitosan/poly(γ‐glutamic acid) polyelectrolyte complex hydrogel. By investigating a set of layered structures made of PCL or PCL/hydroxyapatite composite, biphasic scaffold prototypes with good integration of the two phases at the macroscale and microscale were developed. The biphasic constructs were able to absorb cell culture medium up to 10‐fold of their weight, and the combination of the two phases had a significant influence on compressive mechanical properties compared with hydrogel or PCL scaffold alone. In addition, due to the presence of chitosan in the hydrogel phase, biphasic scaffolds exerted a broad‐spectrum antibacterial activity. The developed biphasic systems appear well suited for application in periodontal bone regenerative approaches in which a biodegradable porous structure providing mechanical stability and a hydrogel phase functioning as absorbing depot of endogenous proteins are simultaneously required. © 2016 Society of Chemical Industry     

Establishment of a New Device for Electrical Stimulation of Non-Degenerative Cartilage Cells In Vitro

In cell-based therapies for cartilage lesions, the main problem is still the formation of fibrous cartilage, caused by underlying de-differentiation processes ex vivo. Biophysical stimulation is a promising approach to optimize cell-based procedures and to adapt them more closely to physiological conditions. The occurrence of mechano-electrical transduction phenomena within cartilage tissue is physiological and based on streaming and diffusion potentials. The application of exogenous electric fields can be used to mimic endogenous fields and, thus, support the differentiation of chondrocytes in vitro. For this purpose, we have developed a new device for electrical stimulation of chondrocytes, which operates on the basis of capacitive coupling of alternating electric fields. The reusable and sterilizable stimulation device allows the simultaneous use of 12 cavities with independently applicable fields using only one main supply. The first parameter settings for the stimulation of human non-degenerative chondrocytes, seeded on collagen type I elastin-based scaffolds, were derived from numerical electric field simulations. Our first results suggest that applied alternating electric fields induce chondrogenic re-differentiation at the gene and especially at the protein level of human de-differentiated chondrocytes in a frequency-dependent manner. In future studies, further parameter optimizations will be performed to improve the differentiation capacity of human cartilage cells.     

Degradable natural polymer hydrogels for articular cartilage tissue engineering

Articular cartilage has poor ability to heal once damaged. Tissue engineering with scaffolds of polymer hydrogels is promising for cartilage regeneration and repair. Polymer hydrogels composed of highly hydrated crosslinked networks mimic the collagen networks of the cartilage extracellular matrix and thus are employed as inserts at cartilage defects not only to temporarily relieve the pain but also to support chondrocyte proliferation and neocartilage regeneration. The biocompatibility, biofunctionality, mechanical properties, and degradation of the polymer hydrogels are the most important parameters for hydrogel‐based cartilage tissue engineering. Degradable biopolymers with natural origin have been widely used as biomaterials for tissue engineering because of their outstanding biocompatibility, low immunological response, low cytotoxicity, and excellent capability to promote cell adhesion, proliferation, and regeneration of new tissues. This review covers several important natural proteins (collagen, gelatin, fibroin, and fibrin) and polysaccharides (chitosan, hyaluronan, alginate and agarose) widely used as hydrogels for articular cartilage tissue engineering. The mechanical properties, structures, modification, and structure–performance relationship of these hydrogels are discussed since the chemical structures and physical properties dictate the in vivo performance and applications of polymer hydrogels for articular cartilage regeneration and repair. © 2012 Society of Chemical Industry     

Compressive properties and creep resistance of a novel,porous, semidegradable poly(vinyl alcohol)/poly(lactic‐co‐glycolic acid) scaffold for articular cartilage repair

Tissue engineering for articular cartilage repair has shown success in ensuring the integration of neocartilage with surrounding natural tissue, but the rapid restoration of biomechanical functions remains a significant challenge. The poly(vinyl alcohol) (PVA) hydrogel is regarded as a potential articular cartilage replacement for its fair mechanical strength, whereas its lack of bioactivity limits its utility. To obtain a scaffold possessing expected bioactivity and initial mechanical properties, we herein report a novel salt‐leaching technique to fabricate a porous PVA hydrogel simultaneously embedded with poly(lactic‐co‐glycolic acid) (PLGA) microspheres. Through the investigation of environmental scanning electron microscopy, we found that the porous PVA/PLGA scaffold was successfully manufactured. The compression and creep properties were also comprehensively studied before and after cell culturing. The relationship between the compressive modulus and strain ratio of the porous PVA/PLGA scaffold showed significant nonlinear behavior. The elastic compressive modulus was influenced a little by the porogen content, whereas it went higher with a higher PLGA microsphere content. The cell‐cultured scaffolds presented higher compressive moduli than the initial ones. The creep resistance of the cell‐cultured scaffolds was much better than that of the initial ones. In all, this new scaffold is a promising material for articular cartilage repair. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40311.     

