Gellan gum as a promising transplantation carrier for differentiated progenitor cells in ophthalmic therapies

Abstract

Stem cell-based therapies for various ocular conditions are increasingly gaining traction in ophthalmic treatments, with hydrogel-based polymers playing a pivotal role. Current stem cell delivery methods face challenges such as limited cell retention, immunological rejection, and uneven dispersion. Hence, there is a critical demand for innovative delivery systems to enhance the viability, localization, and integration of transplanted stem cells while minimizing adverse effects. Central to this advancement is the meticulous selection of appropriate materials. Among the promising options, gellan gum, a versatile polysaccharide, is emerging as a potential carrier for differentiated progenitor cells in regenerative medicine, particularly in ophthalmology. This study explores the utilization of gellan gum hydrogels as carriers, focusing on their biocompatibility, customizable gelation properties, and ability to encapsulate, transplant, and biofunctionalize cells. Through a review of literature, the impact of gellan gum hydrogels on cell viability parameters is investigated, revealing their potential for promoting tissue regeneration and functional recovery in ocular diseases. Furthermore, this study compares gellan gum systems utilizing natural and synthetic polymers, discerning differences in efficacy, biocompatibility, and suitability for diverse applications in regenerative ophthalmology. This review highlights the promising role of gellan gum in ophthalmic therapies, providing valuable insights into future directions and hurdles in this evolving field.

Keywords

Stem cell-based therapy ophthalmic therapies tissue regeneration gellan gum hydrogels

Introduction

The intricacy of the eye and its fragile structures present distinct challenges in treating ophthalmic diseases like age-related macular degeneration (AMD), glaucoma, diabetic retinopathy, and corneal disorders.¹ Despite advances in treatment modalities such as surgery, pharmaceuticals, and laser therapy, many ophthalmic disorders, remain challenging to manage effectively.¹ Stem cell replacement therapy presents a pioneering method for combating ocular pathologies by addressing the underlying causes of vision loss. As depicted in Figure 1, within the field of ophthalmology, this therapy strives to regenerate stem cells both in vitro and through in vivo transplantation, replacing dysfunctional cells, regenerating tissues, and ultimately restoring visual function by transplanting differentiated progenitor cells with the assistance of biomaterials such as gellan gum.²

Human embryonic stem cells possess the remarkable potential to differentiate into crucial cell types like retinal progenitor cells, corneal epithelial progenitor cells, retinal pigment epithelium progenitor cells, and optic nerve progenitor cells, vital for vision.² However, challenges persist in obtaining and differentiating these progenitor cells premortem, despite their promise for ophthalmological applications, as their integration into tissues in vivo remains complex.²

Common methods employed for cell transplantation and cell delivery have demonstrated effectiveness but also present considerable challenges.³ Table 1 outlines the primary issues encountered in stem cell encapsulation using hydrogels, such as optimization challenges, inadequate mechanical properties, and limited scalability and reproducibility. To address these challenges, it is important to understand the common methods used for cell encapsulation, as well as their associated shortcomings.⁴ Therefore, there is an urgent need for innovative encapsulation systems that can improve the viability, functionality, and maintenance of stemness in encapsulated stem cells while minimizing adverse effects.

Table 1.

Encapsulation techniques of cells in hydrogels.

