Hydrogel-Based Strategies to Advance Therapies for Chronic Skin Wounds.

Chronic skin wounds are the leading cause of nontraumatic foot amputations worldwide and present a significant risk of morbidity and mortality due to the lack of efficient therapies. The intrinsic characteristics of hydrogels allow them to benefit cutaneous healing essentially by supporting a moist environment. This property has long been explored in wound management to aid in autolytic debridement. However, chronic wounds require additional therapeutic features that can be provided by a combination of hydrogels with biochemical mediators or cells, promoting faster and better healing. We survey hydrogel-based approaches with potential to improve the healing of chronic wounds by reviewing their effects as observed in preclinical models. Topics covered include strategies to ablate infection and resolve inflammation, the delivery of bioactive agents to accelerate healing, and tissue engineering approaches for skin regeneration. The article concludes by considering the relevance of treating chronic skin wounds using hydrogel-based strategies.

Fucoidan Immobilized at the Surface of a Fibrous Mesh Presents Toxic Effects over Melanoma Cells, But Not over Noncancer Skin Cells.

The use of fucoidan, a marine-origin bioactive polymer, is herein proposed as a component of an innovative and effective strategy against melanoma, one of the most aggressive skin cancers. First, fucoidan antitumor activity, in its soluble form, was assessed presenting increased cytotoxicity over melanoma cells when compared to human dermal fibroblasts and keratinocytes. After this antitumor activity validation and trying to develop a more targeted and local strategy aiming to diminish the cytotoxic effects over noncancer cells, fucoidan was immobilized at the surface of an electrospun nanofiber mesh (NFM_Fu), envisioning the development of a therapeutic patch. The maximum immobilization concentration was 1.2 mg mL-1, determined by the Toluidine Blue Assay and confirmed by XPS. Furthermore, NFM_Fu is more hydrophilic than NFM, presenting a contact angle of 36°, lower than the 121° of the control condition. NFM_Fu was able to reduce human melanoma cell viability by 50% without affecting human dermal fibroblasts and keratinocytes. Taken together, these results set the basis for a valuable approach for melanoma treatment.

Microfluidics for Processing of Biomaterials.

Microfluidics techniques can be used to process a wide range of biomaterials, from synthetic to natural origin ones. This chapter describes microfluidic processing of biomaterials, mainly polymeric materials of natural origin, focusing on water-soluble polymers that form non-flowing phases after crosslinking. Some polysaccharides and proteins, including agarose, alginate, chitosan, gellan gum, hyaluronic acid, collagen, gelatin, and silk fibroin are emphasized deu to their relevance in the field. The critical characteristics of these materials are discussed, giving particular consideration to those that directly impact its processability using microfluidics. Furthermore, some microfluidic-based processing techniques are presented, describing their suitability to process materials with different sol-gel transition mechanisms.

Bioreactors and Microfluidics for Osteochondral Interface Maturation.

The cell culture techniques are in the base of any biology-based science. The standard techniques are commonly static platforms as Petri dishes, tissue culture well plates, T-flasks, or well plates designed for spheroids formation. These systems faced a paradigm change from 2D to 3D over the current decade driven by the tissue engineering (TE) field. However, 3D static culture approaches usually suffer from several issues as poor homogenization of the formed tissues and development of a necrotic center which limits the size of in vitro tissues to hundreds of micrometers. Furthermore, for complex tissues as osteochondral (OC), more than recovering a 3D environment, an interface needs to be replicated. Although 3D cell culture is already the reality adopted by a newborn market, a technological revolution on cell culture devices needs a further step from static to dynamic already considering 3D interfaces with dramatic importance for broad fields such as biomedical, TE, and drug development. In this book chapter, we revised the existing approaches for dynamic 3D cell culture, focusing on bioreactors and microfluidic systems, and the future directions and challenges to be faced were discussed. Basic principles, advantages, and challenges of each technology were described. The reported systems for OC 3D TE were focused herein.

Stem Cells for Osteochondral Regeneration.

