Accelerate Healing of Severe Burn Wounds by Mouse Bone Marrow Mesenchymal Stem Cell-Seeded Biodegradable Hydrogel Scaffold Synthesized from Arginine-Based Poly(ester amide) and Chitosan.

Severe burns are some of the most challenging problems in clinics and still lack ideal modalities. Mesenchymal stem cells (MSCs) incorporated with biomaterial coverage of burn wounds may offer a viable solution. In this report, we seeded MSCs to a biodegradable hybrid hydrogel, namely ACgel, that was synthesized from unsaturated arginine-based poly(ester amide) (UArg-PEA) and chitosan derivative. MSC adhered to ACgels. ACgels maintained a high viability of MSCs in culture for 6 days. MSC seeded to ACgels presented well in third-degree burn wounds of mice at 8 days postburn (dpb) after the necrotic full-thickness skin of burn wounds was debrided and filled and covered by MSC-carrying ACgels. MSC-seeded ACgels promoted the closure, reepithelialization, granulation tissue formation, and vascularization of the burn wounds. ACgels alone can also promote vascularization but less effectively compared with MSC-seeded ACgels. The actions of MSC-seeded ACgels or ACgels alone involve the induction of reparative, anti-inflammatory interleukin-10, and M2-like macrophages, as well as the reduction of inflammatory cytokine TNFα and M1-like macrophages at the late inflammatory phase of burn wound healing, which provided the mechanistic insights associated with inflammation and macrophages in burn wounds. For the studied regimens of these treatments, no toxicity was identified to MSCs or mice. Our results indicate that MSC-seeded ACgels have potential use as a novel adjuvant therapy for severe burns to complement commonly used skin grafting and, thus, minimize the downsides of grafting.

High-Content Assay Multiplexing for Vascular Toxicity Screening in Induced Pluripotent Stem Cell-Derived Endothelial Cells and Human Umbilical Vein Endothelial Cells.

Endothelial cells (ECs) play a major role in blood vessel formation and function. While there is longstanding evidence for the potential of chemical exposures to adversely affect EC function and vascular development, the hazard potential of chemicals with respect to vascular effects is not routinely evaluated in safety assessments. Induced pluripotent stem cell (iPSC)-derived ECs promise to provide a physiologically relevant, organotypic culture model that is amenable for high-throughput (HT) EC toxicant screening and may represent a viable alternative to traditional in vitro models, including human umbilical vein endothelial cells (HUVECs). To evaluate the utility of iPSC-ECs for multidimensional HT toxicity profiling of chemicals, both iPSC-ECs and HUVECs were exposed to selected positive (angiogenesis inhibitors, cytotoxic agents) and negative compounds in concentration response for either 16 or 24 h in a 384-well plate format. Furthermore, chemical effects on vascularization were quantified using EC angiogenesis on biological (Geltrex™) and synthetic (SP-105 angiogenesis hydrogel) extracellular matrices. Cellular toxicity was assessed using high-content live cell imaging and the CellTiter-Glo® assay. Assay performance indicated good to excellent assay sensitivity and reproducibility for both cell types investigated. Both iPSC-derived ECs and HUVECs formed tube-like structures on Geltrex™ and hydrogel, an effect that was inhibited by angiogenesis inhibitors and cytotoxic agents in a concentration-dependent manner. The quality of HT assays in HUVECs was generally higher than that in iPSC-ECs. Altogether, this study demonstrates the capability of ECs for comprehensive assessment of the biological effects of chemicals on vasculature in a HT compatible format.

The comparison of the effects of a novel hydrogel compound and traditional hyaluronate following micro-fracture procedure in a rat full-thickness chondral defect model.