Methods	Description	Advantages	Disadvantages	Reference
Dropwise addition	Cells are suspended in a hydrogel precursor solution, added dropwise to a crosslinking agent, and encapsulated as droplets solidify into hydrogel beads or capsules.	Offers precise control over droplet size and composition, crucial for uniformly encapsulating cells. High throughput, scalability and ideal for large-scale applications.	Shear stress during droplet formation can affect cell viability and functionality. Achieving consistent droplet size distribution poses challenges,	Vilabril et al.⁵
Microfluidic encapsulation	Uses microfluidics to control cell and hydrogel precursor flow, enabling single-cell or cluster encapsulation in microscale hydrogels.	Ensures precise cell and hydrogel encapsulation, achieving uniform capsule size and distribution.	Microfluidic channels may clog from cell aggregation or hydrogel polymerization. Maintaining cell viability and functionality is critical in this method. Limited diffusion of nutrients and oxygen to encapsulated cell	Li et al.⁶
Emulsion crosslinking	Cells and hydrogel precursors are emulsified and crosslinked in oil, forming hydrogel microspheres or capsules encapsulating cells	Enables controlled cell encapsulation in hydrogels, providing a protective environment and forming uniform microcapsules	Controlling microcapsule size and distribution, optimizing emulsion conditions for cell viability, and managing crosslinking are complex tasks. Removal of residual chemicals or surfactants may be required to maintain cell behavior.	Chang et al.⁷
Electrostatic interaction	Cells mixed with charged hydrogel precursors (e.g. polyelectrolytes) form stable hydrogel-cell complexes via electrostatic interactions which encapsulate the cells.	Gentle on cells, straightforward, and avoids complex chemical modifications or harsh conditions. Provides precise control over encapsulation, ensuring uniform cell distribution within the hydrogel matrix.	Encapsulated cells may experience limited stability due to reversible electrostatic interactions. Factors such as pH and ionic strength can also influence encapsulation efficiency, affecting technique reproducibility and reliability in diverse experimental or clinical settings.	Jeon et al.⁸
Coaxial electrospinning	A core-shell electrospinning technique creates fibers with cells encapsulated in the core by electrospinning a cell solution within a hydrogel precursor shell.	Enables precise control over core-shell structures, providing a protective hydrogel core that supports cell viability. It also facilitates incorporation of bioactive molecules into the hydrogel matrix, boosting therapeutic potential.	Setup complexity, optimization challenges, and scalability issues. Ensuring compatibility between electrospinning conditions and cell viability is crucial	Nguyen et al.⁹
Layer-by-layer assembly	Hydrogel precursors and cells are layered on a substrate, forming a multilayered structure with encapsulated cells.	Precise control over layer thickness and composition for tailored cell encapsulation, minimizing impact on viability and function. It also incorporates bioactive molecules to customize cell environments for enhanced therapeutic outcomes.	Hindered by its sequential process, limiting scalability for large-scale applications. Ensuring consistent layer thickness and stability over time poses challenges for the long-term performance of encapsulated cells.	Li et al.¹⁰
Photo polymerization	Cells mixed with a photosensitive hydrogel precursor are encapsulated and exposed to light, triggering polymerization to form a crosslinked hydrogel network.	Light-induced gelation enables precise and rapid cell encapsulation in a minimally invasive manner, accommodating diverse cell types and biomolecules within the hydrogel matrix.	Addressing light penetration and photoinitiator cytotoxicity is crucial. Optimizing these factors ensures high cell viability and hydrogel integrity. Photo initiating agents might be toxic.	Kumar et al.¹¹
Enzymatic crosslinking	Enzymatically reactive hydrogel precursors mix with cells, triggering crosslinking via enzymes to encapsulate the cells within the hydrogel.	It offers a biocompatible method that preserves cell viability under mild conditions, allowing precise control over gelation kinetics for tailored encapsulation.	Enzymatic crosslinking, though versatile, can be slower and less efficient due to variability in enzyme availability and specificity.	Hasturk et al.¹²

Once encapsulated, several common cell delivery issues must be addressed. As shown in Table 2, current stem cell transplantation methods face challenges such as insufficient homing to target tissues, leading to reduced therapeutic impact, and significant cell loss during the delivery process.¹³ Additionally, issues such as inadequate integration and survival of cells, potential inflammation or immune responses to scaffold materials, and limited control over cell differentiation can affect the effectiveness of the therapy. Addressing these issues is crucial for improving the success and safety of stem cell treatments.¹³

Table 2.

Cell delivery techniques of stem cells using hydrogels.

Methods	Description	Advantages	Disadvantages	Reference
Direct injection	Hydrogel-cell suspensions or pre-formed hydrogel-cell constructs are injected into target tissues or organs to achieve localized cell delivery.	This technique enables minimally invasive delivery of cells directly to target tissues or organs, bypassing complex surgical procedures and reducing recovery times and potential complications.	A significant portion of the injected cells may be lost due to leakage or being carried away by bodily fluids. Mechanical forces during injection can damage cells, reducing their survival. Ensuring even cell distribution within the target tissue is challenging, leading to uneven concentrations.	Aguado et al.¹⁴
Surgical implantation	Hydrogel-cell constructs are surgically implanted to provide a stable environment for cell survival and integration within tissues or organs.	Enables precise placement into target tissues, ensuring localized therapy and potential tissue integration.	Invasive and carries risks such as infection, tissue damage, and immune rejection. Its effectiveness hinges on surgical skill and material compatibility with the host environment, necessitating careful patient selection and monitoring.	Wang et al.¹⁵
Topical application	Hydrogel-cell preparations are applied topically to wounds to deliver cells and aid healing.	Minimally invasive approach, reducing patient discomfort and recovery time, while enabling targeted delivery of therapeutic substances for conditions like burns, wounds, and skin disorders.	Limited for conditions needing deeper tissue penetration or systemic effects, potentially affecting effectiveness. Cell stability and retention within skin layers can vary, influencing treatment outcomes and requiring optimization for long-term success	Zhou et al.¹⁶
Catheter-based delivery	Hydrogel-cell suspensions are catheter-delivered for precise therapy in blood vessels or hollow organs.	Minimally invasive access to target sites like blood vessels or hollow organs, enabling precise therapeutic interventions with reduced recovery times and lower complication risks compared to open surgery.	Uniform distribution of cells and hydrogels in the target area can be challenging, impacting treatment efficacy. Catheter-related complications like infection or vessel damage require vigilant monitoring and skilled execution.	Martens et al.¹⁷

