The impact of copper nanoparticles surfactant on the structural and biological properties of chitosan/sodium alginate wound dressings.

Multifunctional wound dressings based on hydrogels are an efficacious and practicable strategy in therapeutic processes and accelerated chronic wound healing. Here, copper (Cu) nanoparticles were added to chitosan/sodium alginate (CS/SA) hydrogels to improve the antibacterial properties of the prepared wound dressings. Due to the super-hydrophobicity of Cu nanoparticles, polyethylene glycol (PEG) was used as a surfactant, and then added to the CS/SA-based hydrogels. The CS/SA/Cu hydrogels were synthesized with 0, 2, 3.5, and 5 wt% Cu nanoparticles. The structural and morphological properties in presence of PEG were evaluated using Fourier-transform infrared spectroscopy (FTIR), Differential scanning calorimetry (DSC), and field emission scanning electron microscopy (FESEM). The biodegradation and swelling properties of the hydrogels were investigated in phosphate buffer saline (PBS) at 37 °C for up to 30 days. Cell viability and adhesion, as well as antibacterial behavior, were investigated via MTT assay, FESEM, and disk diffusion method, respectively. The obtained results showed that PEG provided new intra- and intermolecular bonds that affected significantly the hydrogels' degradation and swelling ratio, which increased up to ~1200 %. Cell viability reached ~110 % and all samples showed remarkable antibacterial behavior when CS/SA/Cu containing 2 wt% was introduced. This study provided new insights regarding the use of PEG as a surfactant for Cu nanoparticles in CS/SA hydrogel wound dressing, ultimately affecting the chemical bonding and various properties of the prepared hydrogels.

Suppression of SOCS3 expression in macrophage cells: Potential application in diabetic wound healing.

Impaired polarization of M1 to M2 macrophages has been reported in diabetic wounds. We aimed to improve this polarization by down-regulation of expression of the "Suppressor of Cytokine Signaling 3" (SOCS3) gene in macrophages. Two oligodeoxynucleotide (ASO) sequences were designed against SOC3 mRNA and were loaded to mannosylated-polyethyleneimine (Man-PEI). The optimum N/P ratio for Man-PEI-ASO was determined to be 8 based on loading efficiency, particle size, zeta potential, cellular uptake and cytotoxicity assay. pH stability of ASO in Man-PEI-ASO and its protection from DNase I was confirmed. After in vitro treatment of macrophages with Man-PEI-ASO, SOCS3 was downregulated, SOCS1 upregulated, and SOCS1/SOCS3 ratio increased. Also, expressions of macrophage markers of M2 (IL-10, Arg1, CD206) increased and those of M1 (IL-1ß, NOS2, CD68) decreased, and secretion of pro-inflammatory cytokines (TNF-α and IL-1ß) decreased while that of anti-inflammatory cytokine IL-4 increased. All suggested a polarization into M2 phenotype. Finally, the Man-PEI-ASO was loaded in hydrogel and applied to a diabetic wound model in mice. It improved the healing to the level observed in non-diabetic wounds. We show that using antisense sequences against SOC3 mRNA, macrophage polarization could be directed into the M2 phenotype and healing of diabetic wound could be highly improved.

Enhancing Diabetic Wound Healing Through Improved Angiogenesis: The Role of Emulsion-Based Core-Shell Micro/Nanofibrous Scaffold with Sustained CuO Nanoparticle Delivery.

Attempts are made to design a system for sustaining the delivery of copper ions into diabetic wounds and induce angiogenesis with minimal dose-dependent cytotoxicity. Here, a dual drug-delivery micro/nanofibrous core-shell system is engineered using polycaprolactone/sodium sulfated alginate-polyvinyl alcohol (PCL/SSA-PVA), as core/shell parts, by emulsion electrospinning technique to optimize sustained delivery of copper oxide nanoparticles (CuO NP). Herein, different concentrations of CuO NP (0.2, 0.4, 0.8, and 1.6%w/w) are loaded into the core part of the core-shell system. The morphological, biomechanical, and biocompatibility properties of the scaffolds are fully determined in vitro and in vivo. The 0.8%w/w CuO NP scaffold reveals the highest level of tube formation in HUVEC cells and also upregulates the pro-angiogenesis genes (VEGFA and bFGF) expression with no cytotoxicity effects. The presence of SSA and its interaction with CuO NP, and also core-shell structure sustain the release of the nanoparticles and provide a non-toxic microenvironment for cell adhesion and tube formation, with no sign of adverse immune response in vivo. The optimized scaffold significantly accelerates diabetic wound healing in a rat model. This study strongly suggests the 0.8%w/w CuO NP-loaded PCL/SSA-PVA as an excellent diabetic wound dressing with significantly improved angiogenesis and wound healing.

