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Fabrication of cellulosic composite scaffolds for cartilage tissue engineering
... Besides, physical properties of these materials resemble properties of living tissues; therefore, they are used as biomaterials for various applications, such as soft wound dressings, contact lenses, drug delivery systems, etc. (Hoffman et al. 2012;Chai et al. 2017). Hydrogels have also been the subject of extensive research as replacement materials for damaged cartilage tissue (Oka et al. 2000;Stammen et al. 2001;Ku 2008;Kobayashi and Hyu 2010;Batista et al. 2012;Gonzales and Alvarez 2014;Nandgaonkar et al. 2016;Luo et al. 2022;Qiu et al. 2023). In the experiments involving animals, replacement of cartilage tissues of various localizations with hydrogels was studied, including the knee and hip (Oka et al. 2000;Kobayashi and Hyu 2010;Batista et al. 2012;Gonzales and Alvarez 2014). ...
... The hydrogels with improved mechanical properties can be obtained while using cellulose as a reinforcing component in the compositions with other polymers (Nandgaonkar et al. 2016). Cellulose is an abundant natural polymer characterized by high mechanical stiffness, large surface area, a large number of reactive surface functional groups and excellent biocompatibility (Sannino et al. 2009;Rose and Palkovits 2011;Nandgaonkar et al. 2016;Cordeiro et al. 2022), but the major problem in preparing cellulose hydrogels is a lack of appropriate solvents due to its highly extended hydrogen-bonded structure (Ciolacu and Suflet 2018). ...
... The hydrogels with improved mechanical properties can be obtained while using cellulose as a reinforcing component in the compositions with other polymers (Nandgaonkar et al. 2016). Cellulose is an abundant natural polymer characterized by high mechanical stiffness, large surface area, a large number of reactive surface functional groups and excellent biocompatibility (Sannino et al. 2009;Rose and Palkovits 2011;Nandgaonkar et al. 2016;Cordeiro et al. 2022), but the major problem in preparing cellulose hydrogels is a lack of appropriate solvents due to its highly extended hydrogen-bonded structure (Ciolacu and Suflet 2018). For this reason, only a few examples of the successful formation of cellulose hydrogels are known. ...
High-strength composite hydrogels “cellulose–polyacrylamide” were synthesized by free-radical polymerization of acrylamide conducted inside the previously formed physical network of regenerated plant cellulose. Partial hydrolysis of the amide groups of these hydrogels yielded their ionic forms with a degree of hydrolysis of 0.1 and 0.25. The cylindrical hydrogel samples of three compositions were implanted in the preformed osteochondral defects of the rabbit’s femoral knee joints. No signs of migration or disintegration of the tested implants were revealed in the course of in vivo tests as long as 90 and 120 days after the implantation. The mechanical behavior of hydrogel samples-implants before implantation and after their removal from the joints of laboratory animals was studied in detail. The morphology and chemical composition of the removed implants were studied by SEM combined with the EDX method. The results showed that the mechanical characteristics of hydrogel implants remained practically unchanged after in vivo tests. The removed implants, as well as the initial hydrogels, endured cyclic compression loading at the amplitude up to 50%. Compression stresses up to 3–10 MPa were recorded in these tests, which is close to the data obtained by several authors for natural articular cartilages in the same conditions of loading. The principal differences in the chemical composition and morphology of the implant area adjacent to the subchondral bone for non-ionic and ionic types of implants have been revealed. For non-ionic implants in this area intensive mineralization with formation of calcium phosphates inside the polymeric hydrogel network is observed, while the border area of ionic implants practically does not undergo mineralization.
... The hydrogels with improved mechanical properties can be obtained while using cellulose as a reinforcing component in the compositions with other polymers (Nandgaonkar et al. 2016). Cellulose is a natural polymer characterized by high mechanical stiffness and excellent biocompatibility (Sannino et al. 2009; Nandgaonkar et al. 2016), but the major problem in preparing cellulose hydrogels is a lack of appropriate solvents due to its highly extended hydrogen-bonded structure (Ciolacu and Su et 2018). ...
... The hydrogels with improved mechanical properties can be obtained while using cellulose as a reinforcing component in the compositions with other polymers (Nandgaonkar et al. 2016). Cellulose is a natural polymer characterized by high mechanical stiffness and excellent biocompatibility (Sannino et al. 2009; Nandgaonkar et al. 2016), but the major problem in preparing cellulose hydrogels is a lack of appropriate solvents due to its highly extended hydrogen-bonded structure (Ciolacu and Su et 2018). For this reason, only few examples of the successful formation of cellulose hydrogels are known. ...
High-strength composite hydrogels “cellulose-polyacrylamide” were synthesized by free-radical polymerization of acrylamide conducted inside the previously formed physical network of regenerated plant cellulose. Partial hydrolysis of the amide groups of these hydrogels yielded their ionic forms with a degree of hydrolysis of 0.1 and 0.25. The cylindrical hydrogel samples of three compositions were implanted in the preformed osteochondral defects of the rabbit’s femoral knee joints. No signs of migration or disintegration of the tested implants were revealed in the course of in vivo tests as long as 90 and 120 days after the implantation. The mechanical behavior of hydrogel samples-implants before implantation and after their removal from the joints of laboratory animals was studied in detail. The morphology and chemical composition of the removed implants were studied by SEM combined with the EDX method. The results obtained were shown that the mechanical characteristics of hydrogel implants remained practically unchanged after in vivo tests. The removed implants, as well as the initial hydrogels, endured cyclic compression loading at the amplitude up to 50 %. Compression stresses up to 3 – 10 MPa were recorded in these tests, which is close to the data obtained by several authors for natural articular cartilages in the same conditions of loading. The principal differences in the chemical composition and morphology of the implant area adjacent to the subchondral bone for non-ionic and ionic types of implants have been revealed. For non-ionic implants in this area intensive mineralization with formation of calcium phosphates inside the polymeric hydrogel network is observed, while the border area of ionic implants practically does not undergo mineralization.
... Among this diversity, polymeric hydrogels of various compositions occupy an important place (Berradi et al. 2023;Calo and Khutoryanskiy 2015;Chamkouri and Chamkouri 2021;Pearce and O'Reilly 2021). Analysis of the flow of publications describing the directions of hydrogels use in biomedical applications allows us to distinguish three most popular areas in which impressive results have been achieved: (1) soft medical dressings, (2) scaffolds for tissue enginery, and (3) implants to repair the damage of different human body tissues, such as, for example, the injured hyaline cartilage (Batista et al. 2012;Bustamante-Torres et al. 2021;Chamkouri and Chamkouri 2021;Correa et al. 2021;Gonzales and Alvarez 2014;Guillén-Carvajal et al.2023;Hago and Li 2013;Ku 2008;Nandgaonkar et al. 2016;Peters et al. 2021;Sarangi et al. 2023;Sciaretta 2013;Slaughter et al. 2009). ...
A group of new hydrogel materials combining high physical properties and pronounced antibacterial activity has been developed. These are composite hydrogels “cellulose-polyacrylamide” based on cellulose matrices of two types: bacterial or regenerated plant cellulose. To form biologically active materials, a method of introducing cerium oxide nanoparticles with sizes less than 5 nm was elaborated. The developed technology allows to obtain hydrogels with the content of cerium oxide (in swollen material) up to 0.4–0.5 wt%. Variations of the ratio of gel components concentrations, type of matrix cellulose and synthesis conditions allow to change the complex of mechanical properties of the material within a wide range, in particular, to obtain both soft, low-modular nanocomposites and hydrogels with record high rigidity. Significant differences in mechanical properties of hydrogels based on different types of cellulose fully correlate with the difference in morphological characteristics of these two groups of materials, revealed by SEM. No palpable effect of nanoparticles on the morphological characteristics of the material was revealed. Both cerium oxide nanoparticles and hydrogels containing cerium oxide showed antibacterial activity against S. aureus ATCC 29213, S. aureus ATCC 43300, P. aeruginosa ATCC 27853, K. pneumoniae ATCC 33495. Different intensity of growth depression of the bacterial cells was determined depending on the samples composition and of the bacteria species.
... In recent years, cellulose, in particular the specific type of this polymer, namely bacterial cellulose (BC), has been used for the synthesis of various types of advanced composite materials including hydrogel compositions for biomedical applications (Figueiredo et al., 2013, 2015, Lin, Dufresne, 2014, Nandgaonkar et al., 2016, Ullah et al., 2016. Cellulose is a natural polymer characterized by the high mechanical stiffness and excellent biocompatibility (Sannino et al, 2009). ...