Stem cell research plays a central role in the future of medicine, which is mainly dependent on the advances on regenerative medicine (RM), specifically in the disciplines of tissue engineering (TE) and cellular therapeutics. All RM strategies depend upon the harnessing, stimulation, or guidance of endogenous developmental or repair processes in which cells have an important role. Among the most clinically challenging disorders, cartilage degeneration, which also affects subchondral bone becoming an osteochondral (OC) defect, is one of the most demanding. Although primary cells have been clinically applied, stem cells are currently seen as the promising tool of RM-related research because of its availability, in vitro proliferation ability, pluri- or multipotency, and immunosuppressive features. Being the OC unit, a transition from the bone to cartilage, mesenchymal stem cells (MSCs) are the main focus for OC regeneration. Promising alternatives, which can also be obtained from the patient or at banks and have great differentiation potential toward a wide range of specific cell types, have been reported. Still, ethical concerns and tumorigenic risk are currently under discussion and assessment. In this book chapter, we revise the existing stem cell-based approaches for engineering bone and cartilage, focusing on cell therapy and TE. Furthermore, 3D OC composites based on cell co-cultures are described. Finally, future directions and challenges still to be faced are critically discussed.

Evaluation of the Potential of Collagen from Codfish Skin as a Biomaterial for Biomedical Applications.

Collagen is one of the most widely used biomaterials, not only due its biocompatibility, biodegradability and weak antigenic potential, but also due to its role in the structure and function of tissues. Searching for alternative collagen sources, the aim of this study was to extract collagen from the skin of codfish, previously obtained as a by-product of fish industrial plants, and characterize it regarding its use as a biomaterial for biomedical application, according to American Society for Testing and Materials (ASTM) Guidelines. Collagen type I with a high degree of purity was obtained through acid-extraction, as confirmed by colorimetric assays, SDS-PAGE and amino acid composition. Thermal analysis revealed a denaturing temperature around 16 °C. Moreover, collagen showed a concentration-dependent effect in metabolism and on cell adhesion of lung fibroblast MRC-5 cells. In conclusion, this study shows that collagen can be obtained from marine-origin sources, while preserving its bioactivity, supporting its use in biomedical applications.

Marine Collagen/Apatite Composite Scaffolds Envisaging Hard Tissue Applications.

The high prevalence of bone defects has become a worldwide problem. Despite the significant amount of research on the subject, the available therapeutic solutions lack efficiency. Autografts, the most commonly used approaches to treat bone defects, have limitations such as donor site morbidity, pain and lack of donor site. Marine resources emerge as an attractive alternative to extract bioactive compounds for further use in bone tissue-engineering approaches. On one hand they can be isolated from by-products, at low cost, creating value from products that are considered waste for the fish transformation industry. One the other hand, religious constraints will be avoided. We isolated two marine origin materials, collagen from shark skin (Prionace glauca) and calcium phosphates from the teeth of two different shark species (Prionace glauca and Isurus oxyrinchus), and further proposed to mix them to produce 3D composite structures for hard tissue applications. Two crosslinking agents, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride/N-Hydroxysuccinimide (EDC/NHS) and hexamethylene diisocyanate (HMDI), were tested to enhance the scaffolds' properties, with EDC/NHS resulting in better properties. The characterization of the structures showed that the developed composites could support attachment and proliferation of osteoblast-like cells. A promising scaffold for the engineering of bone tissue is thus proposed, based on a strategy of marine by-products valorisation.

Endothelial cells enhance the in vivo bone-forming ability of osteogenic cell sheets.

Addressing the problem of vascularization is of vital importance when engineering three-dimensional (3D) tissues. Endothelial cells are increasingly used in tissue-engineered constructs to obtain prevascularization and to enhance in vivo neovascularization. Rat bone marrow stromal cells were cultured in thermoresponsive dishes under osteogenic conditions with human umbilical vein endothelial cells (HUVECs) to obtain homotypic or heterotypic cell sheets (CSs). Cells were retrieved as sheets from the dishes after incubation at 20 °C. Monoculture osteogenic CSs were stacked on top of homotypic or heterotypic CSs, and subcutaneously implanted in the dorsal flap of nude mice for 7 days. The implants showed mineralized tissue formation under both conditions. Transplanted osteogenic cells were found at the new tissue site, demonstrating CS bone-inductive effect. Perfused vessels, positive for human CD31, confirmed the contribution of HUVECs for the neovascularization of coculture CS constructs. Furthermore, calcium quantification and expression of osteocalcin and osterix genes were higher for the CS constructs, with HUVECs demonstrating the more robust osteogenic potential of these constructs. This work demonstrates the potential of using endothelial cells, combined with osteogenic CSs, to increase the in vivo vascularization of CS-based 3D constructs for bone tissue engineering purposes.

Fabrication of endothelial cell-laden carrageenan microfibers for microvascularized bone tissue engineering applications.