Fabrication and characterization of a hydrocolloid wound dressing functionalized with human placental derived extracellular matrix for management of skin wounds: An animal study.

BACKGROUND: Functionalization of wound dressing is one of the main approaches for promoting wound healing in skin wound management. In this study, our aim is to fabricate a bio-functionalized hydrocolloid wound dressing. METHODS: The extracellular matrix (ECM) was extracted from human placental tissue. A hydrocolloid film was fabricated using Na-CMC, pectin, gelatin, styrene-isoprene-styrene adhesive, glycerol, and 0.5%-2.5% powdered ECM. A polyurethane film and a release liner were used in the hydrocolloid/ECM films. The mechanical, adhesion, swelling rate, and integrity of the films were investigated. Cell proliferation, adhesion, and migration assays, as well as, SEM and FTIR spectroscopy were also conducted. Macroscopic and microscopic evaluations of wound healing process and formation of blood vessels were conducted in mouse animal models. RESULTS: We successfully fabricated a three-layered ECM-functionalized hydrocolloid dressing with a water vapor transmission rate of 371 g/m2 /day and an adhesion peel strength of 176 KPa. Cellular adhesion, proliferation and migration were promoted by ECM. In the animal tests, ECM-functionalized hydrocolloids significantly improved wound closure and re-epithelialization at days 14 and 21. Also, ECM-functionalized hydrocolloids promoted the formation of hair follicles. CONCLUSIONS: Our findings suggest that ECM could enhance the wound healing properties of hydrocolloid wound dressings. This wound dressing could be considered for application in hard-to-heal acute wounds.

Epithelial cells/progenitor cells in developing human lower respiratory tract: Characterization and transplantation to rat model of pulmonary injury.

Introduction: For cell-based therapies of lung injury, several cell sources have been extensively studied. However, the potential of human fetal respiratory cells has not been systematically explored for this purpose. Here, we hypothesize that these cells could be one of the top sources and hence, we extensively updated the definition of their phenotype. Methods: Human fetal lower respiratory tissues from pseudoglandular and canalicular stages and their isolated epithelial cells were evaluated by immunostaining, electron microscopy, flow cytometry, organoid assay, and gene expression studies. The regenerative potential of the isolated cells has been evaluated in a rat model of bleomycin-induced pulmonary injury by tracheal instillation on days 0 and 14 after injury and harvest of the lungs on day 28. Results: We determined the relative and temporal, and spatial pattern of expression of markers of basal (KRT5, KRT14, TRP63), non-basal (AQP3 and pro-SFTPC), and early progenitor (NKX2.1, SOX2, SOX9) cells. Also, we showed the potential of respiratory-derived cells to contribute to in vitro formation of alveolar and airway-like structures in organoids. Cell therapy decreased fibrosis formation in rat lungs and improved the alveolar structures. It also upregulated the expression of IL-10 (up to 17.22 folds) and surfactant protein C (up to 2.71 folds) and downregulated the expression of TGF-ß (up to 5.89 folds) and AQP5 (up to 3.28 folds). Conclusion: We provide substantial evidence that human fetal respiratory tract cells can improve the regenerative process after lung injury. Also, our extensive characterization provides an updated phenotypic profile of these cells.

Integrated Bioinformatic Analysis of Differentially Expressed Genes Associated with Wound Healing.

