In-Hydrogel Cell-Free Protein Expression System as Biocompatible and Implantable Biomaterial.

Biomaterials capable of delivering therapeutic proteins are relevant in biomedicine, yet their manufacturing relies on centralized manufacturing chains that pose challenges to their remote implementation at the point of care. This study explores the viability of confined cell-free protein synthesis within porous hydrogels as biomaterials that dynamically produce and deliver proteins to in vitro and in vivo biological microenvironments. These functional biomaterials have the potential to be assembled as implants at the point of care. To this aim, we first entrap cell-free extracts (CFEs) from Escherichia coli containing the transcription-translation machinery, together with plasmid DNA encoding the super folded green fluorescence protein (sGFP) as a model protein, into hydrogels using various preparation methods. Agarose hydrogels result in the most suitable biomaterials to confine the protein synthesis system, demonstrating efficient sGFP production and diffusion from the core to the surface of the hydrogel. Freeze-drying (FD) of agarose hydrogels still allows for the synthesis and diffusion of sGFP, yielding a more attractive biomaterial for its reconstitution and implementation at the point of care. FD-agarose hydrogels are biocompatible in vitro, allowing for the colonization of cell microenvironments along with cell proliferation. Implantation assays of this biomaterial in a preclinical mouse model proved the feasibility of this protein synthesis approach in an in vivo context and indicated that the physical properties of the biomaterials influence their immune responses. This work introduces a promising avenue for biomaterial fabrication, enabling the in vivo synthesis and targeted delivery of proteins and opening new paths for advanced protein therapeutic approaches based on biocompatible biomaterials.

Polycaprolactone with multiscale porosity and patterned surface topography prepared using sacrificial 3D printed moulds: Towards tailor-made scaffolds.

Biocompatible three-dimensional porous scaffolds are widely used in multiple biomedical applications. However, the fabrication of tailor-made 3D structures with controlled and combined multiscale macroscopic-microscopic, surface and inner porosities in a straightforward manner is still a current challenge. Herein, we use multimaterial fused deposition modeling (FDM) to generate poly (vinyl alcohol) (PVA) sacrificial moulds filled with poly (Ɛ-caprolactone) (PCL) to generate well defined PCL 3D objects. Further on, the supercritical CO2 (SCCO2) technique, as well as the breath figures mechanism (BFs), were additionally employed to fabricate specific porous structures at the core and surfaces of the 3D PCL object, respectively. The biocompatibility of the resulting multiporous 3D structures was tested in vitro and in vivo, and the versatility of the approach was assessed by generating a vertebra model fully tunable at multiple pore size levels. In sum, the combinatorial strategy to generate porous scaffolds offers unique possibilities to fabricate intricate structures by combining the advantages of additive manufacturing (AM), which provides flexibility and versatility to generate large sized 3D structures, with advantages of the SCCO2 and BFs techniques, which allow to finely tune the macro and micro porosity at material surface and material core levels.

Decellularization of xenografted tumors provides cell-specific in vitro 3D environment.

In vitro cell culture studies are common in the cancer research field, and reliable biomimetic 3D models are needed to ensure physiological relevance. In this manuscript, we hypothesized that decellularized xenograft tumors can serve as an optimal 3D substrate to generate a top-down approach for in vitro tumor modeling. Multiple tumor cell lines were xenografted and the formed solid tumors were recovered for their decellularization by several techniques and further characterization by histology and proteomics techniques. Selected decellularized tumor xenograft samples were seeded with the HCC1806 human triple-negative breast cancer (TNBC) basal-like subtype cell line, and cell behavior was compared among them and with other control 2D and 3D cell culture methods. A soft treatment using Freeze-EDTA-DNAse allows proper decellularization of xenografted tumor samples. Interestingly, proteomic data show that samples decellularized from TNBC basal-like subtype xenograft models had different extracellular matrix (ECM) compositions compared to the rest of the xenograft tumors tested. The in vitro recellularization of decellularized ECM (dECM) yields tumor-type-specific cell behavior in the TNBC context. Data show that dECM derived from xenograft tumors is a feasible substrate for reseeding purposes, thereby promoting tumor-type-specific cell behavior. These data serve as a proof-of-concept for further potential generation of patient-specific in vitro research models.

Influence of gelatin and collagen incorporation on peroxidase-mediated injectable pectin-based hydrogel and bioactivity of fibroblasts.

Pectin has recently attracted increasing attention for biomedical and pharmaceutical applications. Due to the lack of adhesion molecules in polysaccharides, phenolic hydroxyl conjugated gelatin was added to enzymatically-gellable peroxidase-modified pectin derivative and compared with phenolic hydroxyl -pectin/collagen. Both pectin and gelatin were modified by tyramine hydrochloride in the presence of EDC/NHS. The phenolic hydroxyl -pectin/phenolic hydroxyl -gelatin, phenolic hydroxyl-pectin/collagen, and phenolic hydroxyl -pectin hydrogels were prepared using horseradish peroxidase and hydrogen peroxide. The hydrogels were characterized by gelation time analysis. Morphology, enzymatic biodegradation, mechanical and swelling properties as well as water vapor transmission rate were also evaluated. Fibroblasts were cultured for 7 days, and the survival rate was evaluated using conventional MTT assay. Hydrogels composed of Ph-pectin/Ph-gelatin showed decreased biodegradation rate, and WVTR and further improved mechanical performance in comparison with other groups. Both phenolic hydroxyl -pectin/collagen and phenolic hydroxyl -pectin/phenolic hydroxyl -gelatin hydrogels exhibited porous structures. The hydrogels composed of collagen promoted cell survival rate 1.4 and 3.5 times compared to phenolic hydroxyl -gelatin and phenolic hydroxyl -pectin based hydrogels at the end of 7 days, respectively (p < 0.001). The study demonstrated the potential of enzymatically-gellable pectin-based hydrogels as cost-effective frameworks for use in tissue engineering applications.

