3D nanofiber scaffolds from 2D electrospun membranes boost cell penetration and positive host response for regenerative medicine.

The ideal tissue engineering scaffold should facilitate rapid cell infiltration and provide an optimal immune microenvironment during interactions with the host. Electrospinning can produce two-dimensional (2D) membranes mimicking the extracellular matrix. However, their dense structure hinders cell penetration, and their thin form restricts scaffold utility. In this study, latticed hydrogels were three-dimensional (3D) printed onto electrospun membranes. This technique allowed for layer-by-layer assembly of the membranes into 3D scaffolds, which maintained their resilience impressively under both dry and wet conditions. We assessed the cellular and host responses of these 3D nanofiber scaffolds by comparing random membranes and mesh-like membranes with three different mesh sizes (250, 500, and 750 µm). It was found that scaffolds with a mesh size of 500 µm were superior for M2 macrophage phenotype polarization, vascularization, and matrix deposition. Furthermore, it was confirmed by subsequent experiments such as RNA sequencing that the mesh-like topology may promote polarization to the M2 phenotype by affecting the PI3K/AKT pathway. In conclusion, our work offers a novel method for transforming 2D nanofiber membranes into 3D scaffolds. This method boasts flexibility, allowing for the use of varied electrospun membranes and hydrogels in terms of structure and composition. It has vast potential in tissue repair and regeneration.

Three-Dimensional Objects Consisting of Hierarchically Assembled Nanofibers with Controlled Alignments for Regenerative Medicine.

Assembling electrospun nanofibers with controlled alignment into three-dimensional (3D), complex, and predesigned shapes has proven to be a difficult task for regenerative medicine. Herein, we report a novel approach inspired by solids of revolution that transforms two-dimensional (2D) nanofiber mats of a controlled thickness into once-inaccessible 3D objects with predesigned shapes. The 3D objects are highly porous, consisting of layers of aligned nanofibers separated by gaps ranging from several micrometers to several millimeters. Upon compression, the objects are able to recover their original shapes. The porous objects can serve as scaffolds, guiding the organization of cells and producing highly ordered 3D tissue constructs. Additionally, subcutaneous implantation in rats demonstrates that the 3D objects enable rapid cell penetration, new blood vessel formation, and collagen matrix deposition. This new class of 3D hierarchical nanofiber architectures offers promising advancements in both in vitro engineering of complex 3D tissue constructs/models or organs and in vivo tissue repair and regeneration.

Diatom-guided bone healing via a hybrid natural scaffold.

Bone tissue engineering (BTE) involves the design of three-dimensional (3D) scaffolds that aim to address current challenges of bone defect healing, such as limited donor availability, disease transmission risks, and the necessity for multiple invasive surgeries. Scaffolds can mimic natural bone structure to accelerate the mechanisms involved in the healing process. Herein, a crosslinked combination of biopolymers, including gelatin (GEL), chitosan (CS), and hyaluronic acid (HA), loaded with diatom (Di) and ß-sitosterol (BS), is used to produce GCH-Di-S scaffold by freeze-drying method. The GCH scaffold possesses a uniform structure, is biodegradable and biocompatible, and exhibits high porosity and interconnected pores, all required for effective bone repair. The incorporation of Di within the scaffold contributes to the adjustment of porosity and degradation, as well as effectively enhancing the mechanical property and biomineralization. In vivo studies have confirmed the safety of the scaffold and its potential to stimulate the creation of new bone tissue. This is achieved by providing an osteoconductive platform for cell attachment, prompting calcification, and augmenting the proliferation of osteoblasts, which further contributes to angiogenesis and anti-inflammatory effects of BS.

Biomimetic short fiber reinforced 3-dimensional scaffold for bone tissue regeneration.

Bone defects caused by diseases and trauma are considered serious clinical challenges. Autologous and allogeneic transplantations are the most widely used methods to mitigate bone defects. However, transplantation poses risks such as secondary trauma, immune rejection, and disease transmission to patients. Preparing a biologically active bone tissue engineering scaffold as a bone substitute can overcome this problem. In the current study, a PLGA/gelatin (Gel) short fiber-reinforced composite three-dimensional (3D) scaffold was fabricated by electrospinning for bone tissue defect repair. A hybrid scaffold adding inorganic materials hydrotalcite (CaAl-LDH) and osteogenic factors deferoxamine (DFO) based on PLGA and Gel composite filaments was prepared. The structure, swelling, drug release, and compressive resilience performance of the 3D scaffolds in a wet state were characterized and the osteogenic effect of the crosslinked scaffold (C-DLPG) was also investigated. The scaffold has shown the optimum physicochemical attributes which still has 380 kPa stress after a 60% compression cycle and sustainedly released the drug for about twenty days. Moreover, a promisingIn vivoosteogenic performance was noted with better tissue organization. At 8 weeks after implantation, the C-DLPG scaffold could fill the bone defect site, and the new bone area reached 19 mm2. The 3D microfiber scaffold, in this study, is expected to be a promising candidate for the treatment of bone defects in the future.