OBJECTIVE: Wound healing is a complex process involving the coordinated interaction of various genes and molecular
pathways. The study aimed to uncover novel therapeutic targets, biomarkers and candidate genes for drug development
to improve successful wound repair interventions.
Materials and Methods: This study is a network-meta analysis study. Nine wound healing microarray datasets obtained
from the Gene Expression Omnibus (GEO) database were used for this study. Differentially expressed genes (DEGs)
were described using the Limma package and shared genes were used as input for weighted gene co-expression
network analysis. The Gene Ontology analysis was performed using the EnrichR web server, and construction of a
protein-protein interaction (PPI) network was achieved by the STRING and Cytoscape.
Results: A total of 424 DEGs were determined. A co-expression network was constructed using 7692 shared genes
between nine data sets, resulting in the identification of seven modules. Among these modules, those with the top 20
genes of up and down-regulation were selected. The top down-regulated genes, including TJP1, SEC61A1, PLEK,
ATP5B, PDIA6, PIK3R1, SRGN, SDC2, and RBBP7, and the top up-regulated genes including RPS27A, EEF1A1,
HNRNPA1, CTNNB1, POLR2A, CFL1, CSNk1E, HSPD1, FN1, and AURKB, which can potentially serve as therapeutic
targets were identified. The KEGG pathway analysis found that the majority of the genes are enriched in the "Wnt
signaling pathway".
Conclusion: In our study of nine wound healing microarray datasets, we identified DEGs and co-expressed modules
using WGCNA. These genes are involved in important cellular processes such as transcription, translation, and posttranslational
modifications. We found nine down-regulated genes and ten up-regulated genes, which could serve as
potential therapeutic targets for further experimental validation. Targeting pathways related to protein synthesis and cell
adhesion and migration may enhance wound healing, but additional experimental validation is needed to confirm the
effectiveness and safety of targeted interventions.

A Review of Genes Involved in Wound Healing.

Background: Gene therapy holds immense potential in the field of wound healing. However, we still do not recognize this procedure well enough to give oversight effectively to improve healing processes. A wide range of information has been achieved from the database for gene expression profiling by clinical trials, So we performed this study to gain a better understanding of the mechanisms behind wound healing and how it could be utilized to develop new therapies and treatments. Methods: In this study, we have been focusing on wound-healing genes, conducting a thorough review to explore the various genes and pathways involved in this process. For this purpose, a total of 320 articles were collected. All experimental studies, systematic or narrative reviews, studies and clinical trials included in this paper were searched on PubMed, Medline, Embase, Science Direct, and Scopus databases in English using the following terms: Wound Healing, wound regeneration, Gene Transfer, and Gene Therapy were used to search the mentioned databases. Unfortunately, we didn't find a large sample cohort study on this topic. A total amount of 330 articles were collected based on the guidelines of the PRISMA method. Both inclusion and exclusion criteria were settled. Results: During the last decade, different models of gene delivery have been introduced, which include viral transfection and Non-viral techniques. In this regard, TIMP-2 protein and VEGF mutants such as VEGF165, CARP, and HIF-1 are the genes that accelerate the rate of tissue repair. Conclusion: The process of wound healing is mainly related to the change of expression of genes that have a role in the parts of inflammation and repair. In our study, some of the most suitable genes involved in the wound-healing process are mentioned.

In-situ forming hydrogel based on thiolated chitosan/carboxymethyl cellulose (CMC) containing borate bioactive glass for wound healing.

Suitable wound dressings for accelerating wound healing are actively being designed and synthesised. In this study, thiolated chitosan (tCh)/oxidized carboxymethyl cellulose (OCMC) hydrogel containing Cu-doped borate bioglass (BG) was developed as a wound dressing to improve wound healing in a full-thickness skin defect of mouse animal model. Thiolation was used to incorporate thiol groups into chitosan (Ch) to enhance its water solubility and mucoadhesion characteristics. Here, the in situ forming hydrogel was successfully developed using the Schiff-based reaction, and its physio-chemical and antibacterial characteristics were examined. Borate BG was also incorporated in the generated hydrogel to promote angiogenesis and tissue regeneration at the wound site. Investigations of in vitro cytotoxicity assays demonstrated that the synthesised hydrogels showed good biocompatibility and promoted cell growth. These results inspired us to investigate the effectiveness of skin wound healing in a mouse model. On the backs of animals, two full-thickness wounds were created and treated utilising two different treatment conditions: (1) OCMC/tCh hydrogel, (2) OCMC/tCh/borate BG, and (3) control defect. The wound closure ratio, collagen deposition, and angiogenesis activity were measured after 14 days to determine the healing efficacy of the in situ hydrogels used as wound dressings. Overall, the hydrogel containing borate BG was maintained in the defect site, healing efficiency was replicable, and wound healing was apparent. In conclusion, we found consistent angiogenesis, remodelling, and accelerated wound healing, which we propose may have beneficial effects on the repair of skin defects.

