Versatile Potential of Photo-Cross-Linkable Silk Fibroin: Roadmap from Chemical Processing Toward Regenerative Medicine and Biofabrication Applications.

Over the past two decades, hydrogels have come to the forefront of tissue engineering and regenerative medicine due to their biocompatibility, tunable degradation and low immunogenicity. Due to their porosity and polymeric network built up, it is possible to incorporate inside drugs, bioactive molecules, or other biochemically active monomers. Among biopolymers used for the fabrication of functional hydrogels, silk fibroin (SF) has received considerable research attention owing to its known biocompatibility and tunable range of mechanical properties. However, its relatively simple structure limits the potential usability. One of the emerging strategies is a chemical functionalization of SF, allowing for the introduction of methacrylate groups. This allows the versatile processing capability, including photo-cross-linking, which makes SF a useful polymer as a bioink for 3D printing. The methacrylation reaction has been done using numerous monomers such as methacrylic anhydride (MA), 2-isocyanatoethyl methacrylate (IEM), or glycidyl methacrylate (GMA). In this Review, we summarize the chemical functionalization strategies of SF materials and their resulting physicochemical properties. More specifically, a brief explanation of the different functionalization methods, the cross-linking principles, possibilities, and limitations of methacrylate compound functionalization are provided. In addition, we describe types of functional SF hydrogels and link their design principles to the performance in applications in the broad fields of biofabrication, tissue engineering, and regenerative medicine. We anticipate that the provided guidelines will contribute to the future development of SF hydrogels and their composites by providing the rational design of new mechanisms linked to the successful realization of targeted biomedical application.

Heparin-Mimicking Polymer-Based In Vitro Platform Recapitulates In Vivo Muscle Atrophy Phenotypes.

The cell-cell/cell-matrix interactions between myoblasts and their extracellular microenvironment have been shown to play a crucial role in the regulation of in vitro myogenic differentiation and in vivo skeletal muscle regeneration. In this study, by harnessing the heparin-mimicking polymer, poly(sodium-4-styrenesulfonate) (PSS), which has a negatively charged surface, we engineered an in vitro cell culture platform for the purpose of recapitulating in vivo muscle atrophy-like phenotypes. Our initial findings showed that heparin-mimicking moieties inhibited the fusion of mononucleated myoblasts into multinucleated myotubes, as indicated by the decreased gene and protein expression levels of myogenic factors, myotube fusion-related markers, and focal adhesion kinase (FAK). We further elucidated the underlying molecular mechanism via transcriptome analyses, observing that the insulin/PI3K/mTOR and Wnt signaling pathways were significantly downregulated by heparin-mimicking moieties through the inhibition of FAK/Cav3. Taken together, the easy-to-adapt heparin-mimicking polymer-based in vitro cell culture platform could be an attractive platform for potential applications in drug screening, providing clear readouts of changes in insulin/PI3K/mTOR and Wnt signaling pathways.

Development of Composite Sponge Scaffolds Based on Carrageenan (CRG) and Cerium Oxide Nanoparticles (CeO2 NPs) for Hemostatic Applications.

In this study, a novel absorbable hemostatic agent was developed using carrageenan (CRG) as a natural polymer and cerium oxide nanoparticles (CeO2 NPs). CRG-CeO2-0.5 and CRG-CeO2-1 composites were prepared by compositing CeO2 to CRG + CeO2 at a weight ratio of 0.5:100 and 1:100, respectively. The physicochemical and structural properties of these compounds were studied and compared with pristine CRG. Upon incorporation of CeO2 nanoparticles into the CRG matrix, significant reductions in hydrogel degradation were observed. In addition, it was noted that CRG-CeO2 exhibited better antibacterial and hemostatic properties than CRG hydrogel without CeO2 NPs. The biocompatibility of the materials was tested using the NIH 3T3 cell line, and all samples were found to be nontoxic. Particularly, CRG-CeO2-1 demonstrated superior hemostatic effects, biocompatibility, and a lower degradation rate since more CeO2 NPs were present in the CRG matrix. Therefore, CRG-CeO2-1 has the potential to be used as a hemostatic agent and wound dressing.

Antimicrobial and Antibiofilm Properties of Latvian Honey against Causative Agents of Wound Infections.

