(M1230-07-37) Novel Composite Collagen-Hyaluronic Acid-Sodium Alginate Based Scaffolds Crosslinked with Calcium Chloride for Potential Chronic Wound Healing Applications

Purpose: Medicated biological dressings are one of the most advanced wound management approaches that involve taking an active part in molecular and cellular events to repair, regenerate and restore the damaged tissue. Given this importance of biological dressings in wound healing, herein an innovative biocompatible composite scaffold was designed based on crosslinked sodium alginate combined with two biopolymers (collagen and hyaluronic acid) that form part of the natural skin matrix. In this study features such as mimicking the human extracellular matrix (ECM) and excellent hemocompatibility are the factors that were considered in designing the novel biocompatible scaffolds. In addition, the porous architecture of the scaffolds will be beneficial for effective vascularization as well as the exchange of nutrients that can support and further enhance cell migration and proliferation. The crosslinking of sodium alginate is also expected to improve the mechanical strength and functional performance of the composite scaffolds.

Methods: Scaffolds were prepared by freeze-drying gels combining different weight ratios of sodium alginate (SA), fish collagen (COL) and hyaluronic acid (HA) and crosslinked with 2% CaCl₂. The gels were prepared by dispersing the required weights of each polymer in deionized water at room temperature, with constant stirring until a homogeneous gel was obtained. The resulting gels (1g) were poured into each well of a 24 well plate and freeze-dried using an automated cycle on an Advantage freeze-dryer. The scaffolds obtained were characterized for their mechanical strength and adhesion (texture analyzer), exudate handling properties [(porosity (%), swelling capacity, gel strength, water absorption (AW), equilibrium water content (EWC), evaporative water loss (EWL) and water vapor transmission rate (WVTR)], surface morphology using scanning electron microscopy (SEM), and physical form using X-ray diffraction (XRD). The above physico-chemical characterization tests were used to select the optimized formulation(s) for BSA (model protein) loading. The BSA loaded scaffolds were further characterized for protein content and in vitro release profiles using HPLC. The biocompatibility of the BSA loaded scaffolds was evaluated with MTT assay utilizing human dermal fibroblasts (HDF) and human epidermal keratinocytes (HEKC), the major cellular components of the dermis and epidermis respectively, after incubation with the SA:COL:HA scaffolds for 24 and 48 hours. Clotting measurement on whole blood was performed to investigate the coagulation effect of the SA:COL:HA scaffolds compared to Promogran^TM; a commercially available collagen-based wound dressing used for the treatment of chronic wounds.

Results: Composite gels comprising crosslinked SA, COL and HA resulted in physically elegant scaffolds having a porous microstructure (Figure 1). The hardness ranged from 1.41- 4.02N, which is expected to be able to withstand the external forces exerted during handling and application. The crosslinking had a marked effect on the pore shape and porosity with the latter ranging from 73- 81 % (Table 1) which was significantly (p < 0.05) lower than the non-crosslinked scaffolds (data not shown). The AW ranged from 633-1128 % and EWC from 86-92 % after 24 hours of incubation at 37°C (Table 1). The BSA loaded crosslinked scaffolds were able to swell by more than 900% of their original weight, with the formulations containing high amounts of HA showing relatively higher swelling capacity compared to the others (Figure 2). The incorporation of BSA significantly (p < 0.05) increased the WVTR (Table 1) making these wound dressing scaffolds capable of absorbing about 50% exudate from a heavily exuding chronic wound which could prevent the excessive collection of exudates, whilst maintaining a moist wound environment. The in vitro blood analysis performed on human whole blood confirmed that the SA:COL:HA scaffolds reduced the blood clotting index (BCI) by up to 20% in 5 minutes indicating a short hemostatic time and less blood loss compared to Promogran^Tm the commercially available sponge used for the treatment of chronic wounds. Moreover, the scaffolds illustrated no cellular toxicity towards HDF and HEKC with cell viability between 86-98 % after 48 hours. This is greater than the accepted International Standard Organization (ISO) value of 70%, and therefore will not affect cell proliferation and migration during wound healing.

Conclusion: The results suggest that CaCl₂ was able to physically crosslink with SA and enhanced the mechanical stability and the functional properties of the resulting composite scaffold dressings. Furthermore, the MTT and whole blood assay confirmed their promising biocompatibility and hemocompatibility, respectively. These observed features confirm that SA:COL:HA scaffolds could be applied as multifunctional wound dressings to take an active part in the healing of chronic wounds.

Acknowledgements: The authors are grateful to the University of Greenwich for funding this work. We're also grateful to Andrew Hurt for help with SEM analyses.


Representative SEM images of crosslinked scaffolds showing the porous microstructure for both blank and BSA loaded formulations


Swelling profiles of the different crosslinked scaffolds showing differences in swelling capacity between the blank and BSA loaded formulations


Exudate handling properties of the different scaffolds tested showing differences between the blank and BSA loaded formulations