Two types of high-strength composite hydrogels possessing the structure of interpenetrating polymer networks were synthesized via free-radical polymerization of acrylamide carried out straight within the matrix of plant or bacterial cellulose swollen in the reactive solution. The mechanical behavior of synthesized hydrogels subjected to the action of compressive deformations with different amplitude values was studied. The analysis of the stress-strain curves of compression tests of the hydrogels of both types obtained in different test conditions demonstrates the substantial difference in their mechanical behavior. Both the plant cellulose-based and bacterial cellulose-based hydrogels withstand successfully the compression with the amplitude up to 80 %, but the bacterial cellulose-based compositions demonstrate in the multiple compression tests the substantial decrease of the mechanical stiffness caused by the action of compressions. This effect takes place straightly during the multiple cyclic compression tests or in the course of relaxation of previously compressed samples in water in unloaded state during 2-3 days after the first compressive tests. Being subjected to the action of the extremely sever compression (up to 80 %) the bacterial cellulose-based hydrogel varies greatly its mechanical behavior: during the subsequent compressions the stiffness of the material keeps extremely small (as compared to that registered in the first compression) up to the deformations as high as 50-60 % with the dramatic increase of the stiffness during the further loading. Submicron- and micron-scale specific features of structures of composite hydrogels of both types were studied by cryo-scanning electron microscopy to explain the peculiarities of observed mechanical effects.
https://arxiv.org/abs/1904.00988
... Cellulose is a biopolymer used in numerous industrial applications. This biopolymer has several applications ( Abitbol et al., 2016;Choo, Ching, Chuah, Sabariah, & Liou, 2016;Nandgaonkar, Krause, & Lucia, 2016) as a novel physical and chemical reinforcement in nanocomposite materials ( Cao, Dong, & Li, 2007;De France, Chan, Cranston, & Hoare, 2016;) because of its high mechanical strength ( El Miri et al., 2016;Li, Cao, Cao, Guo, & Lu, 2016;Siqueira, Bras, & Dufresne, 2008), shear assembly and di-electrophoresis behaviour ( Csoka, Hoeger, Peralta, Peszlen, & Rojas, 2011). Cellulose is a linear syndiotactic homopolymer composed of d-anhydroglucopyranose units which are linked by-(1 → 4)-glycosidic bonds ( Qiu & Hu, 2013). ...
Recently, surface functionality and thermal property of the green nanomaterials have received wide attention in numerous applications. In this study, microcrystalline cellulose (MCC) was used to prepare the nanocrystalline celluloses (NCCs) using acid hydrolysis method. The NCCs was treated with TEMPO [(2,2,6,6-tetramethylpiperidin-1-yl)oxy radical]-oxidation to prepare TEMPO-oxidized NCCs. Cellulose nanofibrils (CNFs) also prepared from MCC using TEMPO-oxidation. The effects of rapid cooling and chemical treatments on the thermo-structural property studies of the prepared nanocelluloses were investigated through FTIR, thermogravimetric analysis-derivative thermogravimetric (TGA-DTG), and XRD. A posteriori knowledge of the FTIR and TGA-DTG analysis revealed that the rapid cooling treatment enhanced the hydrogen bond energy and thermal stability of the TEMPO-oxidized NCC compared to other nanocelluloses. XRD analysis exhibits the effect of rapid cooling on pseudo 2I helical conformation. This was the first investigation performed on the effect of rapid cooling on structural properties of the nanocellulose.
The use of fibers into scaffolds is a way to mimic natural tissues, in which fibrils are embedded in a matrix. The use of fibers can improve the mechanical properties of the scaffolds and may act as structural support for cell growth. Also, as the morphology of fibrous scaffolds is similar to the natural extracellular matrix, cells cultured on these scaffolds tend to maintain their phenotypic shape. Different materials and techniques can be used to produce micrfibers- and nanofibers for scaffolds manufacturing; cells, in general, adhere and proliferate very well on PCL, chitosan, silk fibroin, and other nanofibers. One of the most important techniques to produce microfibers/nanofibers is electrospinning. Nanofibrous scaffolds are receiving increasing attention in bone tissue engineering, because they are able to offer a favorable microenvironment for cell attachment and growth. Different polymers can be electrospun, i.e., polyester, polyurethane, PLA, PCL, collagen, and silk. Other materials such as bioglass fibers, nanocellulose, and even carbon fiber and fabrics have been used to help increase bioactivity, mechanical properties of the scaffold, and cell proliferation. A compilation of mechanical properties and most common biological tests performed on fibrous scaffolds is included in this chapter.
Highlights:
The use of microfibers and nanofibers allows for tailoring the scaffold properties. Electrospinning is one of the most important techniques nowadays to produce fibrous scaffolds. Microfibers and nanofibers use in scaffolds is a promising field to improve the behavior of scaffolds in osteochondral applications.
Articular cartilage is a hyaline cartilage that consists primarily of extracellular matrix with a sparse population of cells, lacking blood vessels, lymphatic vessels and nerves. The only cell type within cartilage is the chondrocyte and has a low level of metabolic activity with little or no cell division and is the responsible for maintaining in a low-turnover state the unique composition and organization of the matrix that was determined during embryonic and postnatal development. The biological and mechanical properties of articular cartilage depend on the interactions between the chondrocytes and the matrix that maintain the tissue. Chon‐ drocytes form the macromolecular framework of the tissue matrix from three classes of molecules: collagens, proteoglycans, and non-collagenous proteins and maintain the extrac‐ ellular matrix (ECM) by low-turnover replacement of certain matrix proteins.
Aggrecan and type II collagen are the most abundant proteins found within the ECM in the articular cartilage and they are linked together by a number of collagen-binding proteins including cartilage oligomeric matrix protein (COMP), chondroadherin and other minor collagens on their surface. Aggrecan is a large aggregating proteoglycan which is in association with hyaluronan (HA) and link protein (LP). These aggregates are responsible for the turgid and they provide the osmotic properties to resist compressive loads and retain water. Also contain a variety of small leucine-rich repeat proteoglycans (SLRPs) as decorin, biglycan, fibromodulin and lumican where they help maintain the integrity of the tissue and modulate its metabolism.
The failure of organs and tissues caused by trauma and other injuries is one of the most costly of human health problems. It is estimated that 1.6 million people experience work limitations caused by osteoarthritis and related disorders, representing 8.3% of all main conditions. Joint injuries frequently lead to progressive joint degeneration and post-traumatic osteoarthritis. Articular cartilage has only a limited capacity for self-healing, mainly due to the fact that it is avascular; and once seriously damaged, articular cartilage lesions will not regenerate. There is strong evidence that cartilage lesions may lead to osteoarthritis when left untreated. Numerous animal experiments and clinical studies have shown that early biological reconstruction of circumscribed cartilage defects in the knee is superior to conservative or delayed surgical treatment. Tissue engineering has shown promising therapeutic strategies for repair or regeneration of damaged tissues. Currently, ceramic based and polymeric scaffolds have been developed to bring about the restoration of tissue functions. The bioceramics associated with water-soluble polymers have been developed as substitutes for various orthopedic applications. The objectives of this work are the processing and characterization of a composite of carboxymethylcellulose (CMC) and biphasic calcium phosphate (Biphasic Calcium Phosphate - BCP) in the form of a hydrogel, and a study of its cytotoxicity (in vitro), aimed at its application as an injectable biomaterial in order to repair the extracellular matrix of articular cartilage. The CMC and BCP were characterized by Fourier Transform Infrared Spectrometry (FTIR) and X-Ray Diffraction (XRD), X-ray fluorescence (XRF), respectively, and scanning electron microscopy (SEM) of powders and the composite. To evaluate the biological effect of the composite hydrogel, tests of cytotoxicity (MTT) and rheological tests under real conditions of use were performed. The composite of carboxymethylcellulose (CMC) and bioceramics (biphasic calcium phosphate-BCP) in the form of hydrogel showed an adequate injectability in the conditions studied, and a non-toxic response, presenting potential for use as fillers or to stimulate the healing of cartilage defects in the extracellular matrix of articular cartilage.
Starch/cellulose nanofibers composites with proper porosity pore size, mechanical strength, and biodegradability for cartilage tissue engineering have been reported in this study. The porous thermoplastic starch-based composites were prepared by combining film casting, salt leaching, and freeze drying methods. The diameter of 70% nanofibers was in the range of 40-90nm. All samples had interconnected porous morphology; however an increase in pore interconnectivity was observed when the sodium chloride ratio was increased in the salt leaching. Scaffolds with the total porogen content of 70wt% exhibited adequate mechanical properties for cartilage tissue engineering applications. The water uptake ratio of nanocomposites was remarkably enhanced by adding 10% cellulose nanofibers. The scaffolds were partially destroyed due to low in vitro degradation rate after more than 20 weeks. Cultivation of isolated rabbit chondrocytes on the fabricated scaffold proved that the incorporation of nanofibers in starch structure improves cell attachment and proliferation.
