Development and characterization of carbohydrate-based thermosensitive hydrogels for cartilage tissue engineering
Graphical abstract
Introduction
Rheumatic or musculoskeletal pathologies comprise more than 150 diseases and syndromes. These pathologies are the main causes of morbidity and disability, generating huge costs for the health sector and social assistance systems [1], [2]. Among these, osteoarthritis is the most common disease in adults. With the disease progression, total loss of the joint cartilage may occur, leaving the bone surface exposed to direct friction, which leads to pain and stiffness [3], [4], [5].
The currently available treatments for osteoarthritis are surgical procedures that aim the repair of damaged cartilage, but such an approach is generally not fully effective. Consequently, there is a need to develop biomaterials capable to replace the damaged tissue or stimulate its recovery [6]. These biomaterials can be used together with cells or active compounds, such as drugs and growth factors, for the treatment of the injured site, restoring tissue functions [7], [8]. In this sense, formulations capable of being directly injected into the lesion site to fill the tissue defect with minimally invasive procedures associated with low patient discomfort levels are of particular interest [9], mostly those able to also perform the role as scaffolds for cells administered to promote wound healing.
For this purpose, it is crucial to choose biomaterials capable of showing a plethora of properties, e.g. biocompatibility, capacity of effectively incorporating and releasing bioactive agents and cells at appropriate rates [9], together with thermosensitivity, which would allow the biomaterial gelation in situ under mild physiological conditions [10]. Among the several biomaterials available, hydrogels have become increasingly attractive to fulfill this task in the cartilage tissue engineering area [6], [11].
These hydrogels can be formulated with many types of polymers, being the natural ones appealing sources due to their biochemical similarities to cartilage tissue. Furthermore, they can be degraded by enzymes secreted by chondrocytes [6], [11], which allows their substitution by regular tissue as the lesioned area recovers. Examples of polymers with good potential in cartilage tissue engineering include methylcellulose, xanthan gum, and carboxymethyl chitosan.
Methylcellulose (M), a water-soluble form of cellulose, is obtained by substituting hydrophilic hydroxyl groups of cellulose for hydrophobic methoxy groups. It has been used in many industrial applications, e.g. as a thickening agent, to stabilize solutions and for the production of biomaterials for controlled drug delivery [12], [13], [14].
The thermal behavior of this polymer in aqueous solutions is unique. When the temperature is increased to a critical point (around 29 °C), the viscosity of the solution increases rapidly and a thermoreversible gel is obtained [14], [15]. At temperatures significantly higher, above 60 °C, turbidity increases and phase separation can be observed [14]. This behavior is associated to changes both in the predominant types of interactions occurring in the system and the chemical groups involved in them. At temperatures below the critical point, the hydrogen bonds initially formed between the hydrophilic groups of the polymer and the water improves methylcellulose solubility. As the temperature is increased, competition between polymer–polymer direct hydrogen bonding and polymer–water hydrogen bonding leads to the formation of a gel, as the polymer loses part of its hydration water. At even higher temperatures, the hydrophobic interactions between highly methylated zones of the polymer are further strengthened. Gelation of methylcellulose can be affected by a number of factors, such as the polymer substitution pattern and molar weight, the presence of other components in the aqueous solution, e.g. salts, sugars, alcohols, surfactants and other types of polymers [15].
Xanthan gum (X) also presents relevant rheological properties. At low concentrations (e.g. 1% w/v), aqueous solutions of this polysaccharide have high viscosity, which helps to protect the joints in animal models with induced osteoarthritis [16], [17], [18], [19]. In addition, it is stable at room temperature, shows pseudoplastic behavior and emulsion-stabilizing activity, being approved as a food additive by the Food and Drug Administration (FDA, USA) [17], [20].
Carboxymethyl chitosan (C), a water-soluble derivative of chitosan, has also been extensively explored. Its synthesis is a straight-forward and simple process, it is amphiphilic and widely applicable for pharmaceutical and clinical purposes [21]. Carboxymethyl chitosan presents characteristics such as biocompatibility, biodegradability, and bioadhesiveness, which makes it a good candidate for tissue engineering. In addition to these attractive properties, this compound has a structure similar to that of glycosaminoglycans found in cartilage, being then capable to mimick this tissue [22], [23].
Besides polymer type, the choice of drugs to be incorporated in injectable hydrogel formulations designed for cartilage tissue repair is of paramount importance. Bioactive agents such as gallic acid, dexamethasone, and diclofenac sodium are very attractive for this goal.
Gallic acid (GA) inhibits the expression of proinflammatory genes that cause osteoarthritis, thus exerting a protective action on this tissue [24], [25], [26]. In addition, it can interact with cartilage collagen through hydrophobic associations and hydrogen bonds, crosslinking and stabilizing the tissue matrix, which decreases the enzymatic degradation of collagen by metalloproteinases [26], [27].