Honey is widely used in traditional medicine and modern wound healing biomaterial research as a broad-spectrum antimicrobial, anti-inflammatory and antioxidant agent. The study's objectives were to evaluate the antibacterial activity and polyphenolic profiles of 40 monofloral honey samples collected from beekeepers in the territory of Latvia. The antimicrobial and antifungal activity of Latvian honey samples were compared with commercial Manuka honey and the honey analogue sugar solutions-carbohydrate mixture and tested against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, clinical isolates Extended-Spectrum Beta-Lactamases produced Escherichia coli, Methicillin-resistant Staphylococcus aureus and Candida albicans. Antimicrobial activity was evaluated with the well-diffusion method (80% honey solution w/v) and microdilution method. The honey samples with the highest antimicrobial potential were tested to prevent biofilm development and activity against a preformed biofilm. The principal component analysis of the antimicrobial properties of honey samples vs. polyphenolic profile was performed. Eleven honey samples exhibited antibacterial activity to all investigated bacteria. The antibacterial effect of the samples was most significant on the Gram-positive bacteria compared to the studied Gram-negative bacteria. Latvian honey presents promising potential for use in wound healing biomaterials, opening the possibility of achieving long-term antibacterial effects.

Lipid-Based Nano-Sized Cargos as a Promising Strategy in Bone Complications: A Review.

Bone metastasis has been considered the fatal phase of cancers, which remains incurable and to be a challenge due to the non-availability of the ideal treatment strategy. Unlike bone cancer, bone metastasis involves the spreading of the tumor cells to the bones from different origins. Bone metastasis generally originates from breast and prostate cancers. The possibility of bone metastasis is highly attributable to its physiological milieu susceptible to tumor growth. The treatment of bone-related diseases has multiple complications, including bone breakage, reduced quality of life, spinal cord or nerve compression, and pain. However, anticancer active agents have failed to maintain desired therapeutic concentrations at the target site; hence, uptake of the drug takes place at a non-target site responsible for the toxicity at the cellular level. Interestingly, lipid-based drug delivery systems have become the center of interest for researchers, thanks to their biocompatible and bio-mimetic nature. These systems possess a great potential to improve precise bone targeting without affecting healthy tissues. The lipid nano-sized systems are not only limited to delivering active agents but also genes/peptide sequences/siRNA, bisphosphonates, etc. Additionally, lipid coating of inorganic nanomaterials such as calcium phosphate is an effective approach against uncontrollable rapid precipitation resulting in reduced colloidal stability and dispersity. This review summarizes the numerous aspects, including development, design, possible applications, challenges, and future perspective of lipid nano-transporters, namely liposomes, exosomes, solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLC), and lipid nanoparticulate gels to treat bone metastasis and induce bone regeneration. Additionally, the economic suitability of these systems has been discussed and different alternatives have been discussed. All in all, through this review we will try to understand how far nanomedicine is from clinical and industrial applications in bone metastasis.

In-situ crosslinked hydrogel based on amidated pectin/oxidized chitosan as potential wound dressing for skin repairing.

Hydrogel can provide a favorable moisture environment for skin wound healing. In this study, a novel in-situ crosslinked injectable hydrogel was prepared using the water-soluble amidated pectin (AP) and oxidized chitosan (OC) through Schiff-base reaction without any chemical crosslinker. The influence of AP content on the properties of the hydrogel was systemically investigated. It showed that gelation time, pore structure, swelling capability and degradability of the hydrogel can be tuned by varying the content of amine and aldehyde groups from AP and OC. All the porous hydrogels with various AP contents (65%, 70%, and 80%) presented desirable gelation time, swelling property, high hemocompatibility and biocompatibility. Particularly, AP-OC-65 hydrogel presented superior swelling capability and better hemo- and bio-compatibility, owing to more residual amine sites in the hydrogel. Therefore, the injectable AP-OC-65 hydrogel has a greater potential for application to wound dressing or skin substitute.

Oxidized chitosan modified electrospun scaffolds for controllable release of acyclovir.

Developing a novel scaffold carrier with a sustained and controllable release profile of drug is essential to promote the effective transdermal delivery for acyclovir (ACY). In this work, electrospun polyacrylonitrile nanofibers (PAN NFs) was chemically modified with oxidized chitosan (OC). The modified fibrous scaffold was further loaded with the ACY for drug released investigation. FT-IR and NMR results revealed that the conversion of the functional group for each step has successfully occurred on the surface of the fibers. Through the in-vitro drug release and kinetic study, it demonstrated that ACY could be sustainably and controlled released from the OC modified scaffold following the Korsmeyer-Peppas model with a Fickian diffusion mechanism. The human adipose-derived stem cells and the blood combability evaluation confirmed the obtained scaffold possessed excellent cell biocompatibility and hemocompatibility. It could be concluded that the resultant OC modified scaffold based on electrospun PAN NFs opened a new potential option for the topical/transdermal drug delivery of ACY.

A novel hybrid multichannel biphasic calcium phosphate granule-based composite scaffold for cartilage tissue regeneration.