Every day thousands of surgical procedures are performed to replace or repair tissue that has been damaged through disease or trauma. The developing field of tissue engineering (TE) aims to regenerate damaged tissues by combining cells from the body with highly porous scaffold biomaterials, which act as templates for tissue regeneration, to guide the growth of new tissue. This article describes the functional requirements, and types, of materials used in developing state of the art of scaffolds for tissue engineering applications. Furthermore, it describes the challenges and where future research and direction is required in this rapidly advancing field.
Some bacteria can synthesize cellulose when they are cultivated under adequate conditions. These bacteria produce a mat of cellulose on the top of the culture medium, which is formed by a three-dimensional coherent network of pure cellulose nanofibers. Bacterial cellulose (BC) has been widely used in different fields, such as the paper industry, electronics and tissue engineering due to its remarkable mechanical properties, conformability and porosity. Nanocomposites based on BC have received much attention, because of the possibility of combining the good properties of BC with other materials for specific applications. BC nanocomposites can be processed either in a static or an agitated medium. The fabrication of BC nanocomposites in static media can be carried out while keeping the original mat structure obtained after the synthesis to form the final nanocomposite or by altering the culture media with other components. The present article reviews the issue of biocompatibility of BC and BC nanocomposites. Biomedical aspects, such as surface modification for improving cell adhesion, in vitro and in vivo studies are given along with details concerning the physics of network formation and the changes that occur in the cellulose networks due to the presence of a second phase. The relevance of biocompatibility studies for the development of BC-based materials in bone, skin and cardiovascular tissue engineering is also discussed.
Introduction
Current therapies for articular cartilage defects fail to achieve qualitatively sufficient tissue regeneration, possibly because of a mismatch between the speed of cartilage rebuilding and the resorption of degradable implant polymers. The present study focused on the self-healing capacity of resident cartilage cells in conjunction with cell-free and biocompatible (but non-resorbable) bacterial nanocellulose (BNC). This was tested in a novel in vitro bovine cartilage punch model.
Methods
Standardized bovine cartilage discs with a central defect filled with BNC were cultured for up to eight weeks with/without stimulation with transforming growth factor-β1 (TGF-β1. Cartilage formation and integrity were analyzed by histology, immunohistochemistry and electron microscopy. Content, release and neosynthesis of the matrix molecules proteoglycan/aggrecan, collagen II and collagen I were also quantified. Finally, gene expression of these molecules was profiled in resident chondrocytes and chondrocytes migrated onto the cartilage surface or the implant material.
Results
Non-stimulated and especially TGF-β1-stimulated cartilage discs displayed a preserved structural and functional integrity of the chondrocytes and surrounding matrix, remained vital in long-term culture (eight weeks) without signs of degeneration and showed substantial synthesis of cartilage-specific molecules at the protein and mRNA level. Whereas mobilization of chondrocytes from the matrix onto the surface of cartilage and implant was pivotal for successful seeding of cell-free BNC, chondrocytes did not immigrate into the central BNC area, possibly due to the relatively small diameter of its pores (2 to 5 μm). Chondrocytes on the BNC surface showed signs of successful redifferentiation over time, including increase of aggrecan/collagen type II mRNA, decrease of collagen type I mRNA and initial deposition of proteoglycan and collagen type II in long-term high-density pellet cultures. Although TGF-β1 stimulation showed protective effects on matrix integrity, effects on other parameters were limited.
Conclusions
The present bovine cartilage punch model represents a robust, reproducible and highly suitable tool for the long-term culture of cartilage, maintaining matrix integrity and homoeostasis. As an alternative to animal studies, this model may closely reflect early stages of cartilage regeneration, allowing the evaluation of promising biomaterials with/without chondrogenic factors.
Purpose
Multipotent stromal cell (MSC)-based regenerative strategy has shown promise for the repair of cartilage, an avascular tissue in which cells experience hypoxia. Hypoxia is known to promote the early chondrogenic differentiation of MSC. The aim of our study was therefore to determine whether low oxygen tension could be used to enhance the regenerative potential of MSC for cartilage repair.
Methods
MSC from rabbit or human adipose stromal cells (ASC) were preconditioned in vitro in control or chondrogenic (ITS and TGF-β) medium and in 21 or 5% O2. Chondrogenic commitment was monitored by measuring COL2A1 and ACAN expression (real-time PCR). Preconditioned rabbit and human ASC were then incorporated into an Si-HPMC hydrogel and injected (i) into rabbit articular cartilage defects for 18 weeks or (ii) subcutaneously into nude mice for five weeks. The newly formed tissue was qualitatively and quantitatively evaluated by cartilage-specific immunohistological staining and scoring. The phenotype of ASC cultured in a monolayer or within Si-HPMC in control or chondrogenic medium and in 21 or 5% O2 was finally evaluated using real-time PCR.
Results/Conclusions
5% O2 increased the in vitro expression of chondrogenic markers in ASC cultured in induction medium. Cells implanted within Si-HPMC hydrogel and preconditioned in chondrogenic medium formed a cartilaginous tissue, regardless of the level of oxygen. In addition, the 3D in vitro culture of ASC within Si-HPMC hydrogel was found to reinforce the pro-chondrogenic effects of the induction medium and 5% O2. These data together indicate that although 5% O2 enhances the in vitro chondrogenic differentiation of ASC, it does not enhance their in vivo chondrogenesis. These results also highlight the in vivo chondrogenic potential of ASC and their potential value in cartilage repair.
Osteoarthritis is a common disease in humans, which is caused by progressive degradation of articular cartilage and imbalance of extracellular matrix turnover. It usually causes pain and malfunction of the affected joints, and the inci-dence of this disease increases with aging. Classical treatment for osteoarthritis is pain alleviation and, in progressed dise-ase, surgical operations. New surgical techniques include transplantation of autologous chondrocytes or osteochondral plugs to the lesion area. However, these techniques have the problem of limited cell and tissue sources available for tran-splantation. Therefore, cartilage engineering and use of mesenchymal stem cells have raised a lot of interest as therapeutic approaches. Various strategies are being tested for their ability to provide tissue constructs that could be used as a repla-cement for the damaged cartilage. Growth factors, cytokines and mechanical forces are known to direct chondrogenesis and the maintenance of chondrocytic phenotype, although the cellular signaling events involved are often poorly known. Yet, understanding of the signal transduction mechanisms involved in chondrogenesis and cartilage tissue engineering will be very important, especially to learn how to guide stem cells into the desired differentiation path. This review aims to summarize the known signal transduction pathways involved with osteoarthritis, cartilage mechanobiology and diffe-rentiation of mesenchymal stem cells to chondrocytes.
One of the challenges of tissue engineering, a promising cell-based treatment for damaged or diseased cartilage, is designing One of the challenges of tissue engineering, a promising cell-based treatment for damaged or diseased cartilage, is designing
the scaffold that provides structure while the tissue regenerates. In addition to the scaffold material’s biocompatibility, the scaffold that provides structure while the tissue regenerates. In addition to the scaffold material’s biocompatibility,
mechanical properties, and ease of manufacturing, scaffold interactions with the cells must also be considered. In cartilage mechanical properties, and ease of manufacturing, scaffold interactions with the cells must also be considered. In cartilage
tissue engineering, a range of scaffolds with various degrees of cell attachment have been proposed, but the attachment density tissue engineering, a range of scaffolds with various degrees of cell attachment have been proposed, but the attachment density
and type have yet to be optimized. Several techniques have been developed to modulate cell adhesion to the scaffold. These and type have yet to be optimized. Several techniques have been developed to modulate cell adhesion to the scaffold. These
studies suggest that the need for cell attachment in cartilage tissue engineering may vary with cell type, stage of differentiation, studies suggest that the need for cell attachment in cartilage tissue engineering may vary with cell type, stage of differentiation,
culture condition, and scaffold material. Further studies will elucidate the role of cell attachment in cartilage regeneration culture condition, and scaffold material. Further studies will elucidate the role of cell attachment in cartilage regeneration
and enhance efforts to engineer cell-based cartilage therapies. and enhance efforts to engineer cell-based cartilage therapies.
