MCT4 and CD147 colocalize with MMP14 in invadopodia and support matrix degradation and invasion by breast cancer cells.

Expression levels of the lactate-H+ cotransporter MCT4 (also known as SLC16A3) and its chaperone CD147 (also known as basigin) are upregulated in breast cancers, correlating with decreased patient survival. Here, we test the hypothesis that MCT4 and CD147 favor breast cancer invasion through interdependent effects on extracellular matrix (ECM) degradation. MCT4 and CD147 expression and membrane localization were found to be strongly reciprocally interdependent in MDA-MB-231 breast cancer cells. Overexpression of MCT4 and/or CD147 increased, and their knockdown decreased, migration, invasion and the degradation of fluorescently labeled gelatin. Overexpression of both proteins led to increases in gelatin degradation and appearance of the matrix metalloproteinase (MMP)-generated collagen-I cleavage product reC1M, and these increases were greater than those observed upon overexpression of each protein alone, suggesting a concerted role in ECM degradation. MCT4 and CD147 colocalized with invadopodia markers at the plasma membrane. They also colocalized with MMP14 and the lysosomal marker LAMP1, as well as partially with the autophagosome marker LC3, in F-actin-decorated intracellular vesicles. We conclude that MCT4 and CD147 reciprocally regulate each other and interdependently support migration and invasiveness of MDA-MB-231 breast cancer cells. Mechanistically, this involves MCT4-CD147-dependent stimulation of ECM degradation and specifically of MMP-mediated collagen-I degradation. We suggest that the MCT4-CD147 complex is co-delivered to invadopodia with MMP14.

Poly-L-lactic acid/gelatin electrospun membrane-loaded bone marrow-derived mesenchymal stem cells attenuate erectile dysfunction caused by cavernous nerve injury.

Radical prostatectomy (RP) can cause neurogenic erectile dysfunction (ED), which negatively affects the quality of life of patients with prostate cancer. Currently, there is a dearth of effective therapeutic strategies. Although stem cell therapy is promising, direct cell transplantation to injured cavernous nerves is constrained by poor cell colonization. In this study, poly-L-lactic acid (PLLA)/gelatin electrospun membranes (PGEM) were fabricated to load bone marrow-derived mesenchymal stem cells (BM-MSCs) as a patch to be placed on injured nerves to alleviate ED. This study aimed to establish a promising and innovative approach to mitigate neurogenic ED post-RP and lay the foundation for modifying surgical procedures. Electrospinning and molecular biotechnology were performed in vitro and in vivo, respectively. It was observed that PGEM enhanced the performance of BM-MSCs and Schwann cells due to their excellent mechanical properties and biocompatibility. The transplanted PGEM and loaded BM-MSCs synergistically improved bilateral cavernous nerve injury-related ED and the corresponding histopathological changes. Nevertheless, transplantation of BM-MSCs alone has been verified to be ineffective. Overall, PGEM can serve as an ideal carrier to supply a more suitable survival environment for BM-MSCs and Schwann cells, thereby promoting the recovery of injured cavernous nerves and erectile function.

Exploration of the Delivery of Oncolytic Newcastle Disease Virus by Gelatin Methacryloyl Microneedles.

Oncolytic Newcastle disease virus is a new type of cancer immunotherapy drug. This paper proposes a scheme for delivering oncolytic viruses using hydrogel microneedles. Gelatin methacryloyl (GelMA) was synthesized by chemical grafting, and GelMA microneedles encapsulating oncolytic Newcastle disease virus (NDV) were prepared by micro-molding and photocrosslinking. The release and expression of NDV were tested by immunofluorescence and hemagglutination experiments. The experiments proved that GelMA was successfully synthesized and had hydrogel characteristics. NDV was evenly dispersed in the allantoic fluid without agglomeration, showing a characteristic virus morphology. NDV particle size was 257.4 ± 1.4 nm, zeta potential was -13.8 ± 0.5 mV, virus titer TCID50 was 107.5/mL, and PFU was 2 × 107/mL, which had a selective killing effect on human liver cancer cells in a dose and time-dependent manner. The NDV@GelMA microneedles were arranged in an orderly cone array, with uniform height and complete needle shape. The distribution of virus-like particles was observed on the surface. GelMA microneedles could successfully penetrate 5% agarose gel and nude mouse skin. Optimal preparation conditions were freeze-drying. We successfully prepared GelMA hydrogel microneedles containing NDV, which could effectively encapsulate NDV but did not detect the release of NDV.