Enhancing cartilage regeneration and repair through bioactive and biomechanical modification of 3D acellular dermal matrix.

Acellular dermal matrix (ADM) shows promise for cartilage regeneration and repair. However, an effective decellularization technique that removes cellular components while preserving the extracellular matrix, the transformation of 2D-ADM into a suitable 3D scaffold with porosity and the enhancement of bioactive and biomechanical properties in the 3D-ADM scaffold are yet to be fully addressed. In this study, we present an innovative decellularization method involving 0.125% trypsin and 0.5% SDS and a 1% Triton X-100 solution for preparing ADM and converting 2D-ADM into 3D-ADM scaffolds. These scaffolds exhibit favorable physicochemical properties, exceptional biocompatibility and significant potential for driving cartilage regeneration in vitro and in vivo. To further enhance the cartilage regeneration potential of 3D-ADM scaffolds, we incorporated porcine-derived small intestinal submucosa (SIS) for bioactivity and calcium sulfate hemihydrate (CSH) for biomechanical reinforcement. The resulting 3D-ADM+SIS scaffolds displayed heightened biological activity, while the 3D-ADM+CSH scaffolds notably bolstered biomechanical strength. Both scaffold types showed promise for cartilage regeneration and repair in vitro and in vivo, with considerable improvements observed in repairing cartilage defects within a rabbit articular cartilage model. In summary, this research introduces a versatile 3D-ADM scaffold with customizable bioactive and biomechanical properties, poised to revolutionize the field of cartilage regeneration.

Fibroblasts/three-dimensional scaffolds complexes promote wound healing in rats with skin defects.

We aim to construct a three-dimensional nano-skin scaffold material in vitro and study its promoting effect on wound healing in vivo. In this study, hybrid constructs of three-dimensional (3D) scaffolds were successfully fabricated by combination of type I collagen (COL-1) and polylactic-glycolic acid (PLGA). Fibroblasts and human umbilical cord mesenchymal stem cells (hUCMSCs) were used to implanted into 3D scaffolds and constructed into SD skin scaffolds in vitro. Finally, the fibroblasts/scaffolds complexes were inoculated on the surface of rat wound skin to study the promoting effect of the complex on wound healing. In our study, we successfully built a 3D scaffold, which had a certain porosity. Meanwhile, the content of COL-1 in the cell supernatant of fibroblast/scaffold complexes was increased. Furthermore, the expression of F-actin, CD105, integrin ß, VEGF, and COL-1 was up-regulated in hUCMSC/scaffold complexes compared with the control group. In vivo, fibroblast/scaffold complexes promoted wound healing in rats. Our data suggested that the collagen Ⅳ and vimentin were elevated and collagen fibers were neatly arranged in the fibroblast/scaffold complex group was significantly higher than that in the scaffold group. Taken together, fibroblast/scaffold complexes were expected to be novel materials for treating skin defects.

Stiffer-Matrix-Induced PGC-1α Upregulation Enhanced Mitochondrial Biogenesis and Oxidative Stress Resistance in Non-small Cell Lung Cancer.