Recent achievements in the area of tissue engineering (TE) have enabled the development of three-dimensional (3D) cell-laden hydrogels as in vitro platforms that closely mimic the 3D scenario found in native tissues. These platforms are extensively used to evaluate cellular behavior, cell-cell interactions, and tissue-like formation in highly defined settings. In this study, we propose a scalable and flexible 3D system based on microsized hydrogel fibers that might be used as building blocks for the establishment of 3D hydrogel constructs for vascularized bone TE applications. For this purpose, chitosan (CHT) coated κ-carrageenan (κ-CA) microfibers were developed using a two-step procedure involving ionotropic gelation (for the fiber formation) of κ-CA and its polyelectrolyte complexation with CHT (for the enhancement of fiber stability). The performance of the obtained fibers was assessed regarding their swelling and stability profiles, as well as their ability to carry and, subsequently, promote the outward release of microvascular-like endothelial cells (ECs), without compromising their viability and phenotype. Finally, the possibility of assembling and integrating these cell-laden fibers within a 3D hydrogel matrix containing osteoblast-like cells was evaluated. Overall, the obtained results demonstrate the suitability of the microsized κ-CA fibers to carry and deliver phenotypically apt microvascular-like ECs. Furthermore, it is shown that it is possible to assemble these cell-laden microsized fibers into 3D heterotypic hydrogels constructs. This in vitro 3D platform provides a versatile approach to investigate the interactions between multiple cell types in controlled settings, which may open up novel 3D in vitro culture techniques to better mimic the complexity of tissues.

Generation of 3D melanoma models using an assembloid-based approach.

While 3D tumor models have greatly evolved over the past years, there is still a strong requirement for more biosimilar models which are capable of recapitulating cellular crosstalk within the tumor microenvironment while equally displaying representative levels of tumor aggressiveness and invasion. Herein, we disclose an assembloid melanoma model based on the fusion of individual stromal multicellular spheroids (MCSs). In contrast to more traditional tumor models, we show that it is possible to develop self-organizing, heterotypic melanoma models where tumor cells present stem-cell like features like up-regulated pluripotency master regulators SOX2, POU5F1 and NANOG. Additionally, these assembloids display high levels of invasiveness while embedded in 3D matrices as evidenced by stromal cell promotion of melanoma cell invasion via metalloproteinase production. Furthermore, sensitivity to anticancer drug doxorubicin was demonstrated for the melanoma assembloid model. These findings suggest that melanoma assembloids may play a significant role in the field of 3D cancer models as they more closely mimic the tumor microenvironment when compared to more traditional MCSs, opening the doors to a better understanding of the role of tumor microenvironment in supporting tumor progression. STATEMENT OF SIGNIFICANCE: The development of complex 3D tumor models that better recapitulate the tumor microenvironment is crucial for both an improved comprehension of intercellular crosstalk and for more efficient drug screening. We have herein developed a self-organizing heterotypic assembloid-based melanoma model capable of closely mimicking the tumor microenvironment. Key features recapitulated were the preservation of cancer cell stemness, sensitivity to anti-cancer agents and tumor cell invasion promoted by stromal cells. The approach of pre-establishing distinct stromal domains for subsequent combination into more complex tumor constructs provides a route for developing superior tumor models with a higher degree of similarity to native cancer tissues.

Prevascularized spongy-like hydrogels maintain their angiogenic potential after prolonged hypothermic storage.

The chronic shortage of organs and tissues for transplantation represents a dramatic burden on healthcare systems worldwide. Tissue engineering offers a potential solution to address these shortages, but several challenges remain, with prevascularization being a critical factor for in vivo survival and integration of tissue engineering products. Concurrently, a different challenge hindering the clinical implementation of such products, regards their efficient preservation from the fabrication site to the bedside. Hypothermia has emerged as a potential solution for this issue due to its milder effects on biologic systems in comparison with other cold preservation methodologies. Its impact on prevascularization, however, has not been well studied. In this work, 3D prevascularized constructs were fabricated using adipose-derived stromal vascular fraction cells and preserved at 4 °C using Hypothermosol or basal culture media (α-MEM). Hypothermosol efficiently preserved the structural and cellular integrity of prevascular networks as compared to constructs before preservation. In contrast, the use of α-MEM led to a clear reduction in prevascular structures, with concurrent induction of high levels of apoptosis and autophagy at the cellular level. In vivo evaluation using a chorioallantoic membrane model demonstrated that, in opposition to α-MEM, Hypothermosol preservation retained the angiogenic potential of constructs before preservation by recruiting a similar number of blood vessels from the host and presenting similar integration with host tissue. These results emphasize the need of studying the impact of preservation techniques on key properties of tissue engineering constructs such as prevascularization, in order to validate and streamline their clinical application.