A Model for Membrane Degradation Using a Gelatin Invadopodia Assay.

One of the most crucial and lethal characteristics of solid tumors is represented by the increased ability of cancer cells to migrate and invade other organs during the so-called metastatic spread. This is allowed thanks to the production of matrix metalloproteinases (MMPs), enzymes capable of degrading a type of collagen abundant in the basal membrane separating the epithelial tissue from the connective one. In this work, we employ a synergistic experimental and mathematical modelling approach to explore the invasion process of tumor cells. A mathematical model composed of reaction-diffusion equations describing the evolution of the tumor cells density on a gelatin substrate, MMPs enzymes concentration and the degradation of the gelatin is proposed. This is completed with a calibration strategy. We perform a sensitivity analysis and explore a parameter estimation technique both on synthetic and experimental data in order to find the optimal parameters that describe the in vitro experiments. A comparison between numerical and experimental solutions ends the work.

Successful cryopreservation in biodegradable containers of sperm from aquaculture Mediterranean fishes.

We aimed to evaluate the efficiency of hard-gelatin and hard-hydroxypropyl methylcellulose (HPMC) capsules as biodegradable alternative containers to plastic straws in European eel (Anguilla anguilla), gilthead seabream (Sparus aurata) and European sea bass (Dicentrarchus labrax) sperm cryopreservation. Sperm samples from each European eel (n = 12) were diluted 1:8:1 (sperm: extender P1+5 % egg yolk: methanol). Gilthead seabream (n = 12) samples were individually diluted in a cryoprotectant solution of 5 % Me2SO + NaCl 1 % plus BSA (10 mg mL-1) at a ratio of 1:6 (sperm: cryoprotectant solution). European sea bass (n = 10) sperm from each male was diluted in non-activating medium (NAM) at a ratio of 1:5.7 (sperm: NAM), and 5 % of Me2SO was added. The diluted European eel and sea bass sperm aliquots (0.5 mL) were individually filled in plastic straws (0.5 mL), hard-gelatin, and HPMC capsules (0.68 mL). Gilthead seabream diluted sperm (0.25 mL) were filled in plastic straws (0.25 mL) and identical capsules described. All samples were frozen in liquid nitrogen vapor and stored in a liquid nitrogen tank. Sperm kinetic parameters were evaluated by CASA-Mot software. Sperm membrane integrity was performed using a Live and Dead KIT and an epifluorescence microscope. To quantify DNA damage, the alkaline comet assay was performed and TailDNA (TD-%) and Olive Tail Moment (OTM) were evaluated by CaspLab software. Sperm cryopreservation of the three Mediterranean species in straws, gelatin, or HPMC capsules reduced the kinetic parameters and cell membrane integrity. Generally, the post-thawing samples cryopreserved in straws and capsules did not differ for the kinetic parameters and cell membrane integrity, except for European sea bass sperm, where the samples stored in gelatin capsules showed higher velocities (VCL - 100; VSL - 76; VAP - 90 µm s-1) than the sperm stored in HPMC capsules (VCL - 87; VSL - 59; VAP - 73 µm s-1). The cryopreservation process did not damage the sperm DNA of European eel and European sea bass, regardless of the containers used. On the other hand, gilthead seabream sperm cryopreserved in gelatin (TD - 9.8 %; OTM - 9.7) and HPMC (TD - 11.1 %; OTM - 11.2) capsules showed higher DNA damage than fresh samples (TD - 3.6 %; OTM - 2.7) and the sperm stored in straws (TD - 4.4 %; OTM - 5.2). The hard-gelatin and HPMC biodegradable capsules can be used as an alternative to straws for European eel, gilthead seabream, and European sea bass sperm cryopreservation.

Lyophilized platelet rich fibrin and gelatin incorporated bioadhesive bone cement composite for repair of mandibular continuity defects.