Transplantation carriers, like hydrogel-based polymers, play a vital role in ophthalmic stem cell replacement therapies, ensuring the delivery of therapeutic cells while maintaining viability and functionality.¹⁸ Selecting biomaterials for such therapies involves considering factors like biocompatibility, supportiveness resembling the extracellular matrix, controlled release, porous structure aiding nutrient exchange, biodegradability, suitable mechanical properties, immunomodulation, sterility, and biofunctionality.^3,18 Tailoring biomaterials based on these factors can enhance cell interaction, adhesion, differentiation, proliferation, and tissue regeneration in ophthalmic transplantation applications.^3,18,19

As emphasized in Figure 2, among biomaterial-based delivery methods, hydrogel-based delivery emerges as the most promising.⁴ This approach involves encapsulating stem cells in hydrogel matrices that mimic the natural extracellular matrix, thereby enhancing cell survival and integration. This method is particularly beneficial for treating ophthalmological diseases.^18,19

However, this approach encounters challenges, including ensuring scaffold biocompatibility, controlling degradation rates, promoting stem cell attachment and migration, optimizing mechanical properties, facilitating vascularization, and ensuring scalability and reproducibility of scaffold fabrication.^18,19 Addressing these hurdles necessitates interdisciplinary collaborations and advancements in hydrogel design, fabrication techniques, and tissue engineering strategies.^3,18,19

Gellan gum, derived from microbial fermentation, stands out in various biomedical fields due to its biocompatibility and versatility.²⁰ In addition to ophthalmology, gellan gum has been explored in drug delivery, tissue engineering, wound healing, gene delivery and regenerative medicine.²⁰ According to literature analyses, in tissue engineering, it has been researched as scaffolds for bone and cartilage regeneration amongst other tissue engineering applications.^21,22 In drug delivery, it has proven to enable controlled release of medications for conditions like cancer and diabetes.²⁰ Its ability to maintain a moist environment makes it ideal for wound healing, where studies have shown it promoting faster recovery of burns and ulcers.²⁰ In gene therapy, its hydrogel forms ability to encapsulate nucleic acids and protect it from degradation makes gellan gum hydrogels a promising vehicle for gene therapy applications.²³ In regenerative medicine, injectable gellan gum delivers stem cells and growth factors to damaged tissues, further expanding its applications in advanced medical treatments and technologies.^22,24

Gellan gum has several properties that make it suitable for ophthalmic treatment. It is biocompatible, non-toxic, and well-tolerated by the body, making it safe for use in sensitive areas like the eyes.²⁵ Its gel-forming ability in the presence of cations such as calcium ions, which are naturally present in tear fluid, allows it to create a sustained-release system for drugs, enhancing treatment effectiveness.²⁵ Gellan gum is mucoadhesive, meaning it can adhere to the mucosal surfaces of the eye, prolonging the residence time of drugs on the ocular surface and improving absorption and efficacy.²⁶ Its high transparency is crucial for ocular applications, ensuring it does not obstruct vision.^24,27 Furthermore, gellan gum hydrogels enable controlled and sustained release of ophthalmic drugs, reducing the frequency of administration and improving patient compliance.^28,29 Its stability under various conditions and ability to be sterilized without losing its properties are essential for maintaining the sterility required in ophthalmic treatments. These characteristics make gellan gum an excellent candidate for developing advanced ophthalmic drug delivery systems.²⁸

While gellan gum holds promise as a biomaterial, its formation techniques closely resemble those of other polysaccharide-based biomaterials such as alginate or hyaluronic acid, employing methods like crosslinking or gelation for scaffold fabrication.^3,18,30 Compared with other polysaccharide polymers (such as sodium alginate, hyaluronic acid, etc.), the biggest advantage of gellan gum in ophthalmology is its gelation mechanism.²¹ Gellan gum gels in the presence of divalent cations, facilitating a more controlled and predictable in situ gel formation compared to other polymers like sodium alginate, which gels through ionotropic gelation with calcium ions.^21,31,32 This unique gelation property of gellan gum allows for more precise control over the consistency and stability of the gel, making it particularly advantageous for ophthalmic applications where consistent drug delivery and retention on the ocular surface are critical.³² It also forms stronger and more stable gels, making it more suitable for sustained drug delivery.²⁴ Its balance of viscosity and fluidity ensures ease of application and effective spreading over the ocular surface, unlike the often highly viscous hyaluronic acid.²² Table 3 summarizes recent research on natural and synthetic biopolymers, underscoring gellan gum’s positive impact on ocular tissue regeneration outcomes. Overall, the combination of biocompatibility, transparency, unique gelation properties, and mechanical strength makes gellan gum particularly advantageous for ophthalmic applications compared to other polysaccharide polymers.²¹

Table 3.