The objective of the present study was to develop a novel hybrid multichannel biphasic calcium phosphate granule (MCG)-based composite system for cartilage regeneration. First, hyaluronic acid-gelatin (HG) hydrogel was coated onto MCG matrix (MCG-HG). Poly(lactic-co-glycolic acid) (PLGA) microspheres was separately prepared and modified with polydopamine subsequent to BMP-7 loading (B). The surface-modified microspheres were finally embedded into MCG-HG scaffold to develop the novel hybrid (MCG-HG-PLGA-PD-B) composite system. The newly developed MCG-HG-PLGA-PD-B composite was then subjected to scanning electron microscopy, energy dispersive X-ray spectroscopy, Fourier Transform infrared spectroscopy, porosity, compressive strength, swelling, BMP-7 release and in-vitro biocompatibility studies. Results showed that 60% of BMP-7 retained on the granular surface after 28 days. A hybrid MCG-HG-PLGA-PD-B composite scaffold exhibited higher swelling and compressive strength compared to MCG-HG or MCG. In-vitro studies showed that MCG-HG-PLGA-PD-B had improved cell viability and cell proliferation for both MC3T3-E1 pre-osteoblasts and ATDC5 pre-chondrocytes cell line with respect to MCG-HG or MCG scaffold. Our results suggest that a hybrid MCG-HG-PLGA-PD-B composite scaffold can be a promising candidate for cartilage regeneration applications.

Preparation and characterization of polycaprolactone-polyethylene glycol methyl ether and polycaprolactone-chitosan electrospun mats potential for vascular tissue engineering.

Recently, natural polymers are frequently comingled with synthetic polymers either by physical or chemical modification to prepare numerous tissue-engineered graft with promising biological function, strength, and stability. The aim of this study was to determine the efficiency for vascular tissue engineering of two distinctly different mats, one that comprised polycaprolactone-polyethylene glycol methyl ether and other that comprised polycaprolactone-chitosan. Nano/microfibrous mats prepared from electro-spinning were characterized for fiber diameter, porosity, wettability, and mechanical strength. Biological efficacy on both biodegradable mats was assessed by rat bone marrow mesenchymal stem cells, and polycaprolactone-polyethylene glycol methyl ether showed feasibility for use as an inner layer by inducing endothelial-specific gene expression and polycaprolactone-chitosan as an outer layer on dual layered without sacrificing tensile strength, small-diameter blood vessels. Therefore, scaffolds fabricated from this research could be potential sources for tissue-engineered vascular graft and could also overcome the well-known drawbacks, such as thrombogenicity and stenosis, in managing vascular disease.

In vitro and in vivo evaluation of effectiveness of a novel TEMPO-oxidized cellulose nanofiber-silk fibroin scaffold in wound healing.

In this study, a novel TEMPO-oxidized cellulose nanofiber (TOCN)-silk fibroin scaffold was prepared using a cost effective freeze drying method. Fundamental physical characterizations were carried out by scanning electron microscopy (SEM), pore diameter determination, FT-IR. PBS uptake behavior of the scaffold showed that, silk fibroin can enhance the swelling capacity of TOCN. L929 primary fibroblast cell was selected for in vitro studies, which showed that the scaffolds facilitated growth of cells. In vivo evaluation of TOCN, TOCN-silk fibroin composites was examined using critical sized rat skin excisional model for one and two weeks. The results of rat wound model revealed that, compared to only TOCN scaffold, TOCN-silk fibroin scaffold successfully promoted wound healing by the expression of wound healing markers. TOCN-silk fibroin 2% has the fastest wound healing capacity. Thus, it appears that TOCN-silk fibroin composite scaffolds can be useful as wound healing material in clinical applications.

Examination of In vitro and In vivo biocompatibility of alginate-hyaluronic acid microbeads As a promising method in cell delivery for kidney regeneration.

In this study, alginate (ALG) and alginate-hyaluronic acid (ALG-HA) injectable microbeads, with the purpose of delivering stem cells for tissue engineering, were prepared by a spraying method into a CaCl2 solution that shows high porosity for the exchange of nutrition and waste. In addition, the size distribution and surface morphology was investigated using optical and scanning electron microscopy, respectively. The chemical structural properties of the ALG-HA microbeads were examined by Fourier transform infrared spectroscopy. The biocompatibility of ALG and ALG-HA microbeads was examined in vitro. Rat bone marrow stem cells were encapsulated in microbeads to investigate cell release, cell viability, proliferation, and secretion of growth factors such as VEGF and PDGF. Growth factors were released for the 21day experimental period. Cells were found to be released from the microbeads after 7days. Furthermore, the in vivo biocompatibility of ALG-HA microbeads was examined using microbeads without cell encapsulation in the kidney capsule, in order to assess the foreign body reaction and inflammatory response, for 14days. The desired in vivo response to ALG-HA microbeads hydrogel makes it an exquisite candidate for subcapsular cell and drug delivery to kidney tissue.