Mandibular continuity defects stem from conditions such as malignancies, trauma, cysts, osteomyelitis and osteoradionecrosis, presenting significant challenges. If mandibular reconstruction fails, it can result in facial collapse, causing significant aesthetic and functional concerns for the patient. In the present study we developed a bio-adhesive Bone Cement (BC) enriched with lyophilised PRF and gelatin to enhance bone repair and induce regeneration. The developed BC consisted of a mixture of Tetracalcium Phosphate (TTCP) and O-Phospho-l-serine (OPLS) in addition to lyophilised Platelet Rich Fibrin (PRF) for sustained growth factor release and gelatin (GE) for improved cement resorption. It is primarily designed for in-situ application, conforming to the shape and size of the defect for effective bone repair and regeneration. The study evaluated four groups: (i) BC (control), (ii) BC-GE (control), (iii) BC-PRF, and (iv) BC-GE-PRF. All the four groups were characterised using FTIR, SEM and XRD. The mechanical studies of the prepared beads exhibited a significant increase in the compressive strength of the PRF loaded bone cement composites. In vitro degradation study of the beads over a 60-day period revealed a significantly higher percentage of bone cement resorption in the gelatin-incorporated groups, BC-GE (44 ± 0.5 %), and BC-GE-PRF (45 ± 2 %). The assessment of growth factor release (TGF-ß and VEGF) using ELISA revealed a prolonged and sustained release of both growth factors over a 28-day period. In vitro studies were performed on human Dental Follicle Stem Cells (DFSCs) to assess cell attachment, proliferation, mineralisation and osteogenic differentiation. These studies clearly depicted that BC-PRF and BC-GE-PRF showed significantly greater proliferation of DFSCs. Furthermore, BC-PRF and BC-GE-PRF samples exhibited notably elevated expression of Runx2 and OPN (osteogenic markers), as well as a higher intensity of alizarin red stain (mineralisation). Therefore, it was concluded that PRF incorporated bioadhesive bone cement composites greatly enhance the cell attachment, proliferation, mineralisation and osteogenic differentiation of the DFSCs. Thus, the PRF and gelatin incorporated bone cement composites is expected to facilitate effective and faster bone regeneration and healing in a wide range of dental and maxillofacial defects.

Porous gelatin microspheres implanted with adipose mesenchymal stromal cells promote angiogenesis via protein kinase B/endothelial nitric oxide synthase signaling pathway in bladder reconstruction.

BACKGROUND AIMS: Cell failure and angiogenesis are the key to bladder wall regeneration. Three-dimensional (3D) culture using porous gelatin microspheres (GMs) as a vehicle promotes stem cell proliferation and improves the paracrine capacity of cells. This study aimed to evaluate the therapeutic potential of GMs constructed from adipose-derived mesenchymal stromal cells (ADSCs) (ADSC-GMs) combined with bladder acellular matrix (BAM) in tissue-engineered bladders. METHODS: Isolation of ADSCs, flow cytometry, scanning electron microscopy and cell counting kit-8, ß-galactosidase and enzyme-linked immunosorbent assays were performed in vitro to compare two-dimensional (2D) and 3D cultures. In the in vivo study, male Sprague-Dawley rats were randomly divided into three groups: the BAM replacement alone (BAM) group, ADSCs grown on BAM in replacement (ADSC) group and ADSC-GMs combined with BAM followed by replacement (ADSC-GM) group. Bladder function assessed by urodynamics after 12 weeks of bladder replacement, and the rats were sacrificed at 4 and 12 weeks for further experiments. RESULTS: The in vitro results showed that GM culture promoted ADSC proliferation, inhibited apoptosis and delayed senescence compared with those in the 2D culture. In addition, ADSC-GMs increased the secretion of the angiogenic factors vascular endothelial growth factor, platelet-derived growth factor-BB, and basal fibroblast growth factor. In vivo experiments revealed that ADSC-GMs adhered to the BAM for longer than ADSCs. Moreover, ADSC-GMs significantly promoted the regeneration of bladder vessels and smooth muscle, thereby facilitating the recovery of bladder function. The expression of phosphorylated protein kinase B (AKT) and phosphorylated endothelial nitric oxide synthase (eNOS) was significantly greater in the ADSC-GMs group compared with the BAM and ADSCs groups. CONCLUSIONS: ADSC-GMs increased retention of ADSCs on the BAM, thereby promoting the regeneration and functional recovery of the bladder tissue. ADSC-GMs promoted angiogenesis by activating the AKT/eNOS pathway.

Elastic modulus of hydrogel regulates osteogenic differentiation via liquid-liquid phase separation of YAP.