Ancient fibrous biomaterials from silkworm protein fibroin and spider silk blends: Biomechanical patterns.

Silkworm silk protein fibroin and spider silk spidroin are known biocompatible and natural biodegradable polymers in biomedical applications. The presence of ß-sheets in silk fibroin and spider spidroin conformation improves their mechanical properties. The strength and toughness of pure recombinant silkworm fibroin and spidroin are relatively low due to reduced molecular weight. Hence, blending is the foremost approach of recent studies to optimize silk fibroin and spidroin's mechanical properties. As summarised in the present review, numerous research investigations evaluate the blending of natural and synthetic polymers. The effects of blending silk fibroin and spidroin with natural and synthetic polymers on the mechanical properties are discussed in this review article. Indeed, combining natural and synthetic polymers with silk fibroin and spidroin changes their conformation and structure, fine-tuning the blends' mechanical properties. STATEMENT OF SIGNIFICANCE: Silkworm and spider silk proteins (silk fibroin and spidroin) are biocompatible and biodegradable natural polymers having different types of biomedical applications. Their mechanical and biological properties may be tuned through various strategies such as blending, conjugating and cross-linking. Blending is the most common method to modify fibroin and spidroin properties on demand. This review article aims to categorize and evaluate the effects of blending fibroin and spidroin with different natural and synthetic polymers. Increased polarity and hydrophilicity end to hydrogen bonding triggered conformational change in fibroin and spidroin blends. The effect of polarity and hydrophilicity of the blending compound has been discussed in relation to the structural and mechanical properties of the biomaterials. These effects have been categorized to combinatorial, synergistic and indirect impact on silk fibroin and spidroin properties. This outlook guides us to choose the blending compounds mindfully as this mixing affects the biochemical and biophysical characteristics of the biomaterials.

Transplantation of decellularised human amniotic membranes seeded with mesenchymal stem cell-educated macrophages into animal models.

The reconstruction of chronic skin wounds remains a public health challenge in dermatology. Precisely controlling and monitoring the wound-healing process should result in enhanced outcomes for the patient. Cell-based therapies have shown great potential in medicine due to their immunomodulatory and healing properties. Herein, we produced activated macrophages by treating circulating monocytes with mesenchymal stem cell (MSC) supernatant. We also demonstrated the critical role of activated macrophages transplantation using amniotic membranes in accelerating wound healing in an animal wound model. The activated macrophages not only exhibited immunomodulatory cytokines like transforming growth factorß (TGFß) and interleukin 10 (and IL10) secretion but also showed attachment and proliferation ability on the amniotic membrane scaffold. Moreover, MSCs supernatant-treated cells also displayed significant ARG1, CD206, and IL 10 genes expression. Inspired by the in vitro results, we examined the in vivo therapeutic efficacy of the activated macrophage transplantation using an acellular amniotic membrane carrier in a full-thickness cutaneous wound model. The wound healing rate was significant in the group treated with macrophages generated via mesenchymal cell therapy seeded human amniotic membrane. There was less scarring in the wound sites after placing cell-scaffold constructs in the wound sites in the animal models. Overall, macrophages stimulated with mesenchymal cells' supernatant exhibited improved healing processes in incisional wounds by decreasing the inflammatory phase, increasing angiogenesis, and reducing scar tissue development.

Introduction of an efficient method for placenta decellularization with high potential to preserve ultrastructure and support cell attachment.