Introduction: Metabolic strategies in different microenvironments can affect cancer metabolic adaptation, ultimately influencing the therapeutic response. Understanding the metabolic alterations of cancer cells in different microenvironments is critical for therapeutic success. Methods: In this study, we cultured non-small cell lung cancer cells in three different microenvironments (two-dimensional (2D) plates, soft elastic three-dimensional (3D) porous 2 wt% scaffolds, and stiff elastic 3D porous 4 wt% scaffolds) to investigate the effects of different matrix elasticity as well as 2D and 3D culture settings on the metabolic adaptation of cancer cells. Results: The results revealed that PGC-1α expression is sensitive to the elasticity of the 3D scaffold. PGC-1α expression was markedly increased in cancer cells cultured in stiff elastic 3D porous 4 wt% scaffolds compared with cells cultured in soft elastic 3D porous 2 wt% scaffolds or 2D plates, enhancing mitochondrial biogenesis and oxidative stress resistance of non-small cell lung cancer through increased reactive oxygen species (ROS) detoxification capacity. However, phosphofructokinase-1 (PFK-1) expression, a key rate-limiting enzyme in glycolysis, did not change significantly in the three microenvironments, indicating that microenvironments may not affect the early stage of glycolysis. Conversely, monocarboxylate transporter 1 (MCT1) expression in 3D culture was significantly reduced compared to 2D culture but without significant difference between soft and stiff scaffolds, indicating that MCT1 expression is more sensitive to the shape of the different cultures of 2D and 3D microenvironment surrounding cells but is unaffected by the scaffold elasticity. Conclusions: Together, these results demonstrate that differences in the microenvironment of cancer cells profoundly impact their metabolic response.

3D Polycaprolactone/Gelatin-Oriented Electrospun Scaffolds Promote Periodontal Regeneration.

Periodontitis is a worldwide chronic inflammatory disease, where surgical treatment still shows an uncertain prognosis. To break through the dilemma of periodontal treatment, we fabricated a three-dimensional (3D) multilayered scaffold by stacking and fixing electrospun polycaprolactone/gelatin (PCL/Gel) fibrous membranes. The biomaterial displayed good hydrophilic and mechanical properties. Besides, we found human periodontal ligament stem cell (hPDLSC) adhesion and proliferation on it. The following scanning electron microscopy (SEM) and cytoskeleton staining results proved the guiding function of fibers to hPDLSCs. Then, we further analyzed periodontal regeneration-related proteins and mRNA expression between groups. In vivo results in a rat acute periodontal defect model confirmed that the topographic cues of materials could directly guide cellular orientation and might provide the prerequisite for further differentiation. In the aligned scaffold group, besides new bone regeneration, we also observed that angular concentrated fiber regeneration in the root surface of the defect is similar to the normal periodontal tissue. To sum up, we have constructed electrospun membrane-based 3D biological scaffolds, which provided a new treatment strategy for patients undergoing periodontal surgery.

Cellular Interaction of Bone Marrow Mesenchymal Stem Cells with Polymer and Hydrogel 3D Microscaffold Templates.

Biomimicking biological niches of healthy tissues or tumors can be achieved by means of artificial microenvironments, where structural and mechanical properties are crucial parameters to promote tissue formation and recreate natural conditions. In this work, three-dimensional (3D) scaffolds based on woodpile structures were fabricated by two-photon polymerization (2PP) of different photosensitive polymers (IP-S and SZ2080) and hydrogels (PEGDA 700) using two different 2PP setups, a commercial one and a customized one. The structures' properties were tuned to study the effect of scaffold dimensions (gap size) and their mechanical properties on the adhesion and proliferation of bone marrow mesenchymal stem cells (BM-MSCs), which can serve as a model for leukemic diseases, among other hematological applications. The woodpile structures feature gap sizes of 25, 50, and 100 µm and a fixed beam diameter of 25 µm, to systematically study the optimal cell colonization that promotes healthy cell growth and potential tissue formation. The characterization of the scaffolds involved scanning electron microscopy and mechanical nanoindenting, while their suitability for supporting cell growth was evaluated with live/dead cell assays and multistaining 3D confocal imaging. In the mechanical assays of the hydrogel material, we observed two different stiffness ranges depending on the indentation depth. Larger gap woodpile structures coated with fibronectin were identified as the most promising scaffolds for 3D BM-MSC cellular models, showing higher proliferation rates. The results indicate that both the design and the employed materials are suitable for further assays, where retaining the BM-MSC stemness and original features is crucial, including studies focused on BM disorders such as leukemia and others. Moreover, the combination of 3D scaffold geometry and materials holds great potential for the investigation of cellular behaviors in a co-culture setting, for example, mesenchymal and hematopoietic stem cells, to be further applied in medical research and pharmacological studies.

Carboxymethyl carrageenan immobilized on 3D-printed polycaprolactone scaffold for the adsorption of calcium phosphate/strontium phosphate adapted to bone regeneration.