Craniofacial bone defects induced by congenital malformations, trauma, or diseases frequently challenge the orthodontic or restorative treatment. Stem cell-based bone regenerative approaches emerged as a promising method to resolve bone defects. Microenvironment physical cues, such as the matrix elastic modulus or matrix topography, regulate stem cell differentiation via multiple genes. We constructed gelatin methacryloyl (GelMA), a well-known scaffold, to investigate the impact of elastic modulus on osteogenic differentiation in a three-dimensional environment. Confocal microscope was used to observe and assess the condensates fission and fusion. New bone formation was evaluated by micro-computed tomography at 6 weeks in calvarial defect rat. We found that the light curing increased elastic modulus of GelMA, and the pore size of GelMA decreased. The expression of osteogenic markers was inhibited in hBMSCs cultured in the low-elastic-modulus GelMA. In contrast, the expression of YAP, TAZ and TEAD was increased in the hBMSCs in the low-elastic-modulus GelMA. Furthermore, YAP assembled via liquid-liquid phase separation (LLPS) into condensates that were sensitive to 1'6-hexanediol. YAP recruit TAZ and TEAD4, but not RUNX2 into the condensates. In vivo, we also found that hBMSCs in high-elastic-modulus GelMA was more apt to form new bone. This study provides new insight into the mechanism of osteogenic differentiation. Reagents that can regulate the elastic modulus of substrate or LLPS may be applied to promote bone regeneration.

Collagen-alginate 3D microscaffolds for studying cellular migration.

Metastasis is one of the major causes for cancer mortality. Its early steps comprise of invasion of basement membrane and migration. Thus, it is hypothesized that a platform, that allows quantification and grading of migration capability of cells can potentially be used for predicting metastatic potential. Two-dimensional (2D) models have been rendered inadequate for modelling in-vivo microenvironment due to various reasons. To attenuate homogeneity observed in 2D, three-dimensional (3D) platforms supplemented with bioinspired components have been designed. Unfortunately, till date there are no simple models to capture the migration of cells in 3D along with quantification of the process. In this study, we report an alginate-collagen based 3D model system, which can predict the migratory property of the cells within 72 h. The micron size of the scaffold enabled faster readout and the optimum pore-size provided conducive cellular growth environment. The platform's ability to allow observation of cellular migration was validated by encapsulating cells with transiently upregulated matrix metalloprotease 9 (MMP9), which has been reported to play a significant role in migration of cells during metastasis. The readout for migration was clustering of cells in the microscaffolds detected in a short span of 48 h. The observed clustering in MMP9 upregulated cells was validated by observing changes in the epithelial-mesenchymal transition (EMT) markers. Thus, this simple 3D platform can be used to study migration and predict the metastatic potential of cells.

Neural Tissue Engineering with Rat Adipose-Derived Mesenchymal Stem Cells: The Role of an Injectable, Resorbable Hydrogel Scaffold Derived from Oxidized Alginate and Gelatin.

The central nervous system has limited regeneration potential. The multipotency of adipose-derived mesenchymal stem cells (ADMSC) makes them an ideal autologous cell source for the regeneration of neural tissues. However, the likelihood of their differentiation into unwanted cell lineages when transplanted into a hostile injury environment is a serious disadvantage. Transplanting predifferentiated cells via an injectable carrier may aid in site-specific delivery for better survival of cells. Here, we focus on identifying an appropriate injectable hydrogel system that favors stem/progenitor cell attachment and differentiation for neural tissue engineering. An injectable composition of the hydrogel, derived from alginate dialdehyde (ADA) and gelatin, was formulated for this purpose. This hydrogel promoted proliferation/differentiation of ADMSCs to neural progenitors, visualized from the generation of prominent neurospheres and stage-specific expression of a neural progenitor marker (nestin, day 4), an intermittent neuronal marker (ß-III tub, day 5), and a mature neuronal marker (MAP-2, day 8) with neural branching and networking (>85%). The differentiated cells also expressed the functional marker synaptophysin. There was no negative impact on stem/progenitor cell survival (>95%) or differentiation (∼90%) as compared to two-dimensional (2D) culture. Addition of appropriate quantities of asiatic acid specific for neural niche supported cell growth and differentiation without affecting cell survival (>90%) and improved neural branching and elongation. Optimized interconnected porous hydrogel niche exhibited rapid gelation (3 min) and self-healing properties mimicking native neural tissue. Both ADA-gelatin hydrogel by itself and that incorporated with asiatic acid were found to support stem/neural progenitor cell growth and differentiation and have potential applications as antioxidants and growth promoters upon release at the cell transplantation site. In short, the matrix itself or incorporated with phytomoieties could serve as a potential minimally invasive injectable cell delivery vehicle for cell-based therapies of neural diseases.