Nanoreinforced hydrogels with a unique network structure were prepared from wood cellulose whiskers coated with chemically modified wood hemicelluloses. The hemicelluloses were modified with 2-hydroxyethylmethacrylate prior to adsorption onto the cellulose whiskers in aqueous medium. Synthesis of the hydrogels was accomplished by in situ radical polymerization of the methacrylic groups of the adsorbed coating to form a network of poly(2-hydroxyethylmethacrylate) (PHEMA) matrix reinforced with cellulose whiskers. The mechanical, swelling and viscoelastic properties, of water-swollen hydrogels were investigated. Results indicated that the number of effective crosslinks between polymer chains and the average chain length between crosslinking points was significantly different from PHEMA hydrogels that had been crosslinked by a conventional chemical method, using a cross-linking agent. The resulting hydrogels had enhanced toughness, increased viscoelasticity, and improved recovery behavior. With respect to the mechanical and swelling properties, it can be hypothesized that these nanoreinforced PHEMA hydrogels have potential for use in load-bearing biomedical applications such as articular cartilage replacement.
Metabolic pathways for amino sugars (N-acetylglucosamine; GlcNAc and glucosamine; Gln) are essential and remain largely conserved in all three kingdoms of life, i.e., microbes, plants and animals. Upon uptake, in the cytoplasm these amino sugars undergo phosphorylation by phosphokinases and subsequently deacetylation by the enzyme N-acetylglucosamine 6-phosphate deacetylase (nagA) to yield glucosamine-6-phosphate and acetate, the first committed step for both GlcNAc assimilation and amino-sugar-nucleotides biosynthesis. Here we report the cloning of a DNA fragment encoding a partial nagA gene and its implications with regard to amino sugar metabolism in the cellulose producing bacterium Glucoacetobacter xylinus (formally known as Acetobacter xylinum). For this purpose, nagA was disrupted by inserting tetracycline resistant gene (nagA::tet(r); named as ΔnagA) via homologous recombination. When compared to glucose fed conditions, the UDP-GlcNAc synthesis and bacterial growth (due to lack of GlcNAc utilization) was completely inhibited in nagA mutants. Interestingly, that inhibition occured without compromising cellulose production efficiency and its molecular composition under GlcNAc fed conditions. We conclude that nagA plays an essential role for GlcNAc assimilation by G. xylinus thus is required for the growth and survival for the bacterium in presence of GlcNAc as carbon source. Additionally, G. xylinus appears to possess the same molecular machinery for UDP-GlcNAc biosynthesis from GlcNAc precursors as other related bacterial species.
Scaffolds derived from natural polysaccharides are very promising in tissue engineering applications and regenerative medicine, as they resemble glycosaminoglycans in the extracellular matrix (ECM). In this study, we have prepared freeze-dried composite scaffolds of chitosan (CHT) and hyaluronic acid (HA) in different weight ratios containing either no HA (control) or 1%, 5%, or 10% of HA. We hypothesized that HA could enhance structural and biological properties of CHT scaffolds. To test this hypothesis, physicochemical and biological properties of CHT/HA scaffolds were evaluated. Scanning electron microscopy micrographs, mechanical properties, swelling tests, enzymatic degradation, and Fourier transform infrared (FTIR) chemical maps were performed. To test the ability of the CHT/HA scaffolds to support chondrocyte adhesion and proliferation, live-dead and MTT assays were performed. Results showed that CHT/HA composite scaffolds are noncytotoxic and promote cell adhesion. ECM formation was further evaluated with safranin-O and alcian blue staining methods, and glycosaminoglycan and DNA quantifications were performed. The incorporation of HA enhanced cartilage ECM production. CHT/5HA had a better pore network configuration and exhibited enhanced ECM cartilage formation. On the basis of our results, we believe that CHT/HA composite matrixes have potential use in cartilage repair.
Natural polysaccharides are attractive compounds with which to build scaffolds for bone and cartilage tissue engineering. Here we tested two non-standard ones, HE800 and GY785, for the two-dimensional (2-D) and three-dimensional (3-D) culture of osteoblasts (MC3T3-E1) and chondrocytes (C28/I2). These two glycosaminoglycan-like marine exopolysaccharides were incorporated into an injectable silylated hydroxypropylmethylcellulose-based hydrogel (Si-HPMC) that has already shown its suitability for bone and cartilage tissue engineering. Results showed that, similarly to hyaluronic acid (HA) (the control), HE800 and GY785 significantly improved the mechanical properties of the Si-HPMC hydrogel and induced the attachment of MC3T3-E1 and C28/I2 cells when these were cultured on top of the scaffolds. Si-HPMC hydrogel containing 0.67% HE800 exhibited the highest compressive modulus (11 kPa) and allowed the best cell dispersion, especially of MC3T3-E1 cells. However, these cells did not survive when cultured in 3-D within hydrogels containing HE800, in contrast to C28/I2 cells. The latter proliferated in the microenvironment or concentrically depending on the nature of the hydrogel. Among all the constructs tested the Si-HPMC hydrogels containing 0.34% HE800 or 0.67% GY785 or 0.67% HA presented the most interesting features for cartilage tissue engineering applications, since they offered the highest compressive modulus (9.5–11 kPa) while supporting the proliferation of chondrocytes.
Articular cartilage does not repair itself spontaneously. To promote its repair, the transfer of stem cells from adipose tissue (ATSC) using an injectable self-setting cellulosic-hydrogel (Si-HPMC) appears promising. In this context, the objective of this work was to investigate the influence of in vitro chondrogenic differentiation of ATSC on the in vivo cartilage formation when combined with Si-HPMC. In a first set of experiments, we characterized ATSC for their ability to proliferate, self renew and express typical mesenchymal stem cell surface markers. Then, the potential of ATSC to differentiate towards the chondrogenic lineage and the optimal culture conditions to drive this differentiation were evaluated. Real-time RT-PCR and histological analysis for sulphated glycosaminoglycans and type II collagen revealed that 3-dimensional culture and hypoxic condition favored ATSC chondrogenesis regarding mRNA expression level and the corresponding proteins production. In order to assess the phenotypic stability of chondrogenically-differentiated ATSC, real-time RT-PCR for specific terminal chondrogenic markers and alkaline phosphatase activity assay were performed. In addition to promote chondrogenesis, our culture conditions seem to prevent the terminal differentiation of ATSC. Histological examination of ATSC/Si-HPMC implants suggested that the in vitro chondrogenic pre-commitment of ATSC in monolayer is sufficient to obtain cartilaginous tissue in vivo.
Despite excellent biocompatibility and mechanical properties, the poor in vitro and in vivo degradability of cellulose has limited its biomedical and biomass conversion applications. To address this issue, we report
a metabolic engineering-based approach to the rational redesign of cellular metabolites to introduce N-acetylglucosamine (GlcNAc) residues into cellulosic biopolymers during de novo synthesis from Gluconacetobacter xylinus. The cellulose produced from these engineered cells (modified bacterial cellulose [MBC]) was evaluated and compared with
cellulose produced from normal cells (bacterial cellulose [BC]). High GlcNAc content and lower crystallinity in MBC compared
to BC make this a multifunctional bioengineered polymer susceptible to lysozyme, an enzyme widespread in the human body, and
to rapid hydrolysis by cellulase, an enzyme commonly used in biomass conversion. Degradability in vivo was demonstrated in subcutaneous implants in mice, where modified cellulose was completely degraded within 20 days. We provide
a new route toward the production of a family of tailorable modified cellulosic biopolymers that overcome the longstanding
limitation associated with the poor degradability of cellulose for a wide range of potential applications.
Hydrogels are macromolecular networks able to absorb and release water solutions in a reversible manner, in response to specific environmental stimuli. Such stimuli-sensitive behaviour makes hydrogels appealing for the design of ‘smart’ devices, applicable in a variety of technological fields. In particular, in cases where either ecological or biocompatibility issues are concerned, the biodegradability of the hydrogel network, together with the control of the degradation rate, may provide additional value to the developed device. This review surveys the design and the applications of cellulose-based hydrogels, which are extensively investigated due to the large availability of cellulose in nature, the intrinsic degradability of cellulose and the smart behaviour displayed by some cellulose derivatives.