Pre-selection of fibroblast subsets prompts prevascularization of tissue engineered skin analogues.

The papillary and reticular dermis harbors phenotypically distinct fibroblasts, whose functions such as maintenance of skin's microvasculature are also distinct. Thus, we hypothesized that pre-selection of the subpopulations of fibroblasts would benefit the generation of skin tissue engineered (TE) constructs, promoting their prevascularization in vitro. We first isolated papillary and reticular fibroblasts using fluorescence-activated cell sorting and studied the effect of their secretome and extracellular matrix (ECM) on human dermal microvascular endothelial cell (hDMEC) organization. Subsequently, we developed a bilayered 3D polymeric structure with distinct layer-associated features to house the subpopulations of fibroblasts, to generate a skin analogue. Both papillary and reticular fibroblasts were able to stimulate capillary-like network formation in a Matrigel assay. However, the secretome of the two subpopulations was substantially different, being enriched in VEGF, IGF-1, and Angio-1 in the case of papillary fibroblasts and in HGF and FGF-2 for the reticular subset. In addition, the fibroblast subpopulations deposited varied levels of ECM proteins, more collagen I and laminin was produced by the reticular subset, but these differences did not impact hDMEC organization. Vessel-like structures with lumens were observed earlier in the 3D skin analogue prepared with the sorted fibroblasts, although ECM deposition was not affected by the cell's pre-selection. Moreover, a more differentiated epidermal layer was obtained in the skin analogue formed by the sorted fibroblasts, confirming that its whole structure was not affected. Overall, we provide evidence that pre-selection of papillary and reticular fibroblasts is relevant for promoting the in vitro prevascularization of skin TE constructs.

Gellan gum-based hydrogels support the recreation of the dermal papilla microenvironment.

The dermal papilla (DP), a specialized compartment within the hair follicle, regulates hair growth. However, human DP cells rapidly lose their inductivity in 2D-culture given the loss of positional and microenvironmental cues. Spheroids have been capable of recreating the 3D intercellular organization of DP cells, however, DP cell-matrix interactions are poorly represented. Considering the specific nature of the DP's extracellular matrix (ECM), we functionalized gellan gum (GG) with collagen IV-(HepIII) or fibronectin-(cRGDfC) derived peptide sequences to generate a 3D environment in which the phenotype and physiological functions of DP cells are restored. We further tuned the stiffness of the microenvironments by varying GG amount. Biomimetic peptides in stiffer hydrogels promoted the adhesion of DP cells, while each peptide and amount of polymer independently influenced the type and quantity of ECM proteins deposited. Furthermore, although peptides did not seem to have an influence, stiffer hydrogels improved the inductive capacity of DP cells after short term culture. Interestingly, independently of the peptide, these hydrogels supported the recapitulation of basic hair morphogenesis-like events when incorporated in an organotypic human skin in vitro model. Our work demonstrates that tailored GG hydrogels support the generation of a microenvironment in which both cell-ECM and cell-cell interactions positively influence DP cells towards the creation of an artificial DP.

Vascularization in skin wound healing: where do we stand and where do we go?

Cutaneous healing is a highly complex process that, if altered due to, for example, impaired vascularization, results in chronic wounds or repaired neotissue of poor quality. Significant progress has been achieved in promoting neotissue vascularization during tissue repair/regeneration. In this review, we discuss the strategies that have been explored and how each one of them contributes to regulate vascularization in the context of cutaneous wound healing from two different perspectives - biomaterial-based and a cell-based approaches. Finally, we discuss the implications of these findings on the development of the 'next generation' approaches to target vascularization in wound healing highlighting the importance of going beyond its contribution to regulate vascularization and take into consideration the temporal features of the healing process and of different types of wounds.

Recreation of a hair follicle regenerative microenvironment: Successes and pitfalls.

The hair follicle (HF) is an exquisite skin appendage endowed with cyclical regenerative capacity; however, de novo follicle formation does not naturally occur. Consequently, patients suffering from extensive skin damage or hair loss are deprived of the HF critical physiological and/or aesthetic functions, severally compromising skin function and the individual's psychosocial well-being. Translation of regenerative strategies has been prevented by the loss of trichogenic capacity that relevant cell populations undergo in culture and by the lack of suitable human-based in vitro testing platforms. Here, we provide a comprehensive overview of the major difficulties associated with HF regeneration and the approaches used to overcome these drawbacks. We describe key cellular requirements and discuss the importance of the HF extracellular matrix and associated signaling for HF regeneration. Finally, we summarize the strategies proposed so far to bioengineer human HF or hair-bearing skin models and disclose future trends for the field.