Kappa-Carrageenan/Chitosan/Gelatin Scaffolds Provide a Biomimetic Microenvironment for Dentin-Pulp Regeneration.

This study aims to investigate the impact of kappa-carrageenan on dental pulp stem cells (DPSCs) behavior in terms of biocompatibility and odontogenic differentiation potential when it is utilized as a component for the production of 3D sponge-like scaffolds. For this purpose, we prepared three types of scaffolds by freeze-drying (i) kappa-carrageenan/chitosan/gelatin enriched with KCl (KCG-KCl) as a physical crosslinker for the sulfate groups of kappa-carrageenan, (ii) kappa-carrageenan/chitosan/gelatin (KCG) and (iii) chitosan/gelatin (CG) scaffolds as a control. The mechanical analysis illustrated a significantly higher elastic modulus of the cell-laden scaffolds compared to the cell-free ones after 14 and 28 days with values ranging from 25 to 40 kPa, showing an increase of 27-36%, with the KCG-KCl scaffolds indicating the highest and CG the lowest values. Cell viability data showed a significant increase from days 3 to 7 and up to day 14 for all scaffold compositions. Significantly increasing alkaline phosphatase (ALP) activity has been observed over time in all three scaffold compositions, while the KCG-KCl scaffolds indicated significantly higher calcium production after 21 and 28 days compared to the CG control. The gene expression analysis of the odontogenic markers DSPP, ALP and RunX2 revealed a two-fold higher upregulation of DSPP in KCG-KCl scaffolds at day 14 compared to the other two compositions. A significant increase of the RunX2 expression between days 7 and 14 was observed for all scaffolds, with a significantly higher increase of at least twelve-fold for the kappa-carrageenan containing scaffolds, which exhibited an earlier ALP gene expression compared to the CG. Our results demonstrate that the integration of kappa-carrageenan in scaffolds significantly enhanced the odontogenic potential of DPSCs and supports dentin-pulp regeneration.

Evaluation of in vitro coculture of keratinocytes derived from foreskin and adipose-derived mesenchymal stem cells (AMSCs) on a multilayer oxygen-releasing electrospun scaffold based on PU/PCL.Sodium percarbonate (SPC)-gelatine/PU.

Despite significant advancements in tissue engineering and regenerative medicine during the last two decades, the fabrication of proper scaffolds with appropriate cells can still be considered a critical achievement in this field. Hypoxia is a major stumbling block to chronic wound healing, which restrains tissue engineering plans because a lack of oxygen may cause cell death. This study evaluated the cocultured human keratinocytes and human adipose-derived mesenchymal stem cells (AMSCs) on a multilayer oxygen-releasing electrospun scaffold based on PU/PCL.Sodium percarbonate (SPC)-gelatin/PU. The scaffold was characterized using Fourier transform infrared (FTIR) and scanning electron microscopy (SEM) methods. Flow cytometry confirmed mesenchymal stem cells, and then the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay and DAPI staining were used to assess the in vitro biocompatibility of the scaffold. The experimental results showed that the multilayer electrospun scaffold containing 2.5% SPC could efficiently produce oxygen. Furthermore, according to cell viability results, this structure makes a suitable substrate for the coculture of keratinocytes and AMSCs. Gene expression analysis of various markers such as Involucrin, Cytokeratin 10, and Cytokeratin 14 after 14 days confirmed that keratinocytes and AMSCs coculture on PU/PCL.SPC-gelatin/PU electrospun scaffold promotes dermal differentiation and epithelial proliferation compared to keratinocytes single-cell culture. Therefore, our study supports using oxygen-releasing scaffolds as a potential strategy to hasten skin tissue regeneration. Based on the results, this structure is suggested as a promising candidate for cell-based skin tissue engineering. Given that the developed oxygen-generating polymeric electrospun scaffolds could be used as part of a future strategy for skin tissue engineering, the PU/PCL.SPC-gelatin/PU hybrid electrospun multilayer scaffold in combination with keratinocyte/AMSC coculture is proposed as an effective substrate for skin tissue engineering and regenerative medicine platforms.