The placenta, as a large discarded tissue and rich in extracellular matrix (ECM), is an excellent candidate for biological scaffolds in reconstructive medicine. Considering the importance of ECM structure in cell fate, the aim of this study was to achieve human placenta decellularization protocol that preserve the structure of scaffolds. Thus, human placenta was decellularized by four protocols and decellularization efficacy was compared by hematoxylin and eosin (H&E), 4',6-diamidino-2-phenylindole (DAPI) staining, and DNA measurement. Decellularized placenta structure preservation was assessed by Masson's trichrome staining, scanning electron microscopy (SEM), and immunofluorescence (IF) for collagen I, IV, and fibronectin. Finally, liquid displacement measured scaffolds' porosity. After culturing menstrual blood-derived stem cells (MenSCs) on placenta scaffolds, cell adhesion was investigated by SEM imaging, and cell viability and proliferation were assessed by MTT assay. According to H&E and DAPI staining, only protocols 1 and 3 could completely remove cells from the scaffolds. DNA measurements confirmed a significant reduction in the genetic material of decellularized scaffolds compared to native placenta. According to Masson's trichrome, IF, and SEM imaging, scaffold structure is better preserved in P3 than P1 protocol. Liquid displacement showed higher porosity of P3 scaffold than P1. SEM imaging confirmed cells adhesion to the decellularized placenta, and the attached cells showed good viability and maintained their proliferative capacity, indicating the suitability of the scaffolds for cell growth. Results introduced an optimized protocol for placenta decellularization that preserves the scaffold structure and supports cell adhesion and proliferation.

Cross-linked acellular lung for application in tissue engineering: Effects on biocompatibility, mechanical properties and immunological responses.

The concept of providing tissue engineering scaffolds with natural physical properties and minimal immunogenicity has not been systematically approached for the lungs yet. Here, the rat acellular lung tissue (ALT) was cross-linked to provide either EDC/NHS cross-linked tissue (EDC/NHS-CLT) or tannic acid cross-linked tissue (TA-CLT). Young's modulus revealed that EDC/NHS-CLT had mechanical properties similar to the native lung and culture of lung mesenchymal cells showed a higher potential of cell proliferation on EDC/NHS-CLT versus TA-CLT and ALT. The in vitro immunogenicity tests showed a strong induction of T-cell proliferation by TA-CLT and an attenuated macrophage induction by TA-CLT. Processed rat lungs were implanted xenogenically into the mouse peritoneal cavity and the host-implant interactions showed that tannic acid is not released from TA-CLT in a physiologically effective dose. The profile of peritoneal fluid proinflammatory (TNFα, IL-1ß, IL-12p70 and IL-17) and anti-inflammatory (IL-10 and TGFß1) cytokines, and CD3+ T-lymphocytes and CD11b+ macrophages revealed that apart from induction of high levels of IL-17 during the first week and IL-10 during the second to third weeks after implantation by TA-CLT, other indicators of immune reactions to cross-linked tissues were not significantly different from ALT. Also, a high fibrotic reaction to TA-CLT was observed on the weeks 2-3, but alveolar structures were preserved in EDC/NHS-CLT. Our findings show that by controlled EDC/NHS cross-linking, an acellular lung scaffold could be provided with mechanical properties similar to native lung, which promotes mesenchymal lung cells proliferation and does not stimulate recipient's immune system more than a non-cross-linked tissue.

PLGA/TiO2 nanocomposite scaffolds for biomedical applications: fabrication, photocatalytic, and antibacterial properties.

Introduction: Porous 3D scaffolds synthesized using biocompatible and biodegradable materials could provide suitable microenvironment and mechanical support for optimal cell growth and function. The effect of the scaffold porosity on the mechanical properties, as well as the TiO2 nanoparticles addition on the bioactivity, antimicrobial, photocatalytic, and cytotoxicity properties of scaffolds were investigated. Methods: In the present study, porous scaffolds consisting poly (lactide-co-glycolide) (PLGA) containing TiO2 nanoparticles were fabricated via air-liquid foaming technique, which is a novel method and has more advantages due to not using additives for nucleation compared to former ways. Results: Adjustment of the foaming process parameters was demonstrated to allow for textural control of the resulting scaffolds and their pore size tuning in the range of 200-600 µm. Mechanical properties of the scaffolds, in particular, their compressive strength, revealed an inverse relationship with the pore size, and varied in the range of 0.97-0.75 MPa. The scaffold with the pore size 270 µm, compressive strength 0.97 MPa, and porosity level 90%, was chosen as the optimum case for the bone tissue engineering (BTE) application. Furthermore, 99% antibacterial effect of the PLGA/10 wt.% TiO2 nanocomposite scaffolds against the strain was achieved using Escherichia coli. Besides, no negative effect of the new method was observed on the bioactivity behavior and apatite forming ability of scaffolds in the simulated body fluid (SBF). This nanocomposite also displayed a good cytocompatibility when assayed with MG 63 cells. Lastly, the nanocomposite scaffolds revealed the capability to degrade methylene blue (MB) dye by nearly 90% under the UV irradiation for 3 hours. Conclusion: Based on the results, nanocomposite new scaffolds are proposed as a promising candidate for the BTE applications as a replacement for the previous ones.

