Engineering an Alginate Hydrogel System for Biofilm Disruption in Chronic Wound Infections

Background

Chronic wounds, such as diabetic foot ulcers, are a growing healthcare challenge, often failing to heal due to persistent bacterial infections. A key reason is the formation of biofilms, which are structured bacterial communities protected by a matrix stabilized by metal ions such as calcium. This barrier makes bacteria up to 100–1000 times more resistant to antibiotics, preventing treatments from effectively reaching their targets. As a result, current wound care methods are often ineffective, requiring repeated treatments that increase cost, medical waste, and antibiotic resistance.

Research Question

How do EDTA-loaded biodegradable hydrogels and variations in hydrogel matrix density influence the disruption and eradication of chronic wound biofilms?

Solution: Controlled Hydrogel Release System

This project explores a controlled delivery strategy using biodegradable hydrogels. Ethylenediaminetetraacetic acid (EDTA), a metal-chelating agent, was selected for its ability to bind and remove metal ions, which are essential for maintaining the structural integrity of biofilms. EDTA was incorporated into alginate and gelatin hydrogel matrices to regulate its release over time.

Importance

By directly targeting biofilms and optimizing the timing of treatment delivery, this system addresses the root cause of chronic wound persistence rather than relying on higher antibiotic doses. Current wound care treatments are costly, accounting for up to ~5% of total healthcare expenditures, and rely on materials and mass production processes that contribute to environmental pollution. In contrast, our biodegradable hydrogels offer an effective, lower-cost alternative that is both economically sustainable and environmentally friendly.

Variables

Controlled:

Incubation temperature (37 °C)

Well plate format (96-well)

Bacterial strain (E. coli K-12)

Treatment time

Independent:

EDTA concentration (5 mM, 25 mM, 50 mM)

Treatment type (free EDTA vs hydrogel-embedded EDTA)

Hydrogel type (chemical vs physical)

Alginate concentration (1%, 1.5%, 2%)

Gelatin concentration (3%, 5%, 10%)

Crosslinking conditions (2% CaCl₂ for alginate)

Exposure time (1 hr, 6 hr, 12hr, 24 hr)

Dependent:

Percent removal of biofilm

EDTA release over time (concentration in solution)

Biofilm Formation

E coli. cultured in nutrient broth (37 °C, 24h)

Cultured broth diluted to another nutrient broth (1:100)

Broth added to 96-well plates

Plates were incubated under static conditions to allow biofilm development

After incubation, planktonic cells were removed

Hydrogel Preparation

Chemical Hydrogels (Alginate):

Sodium alginate mixed with the EDTA solution

Crosslinked using 2% calcium chloride

Physical Hydrogels (Gelatin):

Gelatin dissolved in water at 60℃

EDTA is incorporated during preparation.

Allowed to cool and solidify

Experimentation

Biofilms treated with:

EDTA-loaded alginate hydrogels

EDTA-loaded gelatin hydrogels

Control groups included:

Untreated biofilm

Broth-only (no bacteria)

Hydrogels without EDTA

EDTA solutions

Samples were incubated for set time intervals (1, 6, 12, 24 hours).

Biofilm Quantification (Crystal Violet Assay)

Biofilms stained with 0.1% crystal violet

Excess stain is removed through washing and drying

Bound dye solubilized using 95% ethanol

Absorbance measured using a spectrophotometer (570nm)

Release Kinetics Measurement

EDTA-loaded hydrogels were placed in distilled water.

Samples were collected at multiple time points (15 min, 30 min, 1h, 2h, 6h, 24h).

EDTA reacted with copper sulphate to form a measurable complex.

Absorbance recorded to determine concentration over time.

Data Analysis

All trials were repeated four to five times to ensure consistency and reliability

Statistical analysis performed using:

Student's t-tests (unpaired or paired)

ANOVA (One-way or Two-way)

Significance used to compare treatment effectiveness.

Results

1. Overall Effectiveness Against Biofilms

All hydrogel formulations demonstrated strong antibiofilm activity, achieving approximately 70–90% biofilm eradication across tested conditions and groups at the 24-hour mark. This consistent reduction indicates that the hydrogels were highly effective at disrupting established biofilms. The incorporation of EDTA played a key role in enhancing this effect, likely by chelating essential metal ions and weakening the biofilm matrix, allowing for greater structural breakdown.

2. EDTA Concentration

Referring to Figure 2, a strong dose-response relationship was observed. Increasing EDTA from 5 mM to 50 mM significantly improved biofilm eradication across all groups. In 1% alginate, this shift nearly doubled effectiveness at 1 hour, while optimized conditions reached ~85% biofilm removal by 24 hours.