Bacterial cellulose (BC)/Chitosan (Ch) composite has been successfully prepared by immersing wet BC pellicle in Ch solution followed by freeze-drying process. The morphology of BC/Ch composite was examined by scanning electron microscope (SEM) and compared with pristine BC. SEM images show that Ch molecules can penetrate into BC forming three-dimensional multilayered scaffold. The scaffold has very well interconnected porous network structure and large aspect surface. The composite was also characterized by Fourier transform infrared spectrum, X-ray diffraction, thermogravimetric analysis and tensile test. By incorporation of Ch into BC, crystallinity tends to decrease from 82% to 61%, and the thermal stability increases from 263 °C to 296 °C. At the same time, the mechanical properties of BC/Ch composite are maintained at certain levels between BC and Ch. The biocompatibility of composite was preliminarily evaluated by cell adhesion studies. The cells incubated with BC/Ch scaffolds for 48 h were capable of forming cell adhesion and proliferation. It showed much better biocompatibility than pure BC. Since the prepared BC/Ch scaffolds are bioactive and suitable for cell adhesion, these scaffolds can be used for wound dressing or tissue-engineering scaffolds.
Abstract The contribution of chitosan as a scaffold material is quite significant in the field of tissue engineering, which is a multidisciplinary field of research and technology development requiring the involvement of chemists, physicists, chemical engineers, biologists, cell-biologists etc. to regenerate injured or damaged tissue. The advantages of using chitosan as a three-dimensional scaffold for tissue engineering applications are due to its versatile physicochemical and biological properties. Further, owing to its easy processability, it can be molded into the desired shape and size. Therefore, it is no exaggeration to say that chitosan is a promising biomaterial for tissue engineering scaffolds. There is an enormous body of work already published in various journals on chitosan as a tissue engineering scaffold but, to our knowledge, this work has not yet been brought together in one chapter. We have used our best efforts to accumulate the research work already done on chitosan in a single place so that chitosan researchers can easily find information and can therefore escalate their research activities. This chapter highlights different methods for the fabrication of scaffolds, the suitability of chitosan as a good scaffolding material, and its application as a scaffold for tissue engineering of bone, cartilage, skin, liver, corneal, vascular, nerve, and cardiac tissue. Graphical Abstract
Arthritis is the leading cause of disability in the United States, potentially limiting affected persons from walking a few blocks or climbing a flight of stairs. Using Medical Expenditure Panel Survey (MEPS) data, CDC analyzed national and state-specific direct costs (i.e., medical expenditures) and indirect costs (i.e., lost earnings) attributable to arthritis and other rheumatic conditions (AORC) in the United States during 2003. This report describes the results of that analysis, which indicated that, in 2003, the total cost of AORC in the United States was approximately 128 billion dollars (80.8 billion dollars in direct and 47.0 billion dollars in indirect costs), equivalent to 1.2% of the 2003 U.S. gross domestic product. Total costs attributable to AORC, by state/area, ranged from 225.5 million dollars in the District of Columbia to 12.1 billion dollars in California. Total costs attributable to AORC have increased substantially since 1997, and that increase is expected to continue because of the aging of the population and increases in obesity and physical inactivity. These findings signal the need for broader implementation of effective public health interventions, such as arthritis and chronic disease self-management programs, which can reduce medical expenditures among persons with AORC.
This paper reports bacterial cellulose composites made by blending chitosan, poly(ethylene glycol) (PEG), and gelatin for potential biomedical application of tissue- engineering scaffold and wound-dressing material. The bacterial cellulose composites were successfully prepared by immersing a wet bacterial cellulose pellicle into chitosan, PEG, or gelatin solutions followed by freeze-drying. The products look like a foam structure. Scanning electron microscopy images show that chitosan molecules penetrated into bacterial cellulose forming a multilayer and a well interconnected porous network structure with a large aspect surface. The morphology of the bacterial cellulose/gelatin scaffold indicates that the gelatin molecules could penetrate well between the individual nanofibers of the bacterial cellulose. Cell adhesion studies for these composites were carried out using 3T3 fibroblast cells. They showed much better biocompatibility than pure bacterial cellulose. Preparation and material characterization of these composites are explained.
Small size and heterogeneity of the pores in bacterial nanocellulose (BNC) hydrogels limit the ingrowth of cells and their use as tissue-engineered implant materials. Usage of placeholders during BNC biosynthesis or post-processing steps such as (touch-free) laser perforation can overcome this limitation. Since three-dimensionally (3D) arranged channels may be required for homogeneous and functional seeding, 3D laser perforation of never-dried BNC hydrogels was performed. Never-dried BNC hydrogels were produced in different shapes by: i) cultivation of Gluconacetobacter xylinus (DSM 14666; synonym Komagataeibacter xylinus) in nutrient medium; ii) removal of bacterial residues/media components (0.1 M NaOH; 30min; 100°C) and repeated washing (deionized water; pH 5.8); iii) unidirectional or 3D laser perforation and cutting (pulsed CO2 Rofin SCx10 laser; 220 μm channel diameter); and iv) final autoclaving (2 M NaOH; 121°C; 20 min) and washing (pyrogen-free water). In comparison to unmodified BNC, unidirectionally perforated - and particularly 3D-perforated BNC - allowed ingrowth into and movement of vital bovine/human chondrocytes throughout the BNC nanofiber network. Laser perforation caused limited structural modifications (i.e., fiber or globular aggregates), but no chemical modifications, as indicated by FT-IR, XPS, and viability tests. Pre-cultured human chondrocytes seeding surface/channels of laser-perforated BNC expressed cartilage-specific matrix products, indicating chondrocyte differentiation. 3D-perforated BNC showed compressive strength comparable to that of unmodified samples. Unidirectionally or 3D-perforated BNC shows high biocompatibility and provides short diffusion distances for nutrients and extracellular matrix components. Also, the resulting channels support migration into the BNC, matrix production, and phenotypic stabilization of chondrocytes. It may thus be suitable for in vivo application, e.g., as a cartilage replacement material.
Temperature-responsive polymers are attractive candidates for applications related to injectable delivery of biologically active therapeutics. In this study, a novel approach to provide thermally sensitive neutral solutions based on chitosan/carboxymethyl cellulose (Ch/CMC) combinations is described. The potential of thermosensitive Ch/CMC as a biomaterial is evaluated for the culture of chondrocytes, with the goal of using the Ch/CMC polymer as an injectable matrix/cell therapeutic. These formulations possess a physiological pH and can be held as liquid below room temperature for encapsulating living cells. At body temperature they form monolithic gels, thus injected in vivo, the liquid formulations turn into gel implants in situ. The sols/gels have been thoroughly characterized for rheology and morphology analysis. The use of Ch/CMC aqueous solutions as gelling systems is a new discovery of a new family of thermosetting gels highly compatible with biological compounds.
New composite hydrogels based on cellulose and poly(acrylamide) have been synthesized via radical polymerization of acrylamide in cellulose swollen in a reaction solution. In this study, both a plant form of cellulose and a bacterial form—that cultivated by Acetobacter xylinum bacteria—were used. The behavior of synthesized hydrogels during swelling in water, as well as the behavior of the samples swollen at equilibrium
during deformation under uniaxial compression under various test conditions, have been studied. A comparative analysis of the main mechanical characteristics of hydrogels and the appropriate data for various types of articular cartilage, one of which—rabbit knee meniscus—has been tested in this study, has been performed. An average strength hydrogel is very close to articular cartilage in all mechanical characteristics.
The degrees of loading at the highest compression deformations observed during the function of joint cartilage (30–50%) is in the range 4–12 MPa for this hydrogel, and the average values of the compression modulus in the deformation ranges of 10–15 and 25–30% are 8.8 and 23.7 MPa, respectively. The behavior of hydrogels and rabbit meniscus under cyclic compression with the amplitude of 50% has been studied. Hydrogels and meniscus under this test conditions demonstrate clear viscoelastic behavior, evidenced by noticeable hysteresis for the first cycle and a decrease in the value of the maximum load with an increase in the number of cycles. Structural features of hydrogels, which can affect the behavior of the hydrogels under study, have been
considered. On the whole, the results demonstrate the possibility of modeling the mechanical behavior of cartilage with the use of hydrogels of this type.
The culture of multipotent mesenchymal stem cells on natural biopolymers holds great promise for treatments of connective tissue disorders such as osteoarthritis. The safety and performance of such therapies relies on the systematic in vitro evaluation of the developed stem cell-biomaterial constructs prior to in vivo implantation. This study evaluates bacterial cellulose (BC), a biocompatible natural polymer, as a scaffold for equine-derived bone marrow mesenchymal stem cells (EqMSCs) for application in bone and cartilage tissue engineering. An equine model was chosen due to similarities in size, load and types of joint injuries suffered by horses and humans. Lyophilized and critical point dried BC hydrogel scaffolds were characterized using scanning electron microscopy (SEM) to confirm nanostructure morphology which demonstrated that critical point drying induces fibre bundling unlike lyophilisation. EqMSCs positively expressed the undifferentiated pluripotent mesenchymal stem cell surface markers CD44 and CD90. The BC scaffolds were shown to be cytocompatible, supporting cellular adhesion and proliferation, and allowed for osteogenic and chondrogenic differentiation of EqMSCs. The cells seeded on the BC hydrogel were shown to be viable and metabolically active. These findings demonstrate that the combination of a BC hydrogel and EqMSCs are promising constructs for musculoskeletal tissue engineering applications.