Preparation of internally-crosslinked alginate microspheres: Optimization of process parameters and study of pH-responsive behaviors.

In this study, the effects of various parameters of the water-in-oil emulsification/internal gelation method on the properties of calcium-alginate microparticles were evaluated and optimized. Results showed that the spherical-shaped microparticles with the highest circularity and high production yield can be produced by alginate solution with a concentration of 2 wt.%, calcium carbonate/alginate ratio of 10/1 (w/w), water/oil volume ratio of 1/20, emulsifier concentration of 5 % (v/v), and emulsification speed of 1000 rpm. Two model drugs including simvastatin lactone and simvastatin ß-hydroxyacid were loaded into the microspheres with promising encapsulation efficiencies of 73 % and 69 %, respectively. The microspheres showed a pH-responsive swelling behavior with a percentage of 10.60 %, 352.65 %, 690.03 %, and 1211.46 % at the pH values of 2.0, 4.5, 7.4, and 8.5, respectively. The microspheres showed an increasing trend of release rate in direct proportion to pH. These findings would be useful for therapeutic applications which need pH-responsive drug carriers.

Nanofibrillated chitosan coated highly ordered titania nanotubes array/graphene nanocomposite with improved biological characters.

Designing multifunctional surfaces is key to develop advanced materials for orthopedic applications. In this study, we design a double-layer coating, assembled onto the completely regular titania nanotubes (cRTNT) array. Benefiting from the biological and topological characteristics of chitosan nanofibers (CH) and reduced graphene oxide (RGO) through a unique assembly, the designed material features promoted osteoblast cell viability, prolonged antibiotic release profile, as well as inhibited bacterial biofilm formation. The synergistic effect of RGO and CH on the biological performance of the surface is investigatSed. The unique morphology of the nanofibers leads to the partial coverage of RGO-modified nanotubes, providing an opportunity to access the sublayer properties. Another merit of this coating lies in its morphological similarity to the extracellular matrix (ECM) to boost cellular performance. According to the results of this study, this platform holds promising advantages over the bare and bulk biopolymer-modified TNTs.

Conductive chitosan/polyaniline hydrogel with cell-imprinted topography as a potential substrate for neural priming of adipose derived stem cells.

Biophysical characteristics of engineered scaffolds such as topography and electroconductivity have shown potentially beneficial effects on stem cell morphology, proliferation, and differentiation toward neural cells. In this study, we fabricated a conductive hydrogel made from chitosan (CS) and polyaniline (PANI) with induced PC12 cell surface topography using a cell imprinting technique to provide both topographical properties and conductivity in a platform. The engineered hydrogel's potential for neural priming of rat adipose-derived stem cells (rADSCs) was determined in vitro. The biomechanical analysis revealed that the electrical conductivity, stiffness, and hydrophobicity of flat (F) and cell-imprinted (CI) substrates increased with increased PANI content in the CS/PANI scaffold. The conductive substrates exhibited a lower degradation rate compared to non-conductive substrates. According to data obtained from F-actin staining and AFM micrographs, both CI(CS) and CI(CS-PANI) substrates induced the morphology of rADSCs from their irregular shape (on flat substrates) into the elongated and bipolar shape of the neuronal-like PC12 cells. Immunostaining analysis revealed that both CI(CS) and CI (CS-PANI) significantly upregulated the expression of GFAP and MAP2, two neural precursor-specific genes, in rADSCs compared with flat substrates. Although the results reveal that both cell-imprinted topography and electrical conductivity affect the neural lineage differentiation, some data demonstrate that the topography effects of the cell-imprinted surface have a more critical role than electrical conductivity on neural priming of ADSCs. The current study provides new insight into the engineering of scaffolds for nerve tissue engineering.

Design and fabrication of polycaprolactone/gelatin composite scaffolds for diaphragmatic muscle reconstruction.