Pushing the Natural Frontier: Progress on the Integration of Biomaterial Cues toward Combinatorial Biofabrication and Tissue Engineering.

The engineering of fully functional, biological-like tissues requires biomaterials to direct cellular events to a near-native, 3D niche extent. Natural biomaterials are generally seen as a safe option for cell support, but their biocompatibility and biodegradability can be just as limited as their bioactive/biomimetic performance. Furthermore, integrating different biomaterial cues and their final impact on cellular behavior is a complex equation where the outcome might be very different from the sum of individual parts. This review critically analyses recent progress on biomaterial-induced cellular responses, from simple adhesion to more complex stem cell differentiation, looking at the ever-growing possibilities of natural materials modification. Starting with a discussion on native material formulation and the inclusion of cell-instructive cues, the roles of shape and mechanical stimuli, the susceptibility to cellular remodeling, and the often-overlooked impact of cellular density and cell-cell interactions within constructs, are delved into. Along the way, synergistic and antagonistic combinations reported in vitro and in vivo are singled out, identifying needs and current lessons on the development of natural biomaterial libraries to solve the cell-material puzzle efficiently. This review brings together knowledge from different fields envisioning next-generation, combinatorial biomaterial development toward complex tissue engineering.

Keratinocyte Growth Factor-Based Strategies for Wound Re-Epithelialization.

Wound re-epithelialization is a dynamic process that comprises the formation of new epithelium through an active signaling network between several growth factors (GFs) and various cell types. The main players are keratinocytes (KCs) that migrate from the wound edges over the wound bed to restore the epidermal barrier. One of the most important molecules involved in the re-epithelialization process is keratinocyte growth factor (KGF), a central player on promoting both migration and proliferation of KCs. Stromal cells, such as dermal fibroblasts, are the main producers of this factor, acting on KCs through paracrine signaling. Multiple therapeutic strategies to deliver KGF have been proposed to boost wound healing by targeting re-epithelialization. Different approaches have been explored to attain that purpose, such as topical application of this factor, controlled release of KGF from different biomaterials (hydrogels, nanoparticles, and membranes), and also gene delivery techniques. Among these strategies, KGF release via biomaterials- and genetic-based strategies shows great effectiveness in maintaining sustained KGF levels at the wound site, which is reflected in an efficient wound closure. Under this scope, this review aims not only to elucidate the potential of KGF in wound re-epithelialization but also to describe the underlying mechanism of action and further explore the therapeutic approaches using this GF. Impact statement Upon skin injury, wound re-epithelialization is one of the major milestones of the healing process. This is especially difficult to achieve on hard-to-heal wounds that are often open for long periods, as the dysregulation of the growth factors involved in this response contributes to an impaired proliferation and migration of keratinocytes. Keratinocyte growth factor (KGF) plays a central role in this problematic, as it is a potent factor that in the normal healing scenario promotes direct proliferation and migration of epidermal cells, consequently impacting re-epithelialization. Under this context, in the first part of this review, the process of wound healing and the mechanism of action of KGF are described. In the second part, various KGF delivery approaches aiming at skin re-epithelialization are reported and actively discussed. In this sense, it is herein highlighted the role of KGF in wound re-epithelialization and provided a critical overview of potential therapeutic strategies exploited so far.

Bioinks Enriched with ECM Components Obtained by Supercritical Extraction.