Three dimensional (3D) substrates based on natural and synthetic polymers enhance the osteogenic and mechanical properties of the bone tissue engineering scaffolds. Here, a novel bioactive composite scaffolds from polycaprolactone /kappa-carrageenan were developed for bone regeneration applications. 3D PCL scaffolds were fabricated by 3D printing method followed by coating with carboxymethyl kappa-carrageenan. This organic film was used to create calcium and strontium phosphate layers via a modified alternate soaking process in CaCl 2 /SrCl 2 and Na2HPO4 solutions in which calcium ions were replaced by strontium, with different amounts of strontium in the solutions. Various characterization techniques were executed to analyze the effects of strontium ion on the scaffold properties. The morphological results demonstrated the highly porous with interconnected pores and uniform pore sizes scaffolds. It was indicated that the highest crystallinity and compressive strength were obtained when 100% CaCl2 was replaced by SrCl2 in the solution (P-C-Sr). Incorporation of Sr onto the structure increased the degradation rate of the scaffolds. Mesenchymal stem cells (MSCs) culture on the scaffolds showed that Sr effectively improved attachment and viability of the MSCs and accelerated osteogenic differentiation as revealed by Alkaline phosphatase activity, calcium content and Real Time-Reverse transcription polymerase chain reaction assays.

The Application of Polycaprolactone in Three-Dimensional Printing Scaffolds for Bone Tissue Engineering.

Bone tissue engineering commonly encompasses the use of three-dimensional (3D) scaffolds to provide a suitable microenvironment for the propagation of cells to regenerate damaged tissues or organs. 3D printing technology has been extensively applied to allow direct 3D scaffolds manufacturing. Polycaprolactone (PCL) has been widely used in the fabrication of 3D scaffolds in the field of bone tissue engineering due to its advantages such as good biocompatibility, slow degradation rate, the less acidic breakdown products in comparison to other polyesters, and the potential for loadbearing applications. PCL can be blended with a variety of polymers and hydrogels to improve its properties or to introduce new PCL-based composites. This paper describes the PCL used in developing state of the art of scaffolds for bone tissue engineering. In this review, we provide an overview of the 3D printing techniques for the fabrication of PCL-based composite scaffolds and recent studies on applications in different clinical situations. For instance, PCL-based composite scaffolds were used as an implant surgical guide in dental treatment. Furthermore, future trend and potential clinical translations will be discussed.

Biomaterials with Potential Use in Bone Tissue Regeneration-Collagen/Chitosan/Silk Fibroin Scaffolds Cross-Linked by EDC/NHS.

Blending of different biopolymers, e.g., collagen, chitosan, silk fibroin and cross-linking modifications of these mixtures can lead to new materials with improved physico-chemical properties, compared to single-component scaffolds. Three-dimensional scaffolds based on three-component mixtures of silk fibroin, collagen and chitosan, chemically cross-linked, were prepared and their physico-chemical and biological properties were evaluated. A mixture of EDC (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) was used as a cross-linking agent. FTIR was used to observe the position of the peaks characteristic for collagen, chitosan and silk fibroin. The following properties depending on the scaffold structure were studied: swelling behavior, liquid uptake, moisture content, porosity, density, and mechanical parameters. Scanning Electron Microscopy imaging was performed. Additionally, the biological properties of these materials were assessed, by metabolic activity assay. The results showed that the three-component mixtures, cross-linked by EDC/NHS and prepared by lyophilization method, presented porous structures. They were characterized by a high swelling degree. The composition of scaffolds has an influence on mechanical properties. All of the studied materials were cytocompatible with MG-63 osteoblast-like cells.

Gas foaming of electrospun poly(L-lactide-co-caprolactone)/silk fibroin nanofiber scaffolds to promote cellular infiltration and tissue regeneration.

Electrospun nanofibers emulate extracellular matrix (ECM) morphology and architecture; however, small pore size and tightly-packed fibers impede their translation in tissue engineering. Here we exploited in situ gas foaming to afford three-dimensional (3D) poly(L-lactide-co-ε-caprolactone)/silk fibroin (PLCL/SF) scaffolds, which exhibited nanotopographic cues and a multilayered structure. The addition of SF improved the hydrophilicity and biocompatibility of 3D PLCL scaffolds. Three-dimensional scaffolds exhibited larger pore size (38.75 ± 9.78 µm2) and high porosity (87.1% ± 1.5%) than that of their 2D counterparts. 3D scaffolds also improved the deposition of ECM components and neo-vessel regeneration as well as exhibited more numbers of CD163+/CCR7+ cells after 2 weeks implantation in a subcutaneous model. Collectively, 3D PLCL/SF scaffolds have broad implications for regenerative medicine and tissue engineering applications.

Three-dimensional porous gas-foamed electrospun nanofiber scaffold for cartilage regeneration.