Designing of Combined Nano and Microfiber Network by Immobilization of Oxidized Cellulose Nanofiber on Polycaprolactone Fibrous Scaffold.

In this study, the immobilization of oxidized cellulose nanofiber (OCNF) network on the polycaprolactone (PCL) fibrous scaffold was performed by the aminolysis procedure through electrospinning and layer by layer (LBL) techniques. The morphology of the fibrous scaffold was examined by field emission scanning electron microscopy (FE-SEM), and it indicated that after immobilization of OCNF on PCL, the unique nanofiber and nano network was created. Moreover, the physical and chemical properties of samples were examined using X-ray photoelectron spectroscopy (XPS), Fourier transform spectroscopy (FT-IR), thermogravimetric analysis (TGA), water contact angle measurement, and BSA adsorption properties. Furthermore, the cellular responses to PCL scaffold with and without modification by OCNF were examined by seeding rat bone marrow stem cells (RBMSCs) on the fibrous scaffold for assessing cell attachment, cell viability, and proliferation. Thus, the present study focused on preparation and characterization of a membrane and tubular scaffold with a unique nanostructure, which is an excellent candidate for use as a blood vessel scaffold graft.

HAp granules encapsulated oxidized alginate-gelatin-biphasic calcium phosphate hydrogel for bone regeneration.

Bone repair in the critical size defect zone using 3D hydrogel scaffold is still a challenge in tissue engineering field. A novel type of hydrogel scaffold combining ceramic and polymer materials, therefore, was fabricated to meet this challenge. In this study, oxidized alginate-gelatin-biphasic calcium phosphate (OxAlg-Gel-BCP) and spherical hydroxyapatite (HAp) granules encapsulated OxAlg-Gel-BCP hydrogel complex were fabricated using freeze-drying method. Detailed morphological and material characterizations of OxAlg-Gel-BCP hydrogel (OGB00), 25wt% and 35wt% granules encapsulated hydrogel (OGB25 and OGB35) were carried out for micro-structure, porosity, chemical constituents, and compressive stress analysis. Cell viability, cell attachment, proliferation and differentiation behavior of rat bone marrow-derived stem cell (BMSC) on OGB00, OGB25 and OGB35 scaffolds were confirmed by MTT assay, Live-Dead assay, and confocal imaging in vitro experiments. Finally, OGB00 and OGB25 hydrogel scaffolds were implanted in the critical size defect of rabbit femoral chondyle for 4 and 8 weeks. The micro-CT analysis and histological studies conducted by H&E and Masson's trichrome demonstrated that a significantly higher (***p<0.001) and earlier bone formation happened in case of 25% HAp granules encapsulated OxAlg-Gel-BCP hydrogel than in OxAlg-Gel-BCP complex alone. All results taken together, HAp granules encapsulated OxAlg-Gel-BCP system can be a promising 3D hydrogel scaffold for the healing of a critical bone defect.

Bone formation of a porous Gelatin-Pectin-biphasic calcium phosphate composite in presence of BMP-2 and VEGF.

A composite scaffold of gelatin (Gel)-pectin (Pec)-biphasic calcium phosphate (BCP) was fabricated for the successful delivery of growth factors. Bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) were coated on the Gel-Pec-BCP surface to investigate of effect of them on bone healing. Surface morphology was investigated by scanning electron microscopy, and BCP dispersion in the hydrogel scaffolds was measured by energy dispersive X-ray spectroscopy. The results obtained from Fourier transform infrared spectroscopy showed that BMP-2 and VEGF were successfully coated on Gel-Pec-BCP hydrogel scaffolds. MC3T3-E1 preosteoblasts were cultivated on the scaffolds to investigate the effect of BMP-2 and VEGF on cell viability and proliferation. VEGF and BMP-2 loaded on Gel-Pec-BCP scaffold facilitated increased cell spreading and proliferation compared to Gel-Pec-BCP scaffolds. In vivo, bone formation was examined using rat models. Bone formation was observed in Gel-Pec-BCP/BMP-2 and Gel-Pec-BCP/VEGF scaffolds within 4 weeks, and was greatest with Gel-Pec-BCP/BMP-2 scaffolds. In vitro and in vivo results suggest that Gel-Pec-BCP/BMP-2 and Gel-Pec-BCP/VEGF scaffolds could enhance bone regeneration.