Current hydrogel systems employed for ocular cell encapsulation and transplantation.

Hydrogel materials			Objective	Cell Line	Results	Reference
Natural polymers	With gellan gum	Silk sericin, gellan gum	Development of an injectable hydrogel intended to function as a carrier for differentiated hESCs.	hESCs, ARPE-19	The gellan gum matrix showed superior biocompatibility, incorporating silk sericin into the gellan gum matrix resulted in good RPE growth, with good cell viability observed.	Kim et al.³³
		Hyaluronic acid, gellan gum	Enhancing hydrogel optimization for retinal tissue engineering through 3D matrix cell encapsulation.	ARPE-19	During in vitro experiments, the cellular compatibility of the hydrogel matrix was significantly improved with the addition of gellan gum.	Youn, Jina, et al.³⁴
		Eggshell membrane, gellan gum	Evaluating the effects of integrating an eggshell membrane into a gellan gum hydrogel matrix.	RPE	Gellan gum facilitated the excellent proliferation of RPE cells without causing significant cytotoxic effects. Improved biocompatibility upon addition of eggshell membrane,	Choi et al.³⁵
		Gelatin, chitosan, gellan gum	Injectable cell delivery system	ARPE-19	Gellan gum matrix boosted cell growth, survival, adhesion, and migration.	Rim et al.³⁶
		Naturally derived dopamine, gellan gum	An injectable hydrogel platform for delivering RPE cells.	ARPE	Superior biocompatibility with dopamine and gellan gum	Lee et al.³⁷
	Without gellan gum	RGD-alginate, hyaluronic acid	3D generation of laminated neural retina and/or RPE from hiPSCs/hESCs	hiPSCs/hESCs	Alginate hydrogels promoted retinal tissue development from PSCs, while hyaluronic acid-based hydrogels did not demonstrate the same enhancement.	Hunt et al.³⁸
		Chitosan hydrochloride, oxidized dextran hydrogels	Cell encapsulation and injection	RPC	Cells can retain a high survival ratio (about 90%) with the protection of the self-healing Chitosan-Odex hydrogels post-injection.	Jiang F et al.³⁹
		Hyaluronic acid	Investigating the encapsulation of cells, their delivery via injection, and subsequent differentiation upon transplantation into the retina.	RPC	Hydrogel degradation facilitated even distribution of RPCs within the subretinal space, and the expressed mature photoreceptor marker, recoverin	Liu et al.⁴⁰
		Alginate, curcumin	The produced hydrogel was capable of securely anchoring RPE cells within its structure.	Rabbit RPE	The increase of curcumin promotes cell proliferation.	Park et al.⁴¹
		Gelatin	Development of an injectable hydrogel system for delivering cell sheet encapsulation, aimed at mediating tissue regeneration in the cornea.	Corneal endothelial cell (CEC)	Analysis of rabbit corneal endothelial cell morphology and count in vivo suggests that high-solid-content gelatin discs may trigger unfavorable tissue-material interactions upon implantation.	Lai et al.⁴²
Synthetic polymers	With gellan gum.	Polyethylene glycol, gellan gum	Re-engineering RPE cells using PEG/Gellan gum hydrogels prepared via cryogelation for retinal delivery	ARPE-19	Biocompatibility, as assessed by cell viability and proliferation, was satisfactory. However, observations indicate that higher PEG concentrations in the hydrogel led to decreased porosity.	Kim et al.⁴³
	Without gellan gum	Poly(acrylic acid)-Cysteine	Injectable hydrogels, modified with cysteine were designed via pH adjustments. Cells were seeded on them, to investigate their potential as an injectable therapy for macular and retinal abnormalities.	RPE1	The hydrogels showed good biocompatibility but lacked assessment of long-term stability crucial for sustained cell culture; additionally, their resistance to cellular biodegradation raises concerns for controlled cell release applications.	Bolanta et al.⁴⁴
		PEG-N-hydroxysuccinimide, collagen	Cells were incorporated into PEG-collagen hydrogels, formed by temperature and chemical crosslinking, and then applied to treat corneal injuries via injection into the cornea.	MSCs	The decline in cell proliferation over time raises concerns about the long-term stability and sustained support for cell growth within the hydrogel matrix due to hydrogel degradation.	Na et al.⁴⁵
		Poly(vinyl alcohol) (PVA), cellulose	Composite hydrogel’s biocompatibility with human corneal epithelial cells (HCE-2 cells) was assessed for corneal regeneration.	HCE-2	Quantification of metabolically active cells on lens surfaces revealed a significant difference compared to the control, suggesting potential limitations for certain applications.	Tummala et al.⁴⁶
		Poly(Lactic-Co-Glycolic) Acid	Injectable human conjunctival epithelial sheets were created using a coculture system comprising a human feeder cell layer and PLGA membrane-based transwell inserts.	Human Conjunctival Epithelial Sheets, fibroblasts	Despite satisfactory biocompatibility, xenoparticle contamination from murine feeder layers poses safety concerns for human transplantation.	Hong et al.⁴⁷
		Polyurethane	RPE cells were encapsulated for retinal regeneration within an injectable hydrogel fabricated through electrospinning and cryogelation techniques.	ARPE-19, bovine retinal pigment epithelium (bRPE), iris pigment epithelium (bIPE), and human retinal pigment epithelium	Cells exhibited good epithelial phenotype, demonstrated functional capabilities like phagocytosis, and showed reduced dextran transport through the membrane-cell complex. However, initial tests of an in vitro injury model revealed inconsistent cell integration, indicating the need for further refinement.	Alias⁴⁸