Live Imaging of Monocyte/Macrophage Differentiation with Cationized Gelatin Nanospheres Incorporating a Molecular Beacon.

As one imaging method to evaluate monocyte-macrophage differentiation, cationized gelatin nanospheres incorporating a molecular beacon (MB) (cGNSMB) were designed. Cationized gelatin nanospheres (cGNS) of different apparent sizes were prepared by the conventional coacervation method, and then the MB of CD204 was incorporated into cGNS to prepare cGNSMB. When three types of cGNSMB were cultured with human monocytoma (THP-1) cells, the cGNSMB with a 110 nm diameter showed the highest MB delivery efficiency. In addition, no influence on the monocyte-macrophage differentiation was observed in terms of CD204 gene expression and cell viability. After incubation with cGNS incorporating CD204 MB (cGNSCD204), THP-1 cells were stimulated by phorbol 12-myristate 13-acetate (PMA) for monocyte differentiation into macrophages. The fluorescence intensity of macrophages increased with the incubation time. In contrast, the fluorescence intensity of macrophages incubated with MB alone was not changed. On the other hand, there was no change in the fluorescence intensity of original THP-1 cells cultured with cGNSCD204. It is concluded that the cGNSCD204 are promising to trace the differentiation of THP-1 cells into macrophages in their live condition.

The Transfer of the Hepatocyte Growth Factor Gene by Macrophages Ameliorates the Progression of Peritoneal Fibrosis in Mice.

Growing evidence indicates that hepatocyte growth factor (HGF) possesses potent antifibrotic activity. Furthermore, macrophages migrate to inflamed sites and have been linked to the progression of fibrosis. In this study, we utilized macrophages as vehicles to express and deliver the HGF gene and investigated whether macrophages carrying the HGF expression vector (HGF-M) could suppress peritoneal fibrosis development in mice. We obtained macrophages from the peritoneal cavity of mice stimulated with 3% thioglycollate and used cationized gelatin microspheres (CGMs) to produce HGF expression vector-gelatin complexes. Macrophages phagocytosed these CGMs, and gene transfer into macrophages was confirmed in vitro. Peritoneal fibrosis was induced by intraperitoneal injection of chlorhexidine gluconate (CG) for three weeks; seven days after the first CG injection, HGF-M was administered intravenously. Transplantation of HGF-M significantly suppressed submesothelial thickening and reduced type III collagen expression. Moreover, in the HGF-M-treated group, the number of α-smooth muscle actin- and TGF-ß-positive cells were significantly lower in the peritoneum, and ultrafiltration was preserved. Our results indicated that the transplantation of HGF-M prevented the progression of peritoneal fibrosis and indicated that this novel gene therapy using macrophages may have potential for treating peritoneal fibrosis.

An Innovative Approach for Formulation of Rutin Tablets Targeted for Colon Cancer Treatment.

The aim of this study was the improvement of rutin solubility along with targeting its release to colon for effective treatment of colon cancer. Five formulations of compression-coated tablets were prepared with the same core composition including rutin-polyvinyl pyrrolidone K30 solid dispersion (rutin-PVP K30 SD) but differ in being coated with either frankincense alone or different combinations of frankincense with gelatin. The superior formula was selected based on the in vitro drug release then further evaluated in terms of physical properties and in vivo performance in dogs using X-ray. Moreover, in vitro cytotoxicity of rutin, rutin-PVP K30 SD, frankincense, and a mixture of rutin-PVP K30 SD with frankincense in a ratio representing their concentrations in the selected formula was assessed against human colon cancer (HCT-116) cell lines using sulforhodamine B assay. The formula (F4) with the coat consisted of 65%w/w frankincense and 35%w/w gelatin achieved acceptable in vitro controlled drug release. In vivo X-ray in dogs confirmed that F4 tablet could remain intact in the stomach and small intestine until reaching the colon. In vitro cytotoxicity revealed that mixture of rutin-PVP K30 SD with frankincense was more effective in arresting cancer cell growth than rutin or frankincense alone. Moreover, stability studies revealed that F4 tablets were physically and chemically stable. Thus, improving rutin solubility using solid dispersion technique and formulating it into frankincense-based compression-coated (F4) tablets would be a successful approach for colonic delivery of rutin with potential of improving therapeutic efficacy.