To achieve optimal functional recovery of articular cartilage, scaffolds with nanofibrous structure and biological function have been widely pursued. In this study, two-dimensional electrospun poly(l-lactide-co-ε-caprolactone)/silk fibroin (PLCL/SF) scaffolds (2DS) were fabricated by dynamic liquid support (DLS) electrospinning system, and then cross-linked with hyaluronic acid (HA) to further mimic the microarchitecture of native cartilage. Subsequently, three-dimensional PLCL/SF scaffolds (3DS) and HA-crosslinked three-dimensional scaffolds (3DHAS) were successfully fabricated by in situ gas foaming and freeze-drying. 3DHAS exhibited better mechanical properties than that of the 3DS. Moreover, all scaffolds exhibited excellent biocompatibility in vitro. 3DHAS showed better proliferation and phenotypic maintenance of chondrocytes as compared to the other scaffolds. Histological analysis of cell-scaffold constructs explanted 8 weeks after implantation demonstrated that both 3DS and 3DHAS scaffolds formed cartilage-like tissues, and the cartilage lacuna formed in 3DHAS scaffolds was more mature. Moreover, the reparative capacity of scaffolds was discerned after implantation in the full-thickness articular cartilage model in rabbits for up to 12 weeks. The macroscopic and histological results exhibited typical cartilage-like character and well-integrated boundary between 3DHAS scaffolds and the host tissues. Collectively, biomimetic 3DHAS scaffolds may be promising candidates for cartilage tissue regeneration applications.

Advanced fabrication for electrospun three-dimensional nanofiber aerogels and scaffolds.

Electrospinning is a versatile strategy for creating nanofiber materials with various structures, which has broad application for a myriad of areas ranging from tissue engineering, energy harvesting, filtration and has become one of the most important academic and technical activities in the field of material science in recent years. In addition to playing a significant role in the construction of two-dimensional (2D) nanomaterials, electrospinning holds great promise as a robust method for producing three-dimensional (3D) aerogels and scaffolds. This article reviews and summarizes the recent advanced methods for fabricating electrospun three-dimensional nanofiber aerogels and scaffolds, including gas foaming, direct electrospinning of 3D nanofibrous scaffold, short nanofibers assembling into 3D aerogels/scaffolds, 3D printing, electrospray, origami and cell sheet engineering, centrifugal electrospinning, and other methods. Besides, intriguing formation process, crosslinking pathway, properties, and applications of 3D aerogels and scaffolds are also introduced. Taken together, these aerogels and scaffolds with various excellent features present tremendous potential in various fields.

A Polydopamine-Functionalized Carbon Microfibrous Scaffold Accelerates the Development of Neural Stem Cells.

Neuroregenerative medicine has witnessed impressive technological breakthroughs in recent years, but the currently available scaffold materials still have limitations regarding the development of effective treatment strategies for neurological diseases. Electrically conductive micropatterned materials have gained popularity in recent years due to their significant effects on neural stem cell fate. Polydopamine (PDA)-modified materials can also enhance the differentiation of neurons. In this work, we show that PDA-modified carbon microfiber skeleton composites have the appropriate conductivity, three-dimensional structure, and microenvironment regulation that are crucial for the growth of neural stem cells. The design we present is low-cost and easy to make and shows great promise for studying the growth and development of mouse neural stem cells. Our results show that the PDA-mediated formation of electrically conductive and viscous nanofiber webs promoted the adhesion, organization, and intercellular coupling of neural stem cells relative to the control group. PDA induced massive proliferation of neural stem cells and promoted the expression of Ki-67. Together, our results suggest that the composite material can be used as a multifunctional neural scaffold for clinical treatment and in vitro research by improving the structure, conductivity, and mechanical integrity of the regenerated tissues.

Tissue-engineered islet-like cell clusters generated from adipose tissue-derived stem cells on three-dimensional electrospun scaffolds can reverse diabetes in an experimental rat model and the role of porosity of scaffolds on cluster differentiation.