Dexamethasone (DM) has a high affinity to intracellular glucocorticoid receptors, enhancing the transcription of anti-inflammatory cytokines and suppressing transcription of pro-inflammatory cytokines [28], [29]. It also potentializes chondrocyte differentiation and proteoglycan synthesis, in addition to reducing the loss of glycosaminoglycans in injured cartilages in vitro assays [30], [31]. Diclofenac sodium (DS), a nonsteroidal anti-inflammatory drug, acts directly inhibiting pro-inflammatory enzymes, decreasing pain and inflammation in the injured site [32]. DS is widely used in the treatment of osteoarthritis, rheumatoid arthritis and ankylosing spondylitis [33]. As DS and dexamethasone are rapidly absorbed, generally repeated oral administrations are needed, which may cause adverse gastrointestinal effects. To overcome this problem, these compounds could be administered by intra-articular injections [34] and the use of thermosensitive hydrogels as vehicles could contribute to prolonging their local action.
Therefore, three formulations of drug-loaded injectable thermosensitive hydrogels were investigated in the present work. The hydrogels were produced by mixing different proportions of methylcellulose, xanthan gum, and carboxymethyl chitosan, and gallic acid, dexamethasone, and diclofenac sodium were then incorporated to the hydrogels by direct addition. The interactions between the polysaccharides and the drugs, the hydrogels gelling temperature and mechanical properties, their morphology, swelling, degradation and drug release kinetics, as well as their in vitro cytotoxicity to mesenchymal stem cells were analyzed to determine which formulation could be considered the most promising for the treatment of cartilage lesions.
Section snippets
Materials
Xanthan gum from Xanthomonas campestris (product reference: G1253, Sigma-Aldrich), methylcellulose (Methocel A15, product reference: 295603, Dow chemical), carboxymethyl chitosan (product reference: 83512-85-0, Santa Cruz Biotechnology) and glycerol (product reference: G-7893, Sigma-Aldrich) were used for hydrogel production. All materials were used as received from the suppliers. Gallic acid, dexamethasone and diclofenac sodium were purchased from Sigma-Aldrich. Human dental pulp stem cells
Preparation and characterization of hydrogels
The gelling point is defined as the point at which the storage modulus (G’) becomes larger than the loss modulus (G”), in which solid-like behavior dominates the viscoelastic properties of the hydrogel formed[42]. The results attained are shown in Fig. 1.
It can be observed that G’ values are smaller than the corresponding G” values in the low-temperature range, whereas, in the high-temperature range, G’ values are larger than the respective G” values. This demonstrates that all tested hydrogels
Conclusions
Currently, the treatment for osteoarthritis is still a challenge, being in generally not entirely effective, which indicates the need for new approaches. In this work, thermosensitive formulations of hydrogels with good potential for osteoarthritis therapy were obtained, which can be exploited for minimally invasive applications in cartilage tissue engineering.
The hydrogels developed were able to control the drug release rate of dexamethasone and diclofenac sodium for up to 70 h, showed PBS and
Funding sources
The authors would like to acknowledge the support to this research by the National Council for Scientific and Technological Development (Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq, Brazil – Grants #307139/2015-8; 307829/2018-9 and 430860/2018-8), the Coordination for the Improvement of Higher Educational Personnel (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – CAPES, Brazil – Finance Code 001); the Fund for Support to Teaching, Research, and Extension
Data availability
The raw/processed data required to reproduce these findings cannot be shared at this time due to technical or time limitations.
CRediT authorship contribution statement
Cecília B. Westin: Conceptualization, Validation, Investigation, Writing - original draft, Writing - review & editing, Visualization. Mariana H.T. Nagahara: Validation, Investigation, Writing - review & editing. Monize C. Decarli: Validation, Investigation, Writing - review & editing. Daniel J. Kelly: Resources, Supervision, Writing - original draft. Ângela M. Moraes: Conceptualization, Resources, Writing - original draft, Writing - review & editing, Visualization, Supervision, Project
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
References (73)
- et al.
Cell-based tissue engineering strategies used in the clinical repair of articular cartilage
Biomaterials
(2016) - et al.
Mesenchymal stromal cells for cartilage repair in osteoarthritis
Osteoarthr. Cartil.
(2016) - et al.
Osteoarthritis
Best Pract. Res. Clin. Rheumatol.
(2008) - et al.
Osteoarthritis year in review: Mechanics – basic and clinical studies in osteoarthritis
Osteoarthr. Cartil.
(2014) - et al.
Thermosensitive Hydrogels as Scaffolds for Cartilage Tissue Engineering
Biomacromolecules
(2019) - et al.
Superabsorbent hydrogels based on cellulose for smart swelling and controllable delivery
Eur. Polym. J.
(2010) - et al.
Intra-articular injection of xanthan gum reduces pain and cartilage damage in a rat osteoarthritis model
Carbohydr. Polym.
(2013) - et al.
Preparation of xanthan gum injection and its protective effect on articular cartilage in the development of osteoarthritis
Carbohydr. Polym.
(2012) - et al.
The protective effect of xanthan gum on interleukin-1B induced rabbit chondrocytes
Carbohydr. Polym.
(2012) - et al.
Low molecular weight xanthan gum for treating osteoarthritis
Carbohydr. Polym.
(2017)