Injectable hydrogel loaded with 4-octyl itaconate enhances cartilage regeneration by regulating macrophage polarization.

Macrophages play a distinctive role in the early stage of inflammation after cartilage defects. Previous studies have shown that macrophages can express different phenotypes, among which M2 polarization is important to maintain the balance of the inflammatory microenvironment and promote cartilage regeneration. In this study, 4-octyl itaconic acid (4-OI), a derivative of the endogenous metabolite itaconic acid, was used to regulate the polarization behavior of macrophages and enhance cartilage repair. Oxidized sodium alginate (OSA) and gelatin (GEL) were selected as materials to form injectable hydrogels with the function of sustained release of 4-OI. In vivo and in vitro experiments have verified that the OSA/GEL hydrogel system loaded with 4-OI could promote M2 macrophage polarization and inhibit the inflammatory reaction. A rat knee joint cartilage defect model further confirmed its role in promoting cartilage regeneration in the later stage. In this study, the OSA/GEL hydrogel was successfully fabricated as a vehicle for delivering 4-OI, which could evidently alleviate the inflammatory reaction and thus accelerate tissue regeneration. The results of this study provide a new method for promoting subsequent tissue regeneration by regulating the early immune response.

An induced thymic epithelial cell-based high throughput screen for thymus extracellular matrix mimetics.

Thymic epithelial cells (TECs) are key effectors of the thymic stroma and are critically required for T-cell development. TECs comprise a diverse set of related but functionally distinct cell types that are scarce and difficult to isolate and handle. This has precluded TEC-based screening assays. We previously described induced thymic epithelial cells (iTECs), an artificial cell type produced in vitro by direct reprogramming, raising the possibility that iTECs might provide the basis for functional screens related to TEC biology. Here, we present an iTEC-based three-stage medium/high-throughput in vitro assay for synthetic polymer mimics of thymic extracellular matrix (ECM). Using this assay, we identified, from a complex library, four polymers that bind iTEC as well as or better than gelatin but do not bind mesenchymal cells. We show that these four polymers also bind and maintain native mouse fetal TECs and native human fetal TECs. Finally, we show that the selected polymers do not interfere with iTEC function or T-cell development. Collectively, our data establish that iTECs can be used to screen for TEC-relevant compounds in at least some medium/high-throughput assays and identify synthetic polymer ECM mimics that can replace gelatin or ECM components in TEC culture protocols.

Isolation, Identification, and Characterization of Bioactive Peptides in Human Bone Cells from Tortoiseshell and Deer Antler Gelatin.

Tortoiseshell and deer antler gelatin has been used to treat bone diseases in Chinese society. A pepsin-digested gelatin peptide with osteoblast-proliferation-stimulating properties was identified via LC-MS/MS. The resulting pentapeptide, TSKYR, was presumably subjected to further degradation into TSKY, TSK, and YR fragments in the small intestine. The above four peptides were chemically synthesized. Treatment of tripeptide TSK can lead to a significant 30- and 50-fold increase in the mineralized nodule area and density in osteoblast cells and a 47.5% increase in the number of chondrocyte cells. The calcium content in tortoiseshell was relatively higher than in human soft tissue. The synergistic effects of calcium ions and the peptides were observed for changes in osteoblast proliferation and differentiation. Moreover, these peptides can enhance the expression of RUNX2, OCN, FGFR2, and FRFR3 genes in osteoblasts, and aggrecan and collagen type II in chondrocyte (patent pending).

[Research of diclofenac sodium-loaded gelatin scaffold with anti-inflammatory activity for promoting in vivo cartilage regeneration].