Limited studies have evaluated gellan gum’s biocompatibility and encapsulation mechanisms for treating ocular pathologies. However, research on cell encapsulation in ocular treatment has used both natural and synthetic biomaterials, with natural biopolymers being preferred for their superior biocompatibility.⁴⁹

Based on a thorough literature analysis, limited studies have employed gellan gum to encapsulate hESC-derived progenitor cells for treating ocular pathologies. Majority of the current studies predominantly concentrate on drug delivery rather than stem cell delivery.

In this review, the potential of gellan gum as a versatile polymer carrier for progenitor stem cell delivery in ophthalmic therapies is explored. Its inherent properties, recent advancements, and challenges in harnessing this biomaterial to enhance the efficacy of transplantation-based approaches in treating ocular disorders are delved into. Through a focused examination of gellan gum’s role in ophthalmic cell therapies, light is shed on its significance within the broader landscape of polymer-based interventions for ocular health.

Properties of gellan gum

Chemical structure and composition

Gellan gum is a high molecular weight polysaccharide produced by the bacterium Sphingomonas elodea through fermentation.²⁰ It consists of repeating units of glucose, glucuronic acid, and rhamnose, with acyl groups attached to some of the rhamnose residues. There is high acetyl gellan gum, which is the compound in its raw state, and there is low acetyl gellan gum, which is the purified biomaterial used in biomaterial science applications; Figure 3 shows the chemical structure of low acetyl gellan gum.^20,50 The chemical structure of gellan gum imparts unique properties such as thermoreversibility and shear-thinning behavior, which are advantageous for its application in various fields, including biomedicine.²⁰ Gellan gum can indirectly influence biological systems due to its properties and interactions in various biomedical applications, making it a great support for bioactivity.²⁰

Physical properties

Gellan gum can form transparent, thermally reversible gels in the presence of cations such as calcium or magnesium ions.⁵¹ These gels exhibit tunable rheological properties depending on factors such as polymer concentration, ion concentration, and temperature. Gellan gum solutions also demonstrate shear-thinning behavior, wherein viscosity decreases under shear stress and recovers upon cessation of stress, making them suitable for injection-based applications.³⁰

Tunability

Another key advantage of gellan gum is its tunable mechanical properties. Gellan gum hydrogels can be engineered to possess a wide range of stiffness and viscoelasticity, making it possible to tailor the material properties to match those of the surrounding ocular tissues.⁵¹ This is crucial for ensuring proper integration of the transplanted cells and maintaining the structural integrity of the eye. By adjusting parameters such as polymer concentration and crosslinking density, researchers can precisely control the mechanical properties of gellan gum hydrogels to suit the specific requirements of different ophthalmic applications.⁵¹

Injectability

One of the most attractive features of gellan gum hydrogels is their ability to undergo sol-to-gel transition in response to external stimuli, such as changes in temperature or pH.^18,30,39 This property allows for the minimally invasive, injectable delivery of progenitor cells within a liquid precursor solution that solidifies in situ to form a hydrogel scaffold. This injectability enables precise placement of the transplanted cells within the target tissue, facilitating uniform distribution and integration with minimal trauma to the surrounding structures.³³

Porosity and permeability

Gellan gum hydrogels exhibit a highly porous structure, which allows for efficient nutrient and oxygen diffusion to the encapsulated cells.^34,52 This is essential for supporting the survival and function of transplanted progenitor cells, particularly in the avascular environment of the cornea. Additionally, the permeability of gellan gum hydrogels can be modulated to regulate the exchange of signaling molecules between the transplanted cells and the surrounding tissue, enabling precise control over the cellular microenvironment.⁵²

Biocompatibility and biodegradability

One of the key advantages of gellan gum is its excellent biocompatibility, making it suitable for biomedical applications.^30,52,53 Gellan gum is non-toxic and non-immunogenic, minimizing the risk of adverse reactions when used in vivo.^52,54 Furthermore, gellan gum hydrogels can be designed to degrade over time, allowing for controlled release of encapsulated therapeutic agents or cells while gradually resorbing within the body without causing tissue damage.