In the current study, three-dimensional (3D) nanofibrous scaffolds with pore sizes in the range of 24-250 µm and 24-190 µm were fabricated via a two-step electrospinning method to overcome the limitation of obtaining three-dimensionality with large pore sizes for islet culture using conventional electrospinning. The scaffolds supported the growth and differentiation of adipose-derived mesenchymal stem cells to islet-like clusters (ILCs). The pore size of the scaffolds was found to influence the cluster size, viability and insulin release of the differentiated islets. Hence, islet clusters of the desired size could be developed for transplantation to overcome the loss of bigger islets due to hypoxia which adversely impacts the outcome of transplantation. The tissue-engineered constructs with ILC diameter of 50 µm reduced glycemic value within 3-4 weeks after implantation in the omental pouch of diabetic rats. Detection of insulin in the serum of implanted rats demonstrates that the tissue-engineered construct is efficient to control hyperglycemia. Our findings prove that the 3D architecture and pore size of scaffolds regulates the morphology and size of islets during differentiation which is critical in the survival and function of ILCs in vitro and in vivo.

Enhanced Cancer DNA Vaccine via Direct Transfection to Host Dendritic Cells Recruited in Injectable Scaffolds.

Deoxyribonucleic acid (DNA) vaccines are a promising cancer immunotherapy approach. However, effective delivery of DNA to antigen-presenting cells (e.g., dendritic cells (DCs)) for the induction of an adaptive immune response is limited. Conventional DNA delivery via intramuscular, intradermal, and subcutaneous injection by hypodermal needles shows a low potency and immunogenicity. Here, we propose the enhanced cancer DNA vaccine by direct transfection to the high number of DCs recruited into the chemoattractant-loaded injectable mesoporous silica microrods (MSRs). Subcutaneous administration of the MSRs mixed with tumor-antigen coding DNA polyplexes resulted in DC recruitment in the macroporous space of the scaffold formed by the spontaneous assembly of high-aspect-ratio MSRs, thereby allowing for enhanced cellular uptake of antigen-coded DNA by host DCs. The MSR scaffolds delivering the DNA vaccine trigger a more robust DC activation, antigen-specific CD8+ T cell response, and Th1 immune response compared to the bolus DNA vaccine. Additionally, the immunological memory can be induced with a single administration of the vaccine. The combination of the vaccination and antiprogrammed cell death-1 antibody significantly eliminates established lung metastasis. These results indicate that MSRs serve as a powerful platform for DNA vaccine delivery to DCs for effective cancer immunotherapy.

Photocurable Albumin Methacryloyl Hydrogels as a Versatile Platform for Tissue Engineering.

Photopolymerization of protein-derived polymers functionalized with methacryloyl groups has been increasingly used to fabricate three-dimensional tissue constructs for biomedical applications because photocurable protein-based polymers (e.g., gelatin and collagen methacryloyl) feature spatial-temporal controllability of engineering complex constructs as well as inherent biological properties. Herein, we report photocurable albumin-based hydrogels. First, photocurable bovine serum albumin methacryloyl (BSA-MA) with different degrees of substitution (DM) was successfully synthesized in a precise manner, without substantially altering BSA native secondary structure. Resultant photocurable BSA-MA hydrogels exhibited tunable physio-biochemical properties over the swelling, degradation, and mechanical properties. Moreover, photo-cross-linked BSA-MA hydrogels provided a permissible environment to support cell viability and functionality both in two- and three-dimensional culture systems. Photocurable BSA-MA hydrogels may be used as a versatile platform for various bioapplications including tissue engineering and 3D bioprinting.

Development of novel three-dimensional scaffolds based on bacterial nanocellulose for tissue engineering and regenerative medicine: Effect of processing methods, pore size, and surface area.

Despite the efforts focused on manufacturing biological engineering scaffolds for tissue engineering and regenerative medicine, a biomaterial that meets the necessary characteristics for these applications has not been developed to date. Bacterial nanocellulose (BNC) is an outstanding biomaterial for tissue engineering and regenerative medicine; however, BNC's applications have been focused on two-dimensional (2D) medical devices, such as wound dressings. Given the need for three-dimensional (3D) porous biomaterials, this work evaluates two methods to generate (3D) BNC scaffolds. The structural characteristics and physicochemical, mechanical, and cell behaviour properties were evaluated. Likewise, the effects of the pore size and surface area in the mechanical performance of BNC biomaterials and their cell response in a fibroblast cell line are discussed for the first time. In this study, a new method is proposed for the development of 3D BNC scaffolds using paraffin wax. This new method is less time-consuming, more robust in removing the paraffin and less aggressive toward the BNC microstructure. Moreover, the biomaterial had regular porosity with good mechanical behaviour; the cells can adhere and increase in number without overcrowding. Regarding the pore size and surface area, highly interconnected porosities (measuring approximately 60 µm) and high surface area are advantageous for the biomaterial's mechanical properties and cell behaviour. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 348-359, 2019.