3. Hydrogel Concentration

From Figure 3, hydrogel density directly influenced EDTA diffusion. At 1 hour, 1% alginate achieved ~57% removal, while 2% was limited to ~29%, demonstrating diffusion restriction. However, over time, denser hydrogels demonstrated a clear increase in effectiveness that directly corresponds with their release profiles. At the 1-hour mark, 2% alginate hydrogels showed significantly lower removal (~28.97%) compared to 1% hydrogels (~57.68%), reflecting their limited initial EDTA diffusion. However, by the 6-12 hour mark, eradication levels in 2% hydrogels increased substantially, narrowing the gap between concentrations. By 24 hours, the 1.5% and 2% hydrogels overtook the 1% formulation in effectiveness, with the 2% hydrogel achieving an average of approximately 86% biofilm eradication, surpassing both the lower-density systems and free EDTA treatments. This trend directly mirrors the release kinetics data, where higher-density hydrogels exhibited slower but continuous EDTA diffusion. Despite not releasing their full EDTA content, these hydrogels achieved comparable biofilm reduction, demonstrating that prolonged exposure compensates for lower initial availability. This shows that the hydrogel matrix acts as a regulatory filter, where lower concentrations enable rapid release, while higher concentrations promote sustained delivery, ultimately enhancing long-term biofilm disruption.

4. Treated Vs. Untreated Hydrogels

From Figure 4, the control hydrogels without EDTA showed minimal biofilm disruption. In contrast, EDTA-loaded hydrogels demonstrated significantly higher biofilm removal. This confirms that biofilm reduction is not caused by the hydrogel structure itself but by the presence and release of EDTA. This validation is critical, as it eliminates the major counterargument that physical disruption from the material contributes to treatment effectiveness.

5. Alginate Vs. Gelatin (Base)

As shown in Figure 5, there was no statistically significant difference between alginate and gelatin hydrogels. This indicates that the fundamental structure of the hydrogel does not determine biofilm removal. Instead, effectiveness is governed by EDTA concentration and release kinetics, confirming that treatment outcomes depend on delivery behaviour rather than material type.

What our results ultimately show is that biofilm treatment is not just about what you deliver but how you deliver it. While increasing EDTA concentration clearly improves biofilm disruption, our data demonstrates that concentration alone is not the main factor, rather it's the timing and consistency of delivery that are the true drivers of biofilm eradication.

Although both hydrogel types enabled controlled delivery, alginate-based (chemical) hydrogels proved more viable for real-world applications due to their structural stability. In contrast, gelatin-based (physical) hydrogels are more susceptible to degradation, making them less reliable in environments such as chronic wounds, where enzymes and fluid flow can rapidly break down weaker materials.

Beyond mere disruption levels, our results suggest a path toward minimizing cytotoxic risk in wound care. Current clinical practices often rely on "blunt force" and high concentrations of antibiotics, which are not only inefficient but also damaging to tissues and increase antibiotic resistance. By demonstrating that precisely tuned delivery kinetics can outperform high-dose immediate treatment, we can achieve high anti-biofilm efficacy while remaining well below concentrations that might otherwise damage healthy tissue and cells.

This is crucial, as this changes the medical purposes. The future of wound care is not in developing stronger drugs but in mastering the architecture of their delivery. Hence, our alginate hydrogel alternatives for wound dressings are not only biodegradable but also challenging and provide new insights into medical issues, and they are significantly more cost-friendly and accessible.

Limitations and Sources of Errors:

Real biofilms are composed of multiple bacteria and contain complex extracellular components, so our model does not fully represent true clinical biofilm environments.

Inconsistent hydrogel size and composition

Measurement error (absorbance readings)

What's Next:

Using multiple pathogenic bacterial strains found in wounds

Develop a sequential hydrogel system with layered structures to enable timed release (fast EDTA release followed by delayed antibiotic release)

Test on multi-species biofilms to better reflect real-world infections

Simulate real wound conditions (body temperature, pH, moisture, and simulated body fluids)

Thank you to Mr. Michael Patenaude for allowing us to use the Grant Park High School Laboratory for the duration of this project, providing materials and laboratory equipment, and supervising us during experiments.

Thank you to Mrs. Karly Watts for allowing us to use the Grant Park High School Laboratory for the duration of this project, providing bacteria samples, testing material, and laboratory equipment, and supervising us during experiments.

Thank you to our parents for providing materials, inspiring board design, helping when we needed it, driving us to places, and providing encouragement/motivation throughout the entire journey.

Journal/Research Articles:

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Video:

O'Toole, G. A. (2011). Microtiter dish biofilm formation assay. Journal of Visualized Experiments, (47), 2437. ​​https://doi.org/10.3791/2437-v

Pictures:

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Gels. (n.d.). Advances in gelatin-based tissue engineering using HRP/H₂O₂: Schematic representation of wound healing using a hydrogel dressing. ResearchGate. https://www.researchgate.net/figure/Schematic-representation-of-wound-healing-using-a-hydrogel-dressing_fig3_392745807

Pavliv, D., Booker, N., & Lee, S. (n.d.). Some antibiotics are riskier than others: What you should know about quinolones. National Center For Health Research. https://www.center4research.org/antibiotics-riskier-others-know-quinolones/

The Brainy Insights. (2023). Chronic wound care market size by product (surgical & traditional wound care, wound therapy devices, and advanced wound dressing), type (venous ulcers, pressure ulcers, diabetic ulcers, and others) end-use (homecare settings, hospitals, wound care centers, and others), regions, global industry analysis, share, growth, trends, and forecast 2023 to 2031. https://www.thebrainyinsights.com/report/chronic-wound-care-market-13260?srsltid=AfmBOorKJbiSb0QTMbDOVoxo_rgYlU7-UfB9Wchi1CwXO0960xfqsNLT