A current focus of tissue engineering is the use of adult human mesenchymal stem cells (hMSCs) as an alternative to autologous chondrocytes for cartilage repair. Several natural and synthetic polymers (including cellulose) have been explored as a biomaterial scaffold for cartilage tissue engineering. While bacterial cellulose (BC) has been used in tissue engineering, its lack of degradability in vivo and high crystallinity restricts widespread applications in the field. Recently we reported the formation of a novel bacterial cellulose that is lysozyme-susceptible and -degradable in vivo from metabolically engineered Gluconacetobacter xylinus. Here we report the use of this modified bacterial cellulose (MBC) for cartilage tissue engineering using hMSCs. MBC's glucosaminoglycan-like chemistry, combined with in vivo degradability, suggested opportunities to exploit this novel polymer in cartilage tissue engineering. We have observed that, like BC, MBC scaffolds support cell attachment and proliferation. Chondrogenesis of hMSCs in the MBC scaffolds was demonstrated by real-time RT-PCR analysis for cartilage-specific extracellular matrix (ECM) markers (collagen type II, aggrecan and SOX9) as well as histological and immunohistochemical evaluations of cartilage-specific ECM markers. Further, the attachment, proliferation, and differentiation of hMSCs in MBC showed unique characteristics. For example, after 4 weeks of cultivation, the spatial cell arrangement and collagen type-II and ACAN distribution resembled those in native articular cartilage tissue, suggesting promise for these novel in vivo degradable scaffolds for chondrogenesis. Copyright © 2013 John Wiley & Sons, Ltd.
Bacterial cellulose (BC) is a natural and biocompatible material with unique properties, such as high water holding capacity, ultra-fine fibre network and high strength that makes it an attractive material for the repair of articular cartilage lesions. However, data on the tribological properties of BC is very scarce, particularly if natural articular cartilage is involved in the contact. In this work, unmodified BC pellicles were grown from Gluconacetobacter xylinus in order to be used as tribological samples against bovine articular cartilage (BAC) in the presence of phosphate buffered saline (PBS). The tribological assessment of the sliding pairs was accomplished using reciprocating pin-on-flat tests at 37ºC. The reciprocating sliding frequency and stroke length were kept constant at 1 Hz and 8 mm, respectively. Contact pressures ranging from 0.80 to 2.40 MPa were applied. The friction coefficient evolution was continuously monitored during the tests and the release of total carbohydrates into the lubricating solution was followed by means of the phenol-H2SO4 method as an attempt to evaluate wear losses. The morphology of worn surfaces was characterized by SEM/EDS and the main wear mechanisms were identified. Low friction coefficient values (~ 0.05) combined with the preservation of the mating surfaces (BC and BAC) indicate the potential of BC to be used as artificial cartilage for articular joints.
The objective of this chapter is to address timely work in the area of cartilage tissue engineering, with a specific focus
on the regeneration of articular cartilage. Many approaches have been used for the engineering of articular cartilage with
a wide range of materials formed into meshes, sponges, and hydrogels and with a range of cell sources including chondrocytes,
fibroblasts, and stem cells. At present, the current state of the art is the use of mesenchymal stem cells encapsulated in
synthetic hydrogels; an approach which overcomes many of the limitations present in previous material formulations and in
the definition of a clinically relevant and widely available acceptable cell source for tissue repair. Considerable advances
have been made in these areas to generate cartilage constructs with near native properties and histological features. With
the addition of various stimulatory cues (molecules, materials, mechanical loading), we are now poised to develop functional
cartilage replacement tissues.
A family of polysaccharide based scaffold materials, bacterial cellulose/chitosan (BC/CTS) porous scaffolds with various weight
ratios (from 20/80 to 60/40w/w%) were prepared by freezing (−30 and −80°C) and lyophilization of a mixture of microfibrillated
BC suspension and chitosan solution. The microfibrillated BC (MFC) was subjected to 2,2,6,6-tetramethylpyperidine-1-oxyl radical
(TEMPO)-mediated oxidation to introduce surface carboxyl groups before mixing. The integration of MFC within chitosan matrix
was performed by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)-mediated cross-linking. The covalent amide
bond formation was determined by ATR-FTIR. Because of this covalent coupling, the scaffolds retain their original shapes during
autoclave sterilization. The composite scaffolds are three-dimensional open pore microstructure with pore size ranging from
120 to 280μm. The freezing temperature and mean pore size take less effect on scaffold mechanical properties. The compressive
modulus and strength increased with increase in MFC content. The results show that the scaffolds of higher MFC content contribute
to overall better mechanical properties.
KeywordsBacterial cellulose-Chitosan-TEMPO-EDC-Porous scaffolds-Compressive mechanical property
The microfibrillar nature of bacterial cellulose produced by Acetobacter was modified by various chemical reagents in a culture medium. The chemical reagents included antibiotics to inhibit cell division or certain protein synthesis, and reducing reagents that induce reductive cleavage of disulfide bonds in proteins. Among the reagents tested, nalidixic acid and chloramphenicol induced elongation of bacteria, resulting in the formation of wider cellulose ribbons or aggregates of ribbons. The Young's modulus of the sheets made from such cellulose increased, while dithiothreitol, which produced ribbons having only 45% of the width of the control, produced sheets with undiminished Young's modulus. Although further study is necessary to clarify the effect of such modifications, nalidixic acid and chloramphenicol produced a bacterial cellulose with superior mechanical properties.
We fabricated a three-dimensional nanostructured macroporous bacterial cellulose scaffold (3D BC scaffold) and a three-dimensional
nanostructured macroporous bacterial cellulose/agarose scaffold (3D BC/A). Results of scanning electron microscopy (SEM) and
mercury intrusion porosimeter showed that both the 3D BC and the 3D BC/A have interconnected macropores characterized by nanofibrous
pore walls (The diameter of the dominant pores was about 100μm and ranges from <1μm to >1,000μm). 3D BC/A also has high
surface area (80±5m2/g) and sufficient porosity (88.5±0.4%) compare with 3D BC (surface area: 92.81±2.02m2/g; porosity 90.42±0.24%). 3D BC/A do support C5.18 cell and hBMSC attachment, proliferation evaluated with SEM, confocal
microscopy and cell proliferation assay. Furthermore, 3D/ABC has enhanced mechanical property (ultimate compressive strength:
26.26±4.6kPa, Young’s modulus: 39.26±5.72kPa)) than that 3D/BC has (ultimate compressive strength: 7.04±2.34kPa,
Young’s modulus: 10.76±3.52kPa). Overall, the 3D BC/A scaffold had more potential than 3D BC scaffold for using as a scaffold
for tissue engineering and tissue repair applications.
KeywordsAdult human bone marrow derived stromal cells (hBMSCs)–Bacterial cellulose–Chondroblasts–Macropore structure–Nano-biomaterials–Tissue engineering
The response of mesenchymal stem cells (MSCs) to a matrix largely depends on the composition as well as the extrinsic mechanical and morphological properties of the substrate to which they adhere to. Collagen-glycosaminoglycan (CG) scaffolds have been extensively used in a range of tissue engineering applications with great success. This is due in part to the presence of the glycosaminoglycans (GAGs) in complementing the biofunctionality of collagen. In this context, the overall goal of this study was to investigate the effect of two GAG types: chondroitin sulphate (CS) and hyaluronic acid (HyA) on the mechanical and morphological characteristics of collagen-based scaffolds and subsequently on the differentiation of rat MSCs in vitro. Morphological characterisation revealed that the incorporation of HyA resulted in a significant reduction in scaffold mean pore size (93.9 μm) relative to collagen-CS (CCS) scaffolds (136.2 μm). In addition, the collagen-HyA (CHyA) scaffolds exhibited greater levels of MSC infiltration in comparison to the CCS scaffolds. Moreover, these CHyA scaffolds showed significant acceleration of early stage gene expression of SOX-9 (approximately 60-fold higher, p<0.01) and collagen type II (approximately 35-fold higher, p<0.01) as well as cartilage matrix production (7-fold higher sGAG content) in comparison to CCS scaffolds by day 14. Combining their ability to stimulate MSC migration and chondrogenesis in vitro, these CHyA scaffolds show great potential as appropriate matrices for promoting cartilage tissue repair.