Biofunctionalizability

Gellan gum hydrogels can be further functionalized with bioactive molecules to enhance their performance as transplantation carriers.³⁷ For example, growth factors and cell adhesion peptides can be incorporated into the hydrogel matrix to promote cell survival, proliferation, and differentiation. Additionally, gellan gum can be modified to incorporate cell-binding motifs that facilitate adhesion and interaction with specific cell types, further improving the efficacy of the transplantation therapy.⁵⁰ Biofunctionalization aims to impart specific biological functionalities to gellan gum hydrogels to modulate cell behavior and tissue regeneration.^37,50

Gelation mechanisms

The gelation of gellan gum is primarily induced by the presence of divalent cations such as calcium or magnesium ions.^55,56 Upon addition of these ions, gellan gum undergoes a conformational change, forming junction zones that crosslink the polymer chains and stabilize the gel structure.^56,57 The gelation process can be modulated by adjusting parameters such as ion concentration, pH, and temperature, enabling precise control over gel properties and morphology.^49,57

Strategies for loading differentiated progenitor cells into gellan gum hydrogels

By employing a combination of pre-encapsulation, post-encapsulation seeding, covalent immobilization, and gradient loading strategies, researchers can optimize cell viability, functionality, and integration within the hydrogel matrix.⁵⁸

Pre-encapsulation

Pre-encapsulation, a common method for incorporating differentiated progenitor cells into gellan gum hydrogels, involves mixing cells with the gellan gum precursor solution before gelation, enabling uniform cell distribution, and entrapment within the scaffold.⁵⁹ Optimization of cell density, mixing conditions, and rheological properties is crucial to ensure high cell viability and functionality. Microencapsulation techniques may also be employed in pre-encapsulation.⁵⁹

Microencapsulation

Within the pre-encapsulation approach, microencapsulation techniques can be employed to encapsulate individual cells or small cell clusters within gellan gum microspheres.⁶⁰ Microencapsulation offers several advantages, including enhanced protection of encapsulated cells from shear forces and immune responses, as well as improved control over cell distribution and release kinetics. Microspheres can be fabricated using methods such as emulsification, microfluidics, or electrostatic droplet generation, allowing for precise control over size, morphology, and encapsulation efficiency. These microencapsulated cell constructs can then be incorporated into gellan gum hydrogels to create three-dimensional tissue constructs with enhanced cellular distribution and functionality.⁶⁰

Macroencapsulation

Microencapsulation, encapsulating cells in larger hydrogel constructs like beads or sheets, offers advantages over microencapsulation.⁶¹ Cell-laden gellan gum sheets or membranes, fabricated through methods such as solvent casting or layer-by-layer assembly, provide precise control over properties.⁶¹ Additionally, three-dimensional scaffold-based macroencapsulation creates porous scaffolds supporting cell growth and differentiation, facilitating the development of macroscopic tissue constructs with enhanced structural integrity and mechanical stability, ideal for complex tissue architectures and bioactive component integration.^61,62

Post-encapsulation seeding

Alternatively, post-encapsulation seeding involves seeding progenitor cells into pre-formed gellan gum hydrogels after gelation, offering precise control over cell distribution and enabling sequential addition of multiple cell types or bioactive agents.⁶³ Modifying the hydrogel matrix to create cell-friendly microenvironments and utilizing specialized seeding techniques, such as microinjection or micro pipetting, enhance the uniformity and efficiency of cell distribution within the scaffold.⁶³

Gradient loading

Differentiated progenitor cells can be gradient-loaded into gellan gum hydrogels using techniques like controlled release or microfluidic patterning to mimic native tissue organization. This enables the creation of tissue-like structures with spatially defined cell distributions, crucial for regenerating complex tissues like the cornea or retina.⁶⁴

Gelation methods for gellan gum as a carrier of ocular progenitor stem cells: Options for pre and post-encapsulation

Ion-mediated gelation

Pre-encapsulation of progenitor cells in gellan gum hydrogels often relies on ion-mediated gelation, where the addition of divalent cations, such as calcium ions, induces crosslinking of the gellan gum polymer chains to form a stable hydrogel network.⁶⁵ This mechanism involves the coordination of calcium ions with the carboxylate groups on the gellan gum, resulting in a three-dimensional network that supports cell viability and function, as illustrated in Figure 4.