Hollow Alginate-Poly-L-Lysine-Alginate Microspheres Promoted an Epithelial-Mesenchymal Transition in Human Colon Adenocarcinoma Cells.

Purpose: Today, there is an urgent need to develop a three-dimentional culture systems mimicking native in vivo condition in order to screen potency of drugs and possibly any genetic alterations in tumor cells. Due to the existence of limitations in animal models, the development of three dimensional systems is highly recommended. To this end, we encapsulated human colon adenocarcinoma cell line HT29 with alginate-poly-L-lysine (Alg-PLL) microspheres and the rate of epithelial-mesenchymal transition was monitored. Methods: Cells were randomly divided into three groups; control, alginate and Alg-PLL. To encapsulate cells, we mixed HT-29 cells (1 × 106 ) with 1 mL of 0.05% PLL and 1% Alg mixture and electrosprayed into CaCl2 solution by using a high-voltage power. Cells from all groups were maintained at 37˚C in a humidified atmosphere containing 5% CO2 for 7 days. Cell viability was assessed by MTT assay. To monitor the stemness feature, we measured the transcription of genes such as Snail, Zeb, and Vimentin by using real-time PCR analysis. Results: Addition of PLL to Alg in a hallowed state increased the cell survival rate compared to the control and Alg groups (P<0.05). Cells inside Alg-PLL tended to form microcellular aggregates while in Alg microspheres an even distribution of HT-29 cells was found. Real-time PCR analysis showed the up-regulation of Snail, Zeb, and Vimentin in Alg-PLL microspheres compared to the other groups, showing the acquisition of stemness feature (P<0.05). Conclusion: This study showed that hallow Alg-PLL microspheres increased the epithelialmesenchymal transition rate after 7 days in in vitro condition. Such approaches could be touted as appropriate in vitro models for drug screening.

Comparative study of collagen and gelatin in chitosan-based hydrogels for effective wound dressing: Physical properties and fibroblastic cell behavior.

The influence of collagen as an effective substitute for gelatin was investigated on properties of chitosan/gelatin hydrogels for fibroblasts growth and attachment for wound dressing applications. We synthesized hydrogels based on chitosan associated with collagen and gelatin biopolymers (in the ratio of 1:5 and 1:1, respectively). The hydrogels properties such as morphology, swelling ratio, mechanical characteristics, water vapor loss, water vapor transmission rate (WVTR), and biodegradation were analyzed. 1 × 105 human fibroblasts were seeded per ml of hydrogels and maintained for 7 days. Cell viability was assessed by using MTT. The presence of collagen caused reduced swelling ratio, and biodegradation rate compared to chitosan/gelatin hydrogels (p < 0.05). The introduction of collagen into chitosan hydrogels improved the mechanical strength compared to gelatin. Hydrogels with collagen possessed an optimum WVTR compared to the chitosan group and hydrogels with gelatin (p < 0.05). Analyzing the morphology of hydrogels revealed that the addition of collagen leads to a homogenous and interconnected structure. Collagen impregnation promoted cell survival and attachment compared with chitosan hydrogels after 7 days (p < 0.05). Collectively, these results demonstrated the potential of the chitosan/collagen hydrogels for wound dressing applications.

Collagen-alginate-nano-silica microspheres improved the osteogenic potential of human osteoblast-like MG-63 cells.

Modular bone tissue engineering is touted as an alternative approach to replace the damaged bone tissue. Hydrogel microcapsules could promote therapeutic properties by providing 3D condition and an increased cell-to-cell interaction. We investigated the osteogenic properties of alginate-nano-silica hydrogels enriched with collagen and gelatin on human osteoblast-like MG-63 cells. For encapsulation, cells were divided into three groups; control (alginate+ nano-silica), collagen (alginate + collagen + nano-silica), and gelatin (alginate + gelatin + nano-silica) and expanded for 28 days. Cell survival was determined by trypan blue staining and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. To confirm the osteogenic potential, we measured the alkaline phosphatase activity. Alizarin red S staining was used to reveal the existence of hydroxyapatite and transcription BMP-2, osteocalcin and osteonectin evaluated by the real-time polymerase chain reaction. Collagen substrate caused a reduced swelling ratio compared with the control and gelatin groups (P < 0.05). Compared with other groups, collagen had potential to improve mechanical strength and generate porous membrane structure. The addition of collagen (4-fold) and gelatin (1.5-fold) increased cell proliferation rate compared with the control (P < 0.05). Biochemical analysis and Alizarin red S staining showed that collagen-induced osteogenesis by induction of alkaline phosphatase and matrix mineralization. The expression of osteocalcin and BMP-2 was increased in cells from the collagen group. As a result, the combination of natural polymers collagen and gelatin with alginate + nano-silica can increase the osteogenic potential of human osteoblasts.