Bacterial cellulose (BC) is suitable for applications as scaffolds in tissue engineering due to its unique properties. However, BC is not enzymatically degradable in vivo and this has become an essential limiting factor in its potential applications. In this work, BC was modified by periodate oxidation to give rise to a biodegradable 2,3-dialdehyde bacterial cellulose (DABC). After characterization by scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), attenuated total reflectance–Fourier transform infrared (ATR–FTIR) spectroscopy, thin-film X-ray diffractometry (XRD) and X-ray photoelectron spectroscopy (XPS), we demonstrated that the modified DABC nano-network was able to degrade into porous scaffold with micro-sized pores in water, phosphate buffered saline (PBS) and the simulated body fluid (SBF). The degradation process began from the oxidized amorphous part of the network and concurrently hydroxyapatite formed on the scaffold surface during the process in SBF. Our data also demonstrated that the tensile mechanical properties of the DABC nano-network were suitable for its use in tissue engineering scaffolds.
Composite gels, with cellulose nanowhiskers as the reinforcing phase and regenerated cellulose as the matrix, were firstly developed by a rapid thermal-induced phase separation followed by a regenerating process. The gelation behavior, regeneration kinetics, morphology and properties of the all-cellulose composite gel system were investigated by advanced rheometer, potentiometric titration, scanning electron microscopy, Fourier-transform infrared spectroscopy, texture analyzer and swelling testing in the presence of different amount of cellulose nanowhiskers. The results revealed that cellulose nanowhiskers could act as the “bridge” to facilitate the crosslinking of cellulose chains during gel formation process. Moreover, they can significantly improve the dimensional stability and mechanical strength of the regenerated gels. In vitro experiment demonstrated that these gels, featuring membrane–matrix hybrid structures, could be used for controlled release of macromolecules in the simulated body fluid.
Bacterial cellulose (BC) is a unique and promising material for use as implants and scaffolds in tissue engineering. It is composed of a pure cellulose nanofiber mesh spun by bacteria. It is remarkable for its strength and its ability to be engineered structurally and chemically at nano-, micro-, and macroscales. Its high water content and purity make the material biocompatible for multiple medical applications. Its biocompatibility, mechanical strength, chemical and morphologic controllability make it a natural choice for use in the body in biomedical devices with broader application than has yet been utilized. This paper reviews the current state of understanding of bacterial cellulose, known methods for controlling its physical and chemical structure (e.g., porosity, fiber alignment, etc.), biomedical applications for which it is currently being used, or investigated for use, challenges yet to be overcome, and future possibilities for BC.
Recent advances in tissue engineering and regenerative medicine fields can offer alternative solutions to the existing techniques for cartilage repair. In this context, a variety of materials has been proposed, and the injectable hydrogels are among the most promising alternatives. The aim of this work is to explore the ability of poly(N-isopropylacrylamide)-g-methylcellulose (PNIPAAm-g-MC) thermoreversible hydrogel as a three-dimensional support for cell encapsulation toward the regeneration of articular cartilage through a tissue engineering approach. The PNIPAAm-g-MC copolymer was effectively obtained using ammonium-persulfate and N,N,N',N'-tetramethylethylenediamine as initiator as confirmed by Fourier transform infrared spectroscopy and (1) H NMR results. The copolymer showed to be temperature responsive, becoming a gel at temperatures above its lower critical solution temperature (~ 32 °C) while turning into a liquid below it. Results obtained from the MTS test showed that extracts of the hydrogel were clearly noncytotoxic to L929 fibroblast cells. ATDC5 cells, a murine chondrogenic cell line, were used as the in vitro model for this study; they were encapsulated at high cell density within the hydrogel and cultured for up to 28 days. PNIPAAm-g-MC did not affect the cell viability and proliferation, as indicated by both MTS and DNA assays. The results also revealed an increase in synthesis of glycosoaminoglycans within culture time measured by the dimethylmethylene blue quantification assay. These results suggest the viability of using PNIPAAm-g-MC thermoresponsive hydrogel as a three-dimensional scaffold for cartilage tissue engineering using minimal-invasive strategies.
Articular cartilage can tolerate a tremendous amount of intensive and repetitive physical stress. However, it manifests a striking inability to heal even the most minor injury. Both the remarkable functional characteristics and the healing limitations reflect the intricacies of its structure and biology. Cartilage is composed of chondrocytes embedded within an extracellular matrix of collagens, proteoglycans, and noncollagenous proteins. Together, these substances maintain the proper amount of water within the matrix, which confers its unique mechanical properties. The structure and composition of articular cartilage varies three-dimensionally, according to its distance from the surface and in relation to the distance from the cells. The stringent structural and biological requirements imply that any tissue capable of successful repair or replacement of damaged articular cartilage should be similarly constituted. The response of cartilage to injury differs from that of other tissues because of its avascularity, the immobility of chondrocytes, and the limited ability of mature chondrocytes to proliferate and alter their synthetic patterns. Therapeutic efforts have focused on bringing in new cells capable of chondrogenesis, and facilitating access to the vascular system. This review presents the basic science background and clinical experience with many of these methods and information on synthetic implants and biological adhesives. Although there are many exciting avenues of study that warrant enthusiasm, many questions remain. These issues need to be addressed by careful basic science investigations and both short- and long-term clinical trials using controlled, prospective, randomized study design.
Cellulose fibrils with widths in the nanometer range are nature-based materials with unique and potentially useful features. Most importantly, these novel nanocelluloses open up the strongly expanding fields of sustainable materials and nanocomposites, as well as medical and life-science devices, to the natural polymer cellulose. The nanodimensions of the structural elements result in a high surface area and hence the powerful interaction of these celluloses with surrounding species, such as water, organic and polymeric compounds, nanoparticles, and living cells. This Review assembles the current knowledge on the isolation of microfibrillated cellulose from wood and its application in nanocomposites; the preparation of nanocrystalline cellulose and its use as a reinforcing agent; and the biofabrication of bacterial nanocellulose, as well as its evaluation as a biomaterial for medical implants.
The repair of articular cartilage defects remains a significant challenge in orthopedic medicine. Hydrogels, three-dimensional polymer networks swollen in water, offer a unique opportunity to generate a functional cartilage substitute. Hydrogels can exhibit similar mechanical, swelling, and lubricating behavior to articular cartilage, and promote the chondrogenic phenotype by encapsulated cells. Hydrogels have been prepared from naturally derived and synthetic polymers, as cell-free implants and as tissue engineering scaffolds, and with controlled degradation profiles and release of stimulatory growth factors. Using hydrogels, cartilage tissue has been engineered in vitro that has similar mechanical properties to native cartilage. This review summarizes the advancements that have been made in determining the potential of hydrogels to replace damaged cartilage or support new tissue formation as a function of specific design parameters, such as the type of polymer, degradation profile, mechanical properties and loading regimen, source of cells, cell-seeding density, controlled release of growth factors, and strategies to cause integration with surrounding tissue. Some key challenges for clinical translation remain, including limited information on the mechanical properties of hydrogel implants or engineered tissue that are necessary to restore joint function, and the lack of emphasis on the ability of an implant to integrate in a stable way with the surrounding tissue. Future studies should address the factors that affect these issues, while using clinically relevant cell sources and rigorous models of repair.
Tissue engineering is an emerging field of regenerative medicine that holds promise for the restoration of tissues and organs affected by chronic diseases, age-linked degeneration, congenital deformity, and trauma. Tissue engineering consists of building tissue and organs using cells grown on natural or artificial biomaterials outside the body. Recent efforts in bone and cartilage tissue regeneration have turned to tissue engineering, which have shown the proof of concept in clinical situations. Articular cartilage is composed of 70–80% of water retained in the form of a stable macromolecular gels. The extracellular matrix (ECM) and chondrocytes represent 20–30% of the articular cartilage. The lack of vascularization of the articular cartilage, however, prevents the development of an inflammatory response; this severely limits spontaneous repair. Currently, research is being directed to cell therapy associated with specific scaffold-like hydrogels. Articular cartilage, in particular, is considered to be a good candidate for tissue engineering, because it requires less metabolic involvement due to lower cellularity and avascular matrix. Cartilage organization and pathology have been highlighted here with respect to scaffold strategies using synthetic hydrogels as biomimetic extracellular matrices for tissue engineering.