Fabrication of composition-graded collagen/chitosan–polylactide scaffolds with gradient architecture and properties

Chitosan–polylactide (CH–PLA) copolymers with various polylactide percentages changing from around 14 to 40 wt% were synthesized. CH–PLAs were then blended with type-II collagen to fabricate layered collagen/CH–PLA scaffolds that are potentially suitable for the applications in articular cartilage repair. Based on combinatorial processing techniques involving layer-superposition, thermal melting and freeze-drying, two types of stratified collagen/CH–PLA scaffolds were built. The content of collagen inside the scaffolds altered from the top layer to the bottom layer in a trend contrary to that of chitosan. One of them was fabricated using tripolyphosphate (TPP) as a single crosslinker and another type of scaffold was constructed via a dual-crosslinking pathway using TPP and genipin as two crosslinkers in a designated order. These collagen/CH–PLA scaffolds were found to have graded average pore-size and porosity, gradient swelling index and layer-dependent compressive modulus. The resulting scaffolds were thus partially similar to the articular cartilage extracellular matrix in composition, structure and property. In vitro cell culture on some optimized collagen/CH–PLA scaffolds for a period of time up to 3 weeks showed that the scaffolds were able to well support the growth of the seeded cells, suggesting that these collagen/CH–PLA scaffolds have promising potential for articular cartilage repair.     

Reinforcement and chemical cross-linking in collagen-based scaffolds in cartilage tissue engineering: a comparative study

Mechanical strength and biocompatibility are issues of most concern for scaffolds in cartilage tissue engineering. Collagen modification is always used to strengthen scaffolds. There are mainly two ways for collagen modification: inclusion of reinforcing phase to form composites and chemical cross-linking. To explore an alternative approach, the collagen hydrogel modified by a reinforcement phase was compared with cross-linking. Collagen-alginate hydrogel (CAH) and collagen hydrogel cross-linked by genipin (CGH), which were different in modification methods, were chosen candidates. A comprehensive study was carried out on mechanical, structural and biological properties including swelling ratio measurement, in vitro degradation, AFM, mechanical test, thermogravimetric analysis, and in vitro cartilage tissue engineering. The results showed that mechanical strength of collagen was more enhanced for CGH than CAH, as evidenced by analysis of swelling ratio, in vitro degradation, AFM, mechanical test and thermostability. MTT and histological results showed that CGH was superior to CAH with less cytotoxicity and more chondrocytes distributed as well as more aggrecan secreted. With the increase in culture time, the cytotoxicity of cross-linker may be alleviated. CGH may provide a more favorable biomimetic environment for cartilage growth. All these indicated that selecting a cross-linker with a minimal cytotoxicity could be more promising for collagen modification, with improvements observed in both physical and biological properties. For reinforcement, it was required that the incorporated component should be equipped with better or equivalent properties compared with collagen. This study provided important implications to engineering collagen-based hydrogels for cartilage graft applications.     

In vitro characterization of porous calcium phosphate scaffolds capped with crosslinked hydrogels to avoid inherent brittleness

Ductile composite scaffolds can avoid being crushed during filling processes in clinical applications. Thus, this study aimed to modify brittle porous ceramic scaffolds into ductile scaffolds through hydrogel capping. The surfaces of calcium phosphate (CaP) ceramic scaffolds were effectively capped with alginate/gelatin hydrogels. The composite scaffolds were then crosslinked, subjected to vacuum drainage, and washed prior to lyophilization. The detailed controlled approach in this study was proposed with the aim to develop biocompatible composites with anisotropic open pores through the formation of a thin, homogenous, hydrogel film coating on porous ceramic scaffold surfaces. The performances of the hydrogel/CaP composite scaffolds were evaluated on the basis of their morphological characteristics, compressive strengths, and cell viabilities. Results showed that strength, toughness, and specimen integrity after cracking are strongly related to the concentration of the hydrogel cap. Strength testing results showed that the use of 50 vol% alcohol as the crosslinker removal solution yielded scaffolds with high toughness and ductility. Moreover, the cracked specimen dipped in 50 vol% alcohol possessed better integrity than that dipped in water only. This study successfully identified the optimal hydrogel quantity for the fabrication of biocompatible scaffolds with open connective pores. The advantages of the fabricated scaffolds indicate the relevance of the proposed method to clinical applications, such the production of fillers for successful alveolar bone augmentation.     

Development of a novel glucosamine/silk fibroin–chitosan blend porous scaffold for cartilage tissue engineering applications

Silk fibroin–chitosan blend is reported to be an attractive scaffold material for tissue engineering applications. In our earlier study, we developed a scaffold having an optimal silk fibroin–chitosan blend ratio of 80:20 and proved its potentiality for cartilage tissue engineering applications. Glucosamine is one of the major structural components of cartilage tissue. The present work investigates the effect of glucosamine components on the physicochemical and biocompatibility properties of this scaffold. To this end, varied amounts of glucosamine were added to silk fibroin–chitosan blend with the aim of improving various scaffold properties. The addition of glucosamine components did not show any significant change in physicochemical properties of silk fibroin–chitosan blend scaffolds. The composite scaffold showed an open pore structure with desired pore size and porosity. However, cell culture study using human mesenchymal stem cells derived from umbilical cord blood revealed an overall increase in cell supportive properties of glucosamine-added scaffolds. Cell viability, cell proliferation and glycosaminoglycan assays confirmed enhanced cell viability and proliferation of mesenchymal stem cells. Thus, this study demonstrated the beneficial effect of glucosamine on improving the cell supportive property of silk fibroin–chitosan blend scaffolds making it more potential for cartilage tissue regeneration. To the best of our knowledge, this is the first report on the study of glucosamine-added silk fibroin–chitosan blend porous scaffolds seeded with mesenchymal stem cells derived from umbilical cord blood.