Objective: To develop a diclofenac sodium-loaded gelatin scaffold with anti-inflammatory activity and provide a new avenue for alleviating the inflammatory response and enhancing cartilage regeneration in vivo. Methods: Diclofenac sodium was homogeneously mixed with gelatin to prepare a diclofenac sodium-loaded porous gelatin scaffold by freeze-drying method as the experimental group, and a pristine porous gelatin scaffold was served as a control group. The general morphology of the scaffold was observed, the pore size of the scaffold was measured by scanning electron microscopy, the porosity of the scaffold was calculated by drainage method, the loading of diclofenac sodium into the gelatin scaffold was detected by fourier transform infrared spectrometer and X-ray diffraction examinations, and the release kinetics of diclofenac sodium from gelatin scaffold was tested using an in vitro release assay. The two scaffolds were co-cultured with lipopolysaccharide-predisposed RAW264.7 in vitro, and the expressions of interleukin 1ß (IL-1ß) and tumor necrosis factor α (TNF-α) were detected by reverse transcription polymerase chain reaction (RT-PCR), enzyme-linked immuno sorbent assay, and Western blot, to detect the in vitro anti-inflammatory effect of the drug-loaded scaffold. Thereafter, the second generation chondrocytes of New Zealand white rabbits were inoculated on the two groups of scaffolds for in vitro culture, and the cytocompatibility of the scaffold was tested by live/dead staining and cell counting kit 8 assay, the feasibility of in vitro cartilage regeneration of the scaffold was evaluated via gross observation, HE staining, Safranin-O staining, and immunohistochemical collagen type Ⅱ staining, as well as biochemical quantitative analyses. Finally, the two groups of chondrocyte-scaffolds were implanted subcutaneously into New Zealand white rabbits, and after 4 weeks, the general observation, HE staining, safranin O staining, immunohistochemical collagen type Ⅱ staining, and biochemical quantitative analyses were performed to verify the cartilage regeneration in vivo, and the expression of inflammation-related genes CD3 and CD68 was detected by RT-PCR to comprehensively evaluate the anti-inflammatory performance of the scaffolds in vivo. Results: The two scaffolds exhibited similar gross, microporous structure, pore size, and porosity, showing no significant difference (P>0.05). Diclofenac sodium was successfully loaded into gelatin scaffold. Data from in vitro anti-inflammatory assay suggested that diclofenac sodium-loaded gelatin scaffold showed alleviated gene and protein expressions of IL-1ß and TNF-α when compared with gelatin scaffold (P<0.05). The evaluation of cartilage regeneration in vitro showed that the number of living cells increased significantly with the extension of culture time, and there was no significant difference between the two groups at each time point (P>0.05). White cartilage-like tissue was regenerated from the scaffolds in both groups, histological observation showed typical cartilage lacuna structure and specific cartilage extracellular matrix secretion. There was no significant difference in the content of cartilage-specific glycosaminoglycan (GAG) and collagen type Ⅱ between the two groups (P>0.05). In vivo experiments showed that the samples in the experimental group had porcelain white cartilage like morphology, histologic staining showed obvious cartilage lacuna structure and cartilage specific extracellular matrix, the contents of GAG and collagen type Ⅱ were significantly higher than those in the control group, and the protein and mRNA expressions of CD3 and CD68 were significantly lower than those in the control group, with significant differences (P<0.05). Conclusion: The diclofenac sodium-loaded gelatin scaffold presents suitable pore size, porosity, and cytocompatibility, as well as exhibited satisfactory anti-inflammatory ability, providing a reliable scheme for alleviating the inflammatory reaction of regenerated cartilage tissue after in vivo implantation and promoting cartilage regeneration in vivo.

Extracellular matrix protein gelatin provides higher expansion, reduces size heterogeneity, and maintains cell stiffness in a long-term culture of mesenchymal stem cells.

Extracellular matrices (ECM) present in our tissues play a significant role in maintaining tissue homeostasis through various physical and chemical cues such as topology, stiffness, and secretion of biochemicals. They are known to influence the behavior of resident stem cells. It is also known that ECM type and coating density on cell culture plates strongly influence in vitro cellular behavior. However, the influence of ECM protein coating on long-term mesenchymal stem cell expansion has not been studied yet. To address this gap, we cultured bone-marrow derived hMSCs for multiple passages on the tissue culture plastic plates coated with 25 µg/ml of various ECM proteins. We found that cells on plates coated with ECM proteins had much higher proliferation compared to the regular tissue culture plates. Further, gelatin-coated plates helped the cells to grow faster compared to collagen, fibronectin, and laminin coated plates. Additionally, the use of gelatin showed less size heterogeneity among the cells when expanded from passages 3 to 9 (P3 to P9). Gelatin also helped in maintaining cellular stiffness which was not observed across other ECM proteins. In summary, in this research, we have shown that gelatin which is the least expensive compared to other ECM proteins, provides a better platform for mesenchymal stem cell expansion.