In tissue engineering applications or even in 3D cell cultures, the biological cross talk between cells and the scaffold is controlled by the material properties and scaffold characteristics. In order to induce cell adhesion, proliferation, and activation, materials used for the fabrication of scaffolds must possess requirements such as intrinsic biocompatibility and proper chemistry to induce molecular biorecognition from cells. Materials, scaffold mechanical properties and degradation kinetics should be adapted to the specific tissue engineering application to guarantee the required mechanical functions and to accomplish the rate of the new-tissue formation. For scaffolds, pore distribution, exposed surface area, and porosity play a major role, whose amount and distribution influence the penetration and the rate of penetration of cells within the scaffold volume, the architecture of the produced extracellular matrix, and for tissue engineering applications, the final effectiveness of the regenerative process. Depending on the fabrication process, scaffolds with different architecture can be obtained, with random or tailored pore distribution. In the recent years, rapid prototyping computer-controlled techniques have been applied to the fabrication of scaffolds with ordered geometry. This chapter reviews the principal polymeric materials that are used for the fabrication of scaffolds and the scaffold fabrication processes, with examples of properties and selected applications.
Electrospun poly(lactic acid) (PLLA) nanofibers (NF) were modified with cationized gelatin (CG) to improve their compatibility with chondrocytes and to show in vitro and in vivo the potential applications of CG-grafted PLLA nanofibrous membranes (CG-PLLA NFM) as a cartilage tissue engineering scaffold. PLLA NF were first treated with oxygen plasma to introduce -COOH groups on the surface, followed by covalent grafting of CG molecules onto the fiber surface, using water-soluble carbodiimide as the coupling agent. The effects of CG grafting and properties of NFM were characterized by scanning electron microscopy (SEM), transmission electron microscopy, thermogravimetric analysis, atomic force microscope, X-ray photoelectron spectra and Fourier transform infrared spectroscopy. In vitro studies indicated that CG-PLLA NFM could enhance viability, proliferation and differentiation of rabbit articular chondrocytes compared with pristine PLLA NFM. SEM observations of the cell-scaffold construct confirmed the tight attachment of chondrocytes to CG-PLLA NF and in-growth of cells into the interior of the membrane with proper maintenance of cell morphology. Improved cell differentiation in CG-PLLA NFM was confirmed by enhanced glycoaminoglycan and collagen secretion, histological analysis and reverse transcription-polymerase chain reaction studies, which showed that the cells were able to maintain the expression of characteristic markers (collagen II, aggregan and SOX 9) of chondrocytes. Subcutaneous implantation of the cell-scaffold constructs with autologous chondrocytes also confirmed the formation of ectopic cartilage tissues after 28 days by histological examination and immunostaining.
Regeneration of articular cartilage damage is an area of great interest due to the limited ability of cartilage to self-repair. The latest cartilage repair strategies are dependent on access to biomaterials to which chondrocytes can attach and in which they can migrate and proliferate, producing their own extracellular matrix. In the present study, engineered porous bacterial cellulose (BC) scaffolds were prepared by fermentation of Acetobacter xylinum (A. xylinum) in the presence of slightly fused wax particles with a diameter of 150-300 microm, which were then removed by extrusion. This porous material was evaluated as a scaffold for cartilage regeneration. Articular chondrocytes from young adult patients as well as neonatal articular chondrocytes were seeded with various seeding techniques onto the porous BC scaffolds. Scanning electron microscopy (SEM) analysis and confocal microscopy analysis showed that cells entered the pores of the scaffolds and that they increasingly filled out the pores over time. Furthermore, DNA analysis implied that the chondrocytes proliferated within the porous BC. Alcian blue van Gieson staining revealed glycosaminoglycan (GAG) production by chondrocytes in areas where cells were clustered together. With some further development, this novel biomaterial can be a suitable candidate for cartilage regeneration applications.
Silated hydroxypropylmethylcellulose (Si-HPMC) is a modified biopolymer used in biomaterial's domain. A hydroxypropylmethylcellulose (HPMC) was functionalised with silane groups. The macromolecular solution of Si-HPMC (pH 12.9) generates an elastic state at physiological pH (~7.4). In this work we present the gel formation of Si-HPMC resulting from the condensation of silanol groups. The crosslinked hydrogel was characterized using rheological techniques. With the scalar percolation model the gelation time was determined as a function of Si-HPMC concentration at 37 °C.
In this study, genipin-cross-linked collagen/chitosan biodegradable porous scaffolds were prepared for articular cartilage regeneration. The influence of chitosan amount and genipin concentration on the scaffolds physicochemical properties was evaluated. The morphologies of the scaffolds were characterized by scanning electron microscope (SEM) and cross-linking degree was investigated by ninhydrin assay. Additionally, the mechanical properties of the scaffolds were assessed under dynamic compression. To study the swelling ratio and the biostability of the collagen/chitosan scaffold, in vitro tests were also carried out by immersion of the scaffolds in PBS solution or digestion in collagenase, respectively. The results showed that the morphologies of the scaffolds underwent a fiber-like to a sheet-like structural transition by increasing chitosan amount. Genipin cross-linking remarkably changed the morphologies and pore sizes of the scaffolds when chitosan amount was less than 25%. Either by increasing the chitosan ratio or performing cross-linking treatment, the swelling ratio of the scaffolds can be tailored. The ninhydrin assay demonstrated that the addition of chitosan could obviously increase the cross-linking efficiency. The degradation studies indicated that genipin cross-linking can effectively enhance the biostability of the scaffolds. The biocompatibility of the scaffolds was evaluated by culturing rabbit chondrocytes in vitro. This study demonstrated that a good viability of the chondrocytes seeded on the scaffold was achieved. The SEM analysis has revealed that the chondrocytes adhered well to the surface of the scaffolds and contacted each other. These results suggest that the genipin-cross-linked collagen/chitosan matrix may be a promising formulation for articular cartilage scaffolding.
Physiological tissues, including brain and other organs, have three-dimensional (3-D) aspects that need to be supported to model them in vitro. Here we report the use of cellulose microfibers combined with cross-linked gelatin to make biocompatible porous microscaffolds for the sustained growth of brain cell and human mesenchymal stem cells (hMSCs) in 3-D structure. Live imaging using confocal microscopy indicated that 3-D microscaffolds composed of gelatin or cellulose fiber/gelatin both supported brain cell adhesion and growth for 16days in vitro. Cellulose microfiber/gelatin composites containing up to 75% cellulose fibers can withstand a higher mechanical load than gelatin alone, and composites also provided linear pathways along which brain cells could grow compared to more clumped cell growth in gelatin alone. Therefore, the bulk cellulose microfiber provides a novel skeleton in this new scaffold material. Cellulose fiber/gelatin scaffold supported hMSCs growth and extracellular matrix formation. hMSCs osteogenic and adipogenic assays indicated that hMSCs cultured in cellulose fiber/gelatin composite preserved the multilineage differentiation potential. As natural, biocompatible components, the combination of gelatin and cellulose microfibers, fabricated into 3-D matrices, may therefore provide optimal porosity and tensile strength for long-term maintenance and observation of cells.
Bacterial cellulose-polyacrylamide (BC-PAAm) composite hydrogels are prepared by synthesis of PAAm networks inside the BC matrices. The behavior of these gels and of the ionic ones obtained via partial hydrolysis of BC-PAAm gels is studied under swelling and compressive deformation conditions. The dependences of the hydrogels' properties on the BC matrix preparation conditions, gel synthesis conditions and the BC content in the hydrogel compositions are studied. Two types of BC gel pellicle are used in the hydrogel synthesis, namely matrix pellicles subjected to pre-pressing (samples of series A) and those not subjected to any mechanical actions before synthesis (series B samples) containing about 99% water. The effect of anisotropic swelling of type A hydrogels is detected. The type B specimens swell isotropically. Both types of hydrogel exhibit substantial anisotropy of their mechanical properties, apparent in different shapes of compression stress-strain curves of samples cut out from the gel plates in various directions. Composite hydrogels show superb mechanical properties, including compression strength up to 10 MPa and the ability to withstand long-term cyclic stresses (up to 2000-6000 cycles) without substantial reduction of mechanical properties.
Hyaluronic acid (HA) was added into fibroin solution to prepare fibroin-based porous composite scaffolds. HA exhibited important effects on pore formation and hydrophilicity of fibroin-based scaffold. The aqueous-fibroin/HA scaffolds had highly homogeneous and interconnected pores with porosity of above 90% and controllable pore size ranging from 123 to 253 microm. The water take-up of fibroin/HA scaffolds increased significantly with the increase of HA content. Containing HA at a defined content range, such as 3-6%, fibroin-based scaffolds' affinity to primary neural cells was improved. In 6%HA/fibroin scaffolds, neurosphere-forming cell migrated from their original aggregate and adhered tightly to the surface of scaffolds.