This gelation method is particularly suitable for cell encapsulation, as it can be performed under mild physiological conditions without the need for harsh chemicals or high temperatures that could compromise cell viability.⁶⁵ A 2019 study by Kim et al. investigated a novel hydrogel for retinal regeneration. They combined Polyethylene glycol with gellan gum to create a hybrid hydrogel, which was tested at various concentrations. The hydrogel’s ability to support the growth and adhesion of retinal pigment epithelium cells, specifically ARPE-19 cells, was evaluated. Additionally, the study employed calcium ions to induce crosslinking in the hydrogel, facilitating its gelation. Overall, characterization results indicated that ion-mediated gelation is well-suited for producing functional gellan gum hydrogels.⁴³

Temperature-induced gelation

Gellan gum gelation is dependent on both temperature and ionic crosslinking. As shown in Figure 5, when gellan gum is dissolved effectively at temperatures ranging from 70°C to 90°C, it forms a fluid solution that gels upon cooling, a process known as temperature-induced gelation.²¹ The gelation temperature can be relatively high if no ions are present. However, the addition of divalent cations, such as calcium ions, significantly enhances the gelation process. Thus, while gellan gum can gel upon cooling (around 36°C), the presence of ions like calcium greatly facilitates and strengthens the gelation process, indicating that gellan gum gelation is both temperature-dependent and reliant on ionic crosslinking.⁶⁶ Modulating gelation kinetics through temperature adjustment allows precise control over cell seeding timing, but thermal stress on encapsulated cells, especially temperature-sensitive types, must be minimized.⁶⁶

A study by Kim et al. utilized both temperature-induced and ion-mediated gelation mechanisms to prepare gellan gum hydrogels for application as a retinal pigment epithelium cell carrier. Initially, low-acyl gellan gum was dissolved in distilled water under constant stirring at a high temperature of 90°C for 1 h. This high temperature ensured that the gellan gum was completely dissolved. Following this, the temperature of the solution was lowered, where calcium chloride was introduced as a cross-linking agent. The calcium ions facilitated ion-mediated gelation by interacting with the negatively charged carboxyl groups on the gellan gum molecules, promoting the formation of a stable gel network. The cells were added after thorough mixing, as the solution was allowed to cool further to room temperature, where gelation continued to solidify the hydrogel. Thus, the study effectively combined both the temperature-induced sol-to-gel transition and ion-mediated crosslinking to achieve the desired hydrogel structure.³³

Covalent immobilization

Covalent immobilization involves the formation of covalent bonds between gellan gum chains and other molecules or functional groups. This type of gelation provides a more permanent and stable gel network compared to ionic or thermal methods.³² This mechanism is illustrated in Figure 6. Covalently immobilizing progenitor cells within gellan gum hydrogels can prevent post-transplantation leakage or migration, achieved by functionalizing the hydrogel with cell-binding ligands or reactive chemical groups.³² Careful consideration is needed to maintain cell viability and functionality while ensuring sufficient porosity for nutrient exchange; this method can be combined with pre- or post-encapsulation seeding to improve cell retention and integration within the scaffold.³²

In a study by Lee et al., covalent immobilization was utilized to enhance the gelation properties of gellan gum. The researchers prepared a dopamine-functionalized gellan gum hydrogel for retinal pigment epithelium cell delivery through a carbodiimide reaction. This involved the specific use of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to facilitate the coupling of dopamine to the gellan gum. These chemicals facilitate covalent bonding between carboxyl groups on gellan gum and amine groups on dopamine, forming a stable covalent bond. Overall, the hydrogel system demonstrated potential as an effective carrier for retinal pigment epithelium cells, with promising implications for improving visual function.³⁷

Photo-crosslinking

Photo-crosslinking involves the use of light (often UV or visible light) to initiate polymerization or crosslinking reactions in gellan gum hydrogels. This method allows for precise control over the gelation process and spatial patterning of the hydrogel.⁶⁷ This method, performed under mild conditions, minimizes cytotoxicity risks, though optimization of light exposure parameters is crucial to ensure uniform crosslinking and cell viability.⁶⁷

Figure 7 demonstrates the photo-crosslinking process of gellan gum, where ultraviolet light is used to activate photoinitiators or photoactive groups. This activation leads to the formation of covalent bonds between polymer chains, resulting in the creation of a three-dimensional gel network.^67,68 This process begins with photoinitiators, compounds that absorb light and decompose to produce reactive species such as free radicals or cations. Upon ultraviolet exposure, these photoinitiators absorb photons, leading to their excitation and subsequent cleavage into reactive radicals or ions. These reactive species interact with the photoactive groups in the gellan gum, initiating a chain reaction that forms covalent bonds between adjacent polymer chains, resulting in a crosslinked network. To facilitate photo-crosslinking, gellan gum must be chemically modified to incorporate photoactive groups, commonly through methacrylation or acrylation, which introduce methacrylate or acrylate groups, respectively.^67,68