Diaphragmatic wall defects caused by congenital disorders or disease remain a major challenge for physicians worldwide. Polymeric patches have been extensively explored within research laboratories and the clinic for soft tissue and diaphragm reconstruction. However, patch usage may be associated with allergic reaction, infection, granulation, and recurrence of the hernia. In this study, we designed and fabricated a porous scaffold using a combination of 3D printing and freeze-drying techniques. A 3D printed polycaprolactone (PCL) mesh was used to reinforcegelatin scaffolds, representing an advantage over previously reported examples since it provides mechanical strength and flexibility. In vitro studies showed that adherent cells were anchorage-dependent and grew as a monolayer attached to the scaffolds. Microscopic observations indicated better cell attachments for the scaffolds with higher gelatin content as compared with the PCL control samples. Tensile testing demonstrated the mechanical strength of samples was significantly greater than adult diaphragm tissue. The biocompatibility of the specimens was investigated in vivo using a subcutaneous implantation method in Bagg albino adult mice for 20 days, with the results indicating superior cellular behavior and attachment on scaffolds containing gelatin in comparison to pure PCL scaffolds, suggesting that the porous PCL/gelatin scaffolds have potential as biodegradable and flexible constructs for diaphragm reconstruction.

Inhibition of hypertrophy and improving chondrocyte differentiation by MMP-13 inhibitor small molecule encapsulated in alginate-chondroitin sulfate-platelet lysate hydrogel.

BACKGROUND: Mesenchymal stem cells are a promising cell source for chondrogenic differentiation and have been widely used in several preclinical and clinical studies. However, they are prone to an unwanted differentiation process towards hypertrophy that limits their therapeutic efficacy. Matrix metallopeptidase 13 (MMP-13) is a well-known factor regulated during this undesirable event. MMP-13 is a collagen degrading enzyme, which is also highly expressed in the hypertrophic zone of the growth plate and in OA cartilage. Accordingly, we investigated the effect of MMP-13 inhibition on MSC hypertrophy. METHODS: In this study, 5-bromoindole-2-carboxylic acid (BICA) was used as an inhibitory agent for MMP-13 expression. After identifying its optimal concentration, BICA was mixed into a hydrogel and the release rate was studied. To prepare the ideal hydrogel, chondroitin sulfate (CS) and platelet lysate (PL) were mixed with sodium alginate (Alg) at concentrations selected based on synergistic mechanical and rheometric properties. Then, four hydrogels were prepared by combining alginate (1.5%w/v) and/or CS (1%w/v) and/or PL (20%v/v). The chondrogenic potential and progression to hypertrophy of human bone marrow-derived mesenchymal stem cell (hBM-MSC)-loaded hydrogels were investigated under free swelling and mechanical loading conditions, in the presence and absence of BICA. RESULTS: Viability of hBM-MSCs seeded in the four hydrogels was similar. qRT-PCR revealed that BICA could successfully inhibit MMP-13 expression, which led to an inhibition of Coll X and induction of Coll-II, in both free swelling and loading conditions. The GAG deposition was higher in the group combining BICA and mechanical stimulation. CONCLUSIONS: It is concluded that BICA inhibition of MMP-13 reduces MSC hypertrophy during chondrogenesis.

Improvement, scaling-up, and downstream analysis of exosome production.

Exosomes are the most researched extracellular vesicles. In many biological, physiological, and pathological studies, they have been identified as suitable candidates for treatment and diagnosis of diseases by acting as the carriers of both drugs and genes. Considerable success has been achieved regarding the use of exosomes for tissue regeneration, cancer diagnosis, and targeted drug/gene delivery to specific tissues. While major progress has been made in exosome extraction and purification, extraction of large quantities of exosomes is still a major challenge. This issue limits the scope of both exosome-based research and therapeutic development. In this review, we have aimed to summarize experimental studies focused at increasing the number of exosomes. Biotechnological studies aimed at identifying the pathways of exosome biogenesis to manipulate some genes in order to increase the production of exosomes. Generally, two major strategies are employed to increase the production of exosomes. First, oogenesis pathways are genetically manipulated to overexpress activator genes of exosome biogenesis and downregulate the genes involved in exosome recycling pathways. Second, manipulation of the cell culture medium, treatment with specific drugs, and limiting certain conditions can force the cell to produce more exosomes. In this study, we have reviewed and categorized these strategies. It is hoped that the information presented in this review will provide a better understanding for expanding biotechnological approaches in exosome-based therapeutic development.