Extracellular matrix (ECM)-based bioinks have been steadily gaining interest in the field of bioprinting to develop biologically relevant and functional tissue constructs. Herein, we propose the use of supercritical carbon dioxide (scCO2) technology to extract the ECM components of cell-sheets that have shown promising results in creating accurate 3D microenvironments replicating the cell's own ECM, to be used in the preparation of bioinks. The ECM extraction protocol best fitted for cell sheets was defined by considering efficient DNA removal with a minor effect on the ECM. Cell sheets of human dermal fibroblasts (hDFbs) and adipose stem cells (hASCs) were processed using a customised supercritical system by varying the pressure of the reactor, presence, exposure time, and type of co-solvent. A quantification of the amount of DNA, protein, and sulfated glycosaminoglycans (sGAGs) was carried out to determine the efficiency of the extraction in relation to standard decellularization methodologies. The bioinks containing the extracted ECM were fabricated by combining them with alginate as a support polymer. The influence of the alginate (1%, 2% w/vol) and ECM (0.5% and 1.5% w/vol) amounts on the printability of the blends was addressed by analysing the rheological behaviour of the suspensions. Finally, 3D printed constructs were fabricated using an in-house built extrusion-based bioprinter, and the impact of the extrusion process on cell viability was assessed. The optimised scCO2 protocol allowed efficient removal of DNA while preserving a higher number of proteins and sGAGs than the standard methodologies. The characterization of extract's composition also revealed that the ECM produced by hDFbs (fECM) and hASCs (aECM) is distinctively affected by the extraction protocols. Furthermore, rheological analysis indicated an increase in viscosity with increasing ECM composition, an effect even more prominent in samples containing aECM. 3D printing of alginate/ECM constructs demonstrated that cell viability was only marginally affected by the extrusion process, and this effect was also dependent on the ECM source. Overall, this work highlights the benefits of supercritical fluid-based methods for ECM extraction and strengthens the relevance of ECM-derived bioinks in the development of printed tissue-like constructs.

Highly tailorable gellan gum nanoparticles as a platform for the development of T cell activator systems.

BACKGROUND: T cell priming has been shown to be a powerful immunotherapeutic approach for cancer treatment in terms of efficacy and relatively weak side effects. Systems that optimize the stimulation of T cells to improve therapeutic efficacy are therefore in constant demand. A way to achieve this is through artificial antigen presenting cells that are complexes between vehicles and key molecules that target relevant T cell subpopulations, eliciting antigen-specific T cell priming. In such T cell activator systems, the vehicles chosen to deliver and present the key molecules to the targeted cell populations are of extreme importance. In this work, a new platform for the creation of T cell activator systems based on highly tailorable nanoparticles made from the natural polymer gellan gum (GG) was developed and validated. METHODS: GG nanoparticles were produced by a water in oil emulsion procedure, and characterized by dynamic light scattering, high resolution scanning electronic microscopy and water uptake. Their biocompatibility with cultured cells was assessed by a metabolic activity assay. Surface functionalization was performed with anti-CD3/CD28 antibodies via EDC/NHS or NeutrAvidin/Biotin linkage. Functionalized particles were tested for their capacity to stimulate CD4+ T cells and trigger T cell cytotoxic responses. RESULTS: Nanoparticles were approximately 150 nm in size, with a stable structure and no detectable cytotoxicity. Water uptake originated a weight gain of up to 3200%. The functional antibodies did efficiently bind to the nanoparticles, as confirmed by SDS-PAGE, which then targeted the desired CD4+ populations, as confirmed by confocal microscopy. The developed system presented a more sustained T cell activation over time when compared to commercial alternatives. Concurrently, the expression of higher levels of key cytotoxic pathway molecules granzyme B/perforin was induced, suggesting a greater cytotoxic potential for future application in adoptive cancer therapy. CONCLUSIONS: Our results show that GG nanoparticles were successfully used as a highly tailorable T cell activator system platform capable of T cell expansion and re-education.

Development of an antibiotics delivery system for topical treatment of the neglected tropical disease Buruli ulcer.

Skin infection by Mycobacterium ulcerans causes Buruli ulcer (BU) disease, a serious condition that significantly impact patient' health and quality of life and can be very difficult to treat. Treatment of BU is based on daily systemic administration of antibiotics for at least 8 weeks and presents drawbacks associated with the mode and duration of drug administration and potential side effects. Thus, new therapeutic strategies are needed to improve the efficacy and modality of BU therapeutics, resulting in a more convenient and safer antibiotic regimen. Hence, we developed a dual delivery system based on poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) microparticles and a gellan gum (GG) hydrogel for delivery of rifampicin (RIF) and streptomycin (STR), two antibiotics used for BU treatment. RIF was successfully loaded into PHBV microparticles, with an encapsulation efficiency of 43%, that also revealed a mean size of 10 µm, spherical form and rough topography. These microparticles were further embedded in a GG hydrogel containing STR. The resultant hydrogel showed a porous microstructure that conferred a high water retention capability (superior to 2000%) and a controlled release of both antibiotics. Also, biological studies revealed antibacterial activity against M. ulcerans, and a good cytocompatibility in a fibroblast cell line. Thus, the proposed drug delivery system can constitute a potential topical approach for treatment of skin ulcers caused by BU disease.