Gelation of gellan gum using photo-crosslinking has been effectively used to entrap cartilage cells, demonstrating its potential in tissue engineering. This method, is demonstrated in a study by Shin et al., involving a two-step photo-crosslinking process where gellan gum is chemically modified with photoactive methacrylate groups.⁶⁸ This process enables the formation of a robust double network hydrogel capable of supporting and maintaining the viability of cartilage cells, thereby providing a promising scaffold for cartilage tissue engineering. The use of photo-crosslinked gellan gum hydrogels for stem cell delivery in ophthalmic diseases is still largely unexplored. Key challenges include ensuring biocompatibility and safety of photo initiators, achieving suitable mechanical properties for eye tissues, and proving the hydrogel’s effectiveness and safety in treating eye conditions.⁶⁸ In summary, the characterization findings showed that ion-mediated gelation is effective for creating functional gellan gum hydrogels.

Future directions for harnessing gellan gum’s novelty in ophthalmology

As research in this field continues to advance, several future directions can be envisioned to further exploit the novelty of gellan gum in ophthalmic therapies:

Advanced biofunctionalization

Future efforts may focus on enhancing the biofunctionality of gellan gum hydrogels through the incorporation of bioactive molecules, such as growth factors, cytokines, or small peptides. Tailoring the biofunctionalization of gellan gum hydrogels could promote specific cellular responses, such as enhanced cell adhesion, proliferation, and differentiation, leading to improved tissue regeneration outcomes in ophthalmic applications.⁶⁹

Nanotechnology integration

Integration of nanotechnology into gellan gum-based ophthalmic therapies holds promise for achieving precise control over hydrogel properties and cellular behavior.³¹ Future research may explore the development of gellan gum nanocomposites with engineered nanofibers, nanoparticles, or nanosheets to enhance mechanical strength, biological functionality, and drug delivery capabilities for targeted treatment of ocular diseases.

Patient-specific therapies

Advancements in personalized medicine may lead to the development of patient-specific gellan gum hydrogels tailored to individual ocular pathologies and anatomical variations.⁷⁰ Utilizing patient-derived cells and bioengineering techniques, such as 3D bioprinting or organoid culture systems, could enable the fabrication of customized hydrogel implants optimized for enhanced tissue integration and long-term therapeutic efficacy.^69,70

Smart hydrogel platforms

The integration of smart hydrogel platforms with gellan gum could enable dynamic modulation of hydrogel properties in response to external stimuli, such as light, temperature, or pH changes.⁷¹ Future research may explore the design of stimuli-responsive gellan gum hydrogels for on-demand drug release, controlled cell encapsulation, and minimally invasive in situ gelation, offering precise spatiotemporal control over therapeutic interventions in ophthalmology.

Biodegradable implants

The development of biodegradable gellan gum implants with tunable degradation kinetics could address the need for temporary mechanical support and sustained therapeutic delivery in ocular regenerative medicine. Future studies may focus on optimizing the degradation profile of gellan gum hydrogels to match the healing timeline of ocular tissues, allowing for controlled scaffold remodeling and seamless tissue integration without long-term foreign body responses.¹⁵

Clinical translation and commercialization

Accelerating the clinical translation and commercialization of gellan gum-based ophthalmic therapies requires rigorous preclinical validation, regulatory approval, and scalable manufacturing processes.²⁰ Future efforts may involve collaborative partnerships between academia, industry, and regulatory agencies to navigate the complex pathway from bench to bedside, ensuring safe and effective translation of gellan gum innovations into clinical practice for the benefit of patients with ocular diseases and injuries.

Conclusion

This review has highlighted the potential of gellan gum-based carriers in developing ophthalmic therapies by providing a novel platform for the delivery of progenitor cells to treat various ocular disorders. Gellan gum possesses unique properties such as biocompatibility, tunable gelation kinetics, and controlled release capabilities, making it an ideal candidate for encapsulating and delivering therapeutic cells to the eye. Through a comprehensive analysis of the current literature, the applications of gellan gum has been explored in addressing challenges associated with traditional delivery methods and enhancing the efficacy and safety of stem cell-based therapies in ophthalmology.

Moreover, ongoing research and development efforts should focus on addressing remaining challenges, optimizing delivery strategies, and exploring emerging technologies to further enhance the therapeutic potential of gellan gum-based carriers in ophthalmology. By fostering interdisciplinary collaborations and continued innovation, gellan gum-based carriers hold promise for driving forward the next generation of ophthalmic treatments and addressing the growing burden of vision-related disorders.

Footnotes

Acknowledgements

This study does not contain any acknowledgments.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Ethical approval

This study, as a literature review, is exempt from Institutional Review Board approval.

ORCID iD

Mthabisi Talent George Moyo

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