PURPOSE: The aim of this experimental study was to investigate the impact of HA-CS-NAG compound (hyaluronate, sodium chondroitin sulfate, N-acetyl-d-glucosamine) on the quality of repair tissue after micro-fracture and to compare it with HA (hyaluronat), in a rat full-thickness chondral defect model. METHODS: Full-thickness chondral defects were created in a non-weight bearing area by using a handle 2.7-mm drill bit, in the right knees of 33 Sprague-Dawley rats. Each specimen then underwent micro-fracture using a needle. Two weeks after surgery, 3 groups were randomly formed among the rats (n = 33). In Group 1, 0.2 mL of sterile saline solution (0.9%) was injected. In Group 2, 0.2 mL HA with a mean molecular weight of 1.2 Mda was injected. In Group 3, 0.2 mL of HA-CS-NAG compound (hyaluronate, sodium chondroitin sulfate, N-acetyl-d-glucosamine) was injected. The injections were applied on the 14th, the 21st and the 28th postoperative days. All rats were sacrificed on the 42nd postoperative day. Histological analysis of the repair tissue was performed for each specimen by two blinded observers using Wakitani scoring system. RESULTS: There was significantly improved repair tissue in both Group 3 and Group 2 when compared with Group 1. Group 3 showed statistically significant improvement in terms of 'cell morphology' and 'integration of donor with host' when compared to Group 2 (p < 0.001). CONCLUSION: Intra-articular injection of HA-CS-NAG compound after micro-fracture results in significantly improved repair tissue in rats' chondral defects when compared to HA regarding the donor integration and cell morphology.

Physical chitosan microhydrogels as scaffolds for spinal cord injury restoration and axon regeneration.

Recovery from traumatic spinal cord injury (SCI) usually fails due to a cascade of cellular and molecular events that compromise neural tissue reconstitution by giving rise to glial scarring and cavity formation. We designed a scaffold material for SCI treatment containing only chitosan and water as fragmented physical hydrogel suspension (Chitosan-FPHS), with defined degree of acetylation (DA), polymer concentration, and mean fragment size. Implantation of Chitosan-FPHS alone into rat spinal cord immediately after a bilateral dorsal hemisection promoted reconstitution of spinal tissue and vasculature, and diminished fibrous glial scarring: with astrocyte processes primarily oriented towards the lesion, the border between lesion site and intact tissue became permissive for regrowth of numerous axons into, and for some even beyond the lesion site. Growing axons were myelinated or ensheathed by endogenous Schwann cells that migrated into the lesion site and whose survival was prolonged. Interestingly, Chitosan-FPHS also modulated the inflammatory response, and we suggest that this might contribute to tissue repair. Finally, this structural remodeling was associated with significant, long-lasting gain in locomotor function recovery. Because it effectively induces neural tissue repair, Chitosan-FPHS biomaterial may be a promising new approach to treat SCI, and a suitable substrate to combine with other strategies.

Bone augmentation at peri-implant dehiscence defects comparing a synthetic polyethylene glycol hydrogel matrix vs. standard guided bone regeneration techniques.

OBJECTIVES: The aim of the study was to test whether or not the use of a polyethylene glycol (PEG) hydrogel with or without the addition of an arginylglycylaspartic acid (RGD) sequence applied as a matrix in combination with hydroxyapatite/tricalciumphosphate (HA/TCP) results in similar peri-implant bone regeneration as traditional guided bone regeneration procedures. MATERIAL AND METHODS: In 12 beagle dogs, implant placement and peri-implant bone regeneration were performed 2 months after tooth extraction in the maxilla. Two standardized box-shaped defects were bilaterally created, and dental implants were placed in the center of the defects with a dehiscence of 4 mm. Four treatment modalities were randomly applied: i)HA/TCP mixed with a synthetic PEG hydrogel, ii)HA/TCP mixed with a synthetic PEG hydrogel supplemented with an RGD sequence, iii)HA/TCP covered with a native collagen membrane (CM), iv)and no bone augmentation (empty). After a healing period of 8 or 16 weeks, micro-CT and histological analyses were performed. RESULTS: Histomorphometric analysis revealed a greater relative augmented area for groups with bone augmentation (43.3%-53.9% at 8 weeks, 31.2%-42.8% at 16 weeks) compared to empty controls (22.9% at 8 weeks, 1.1% at 16 weeks). The median amount of newly formed bone was greatest in group CM at both time-points. Regarding the first bone-to-implant contact, CM was statistically significantly superior to all other groups at 8 weeks. CONCLUSIONS: Bone can partially be regenerated at peri-implant buccal dehiscence defects using traditional guided bone regeneration techniques. The use of a PEG hydrogel applied as a matrix mixed with a synthetic bone substitute material might lack a sufficient stability over time for this kind of defect.

Novel bioceramic-reinforced hydrogel for alveolar bone regeneration.

UNLABELLED: The osseointegration of dental implants and their consequent long-term success is guaranteed by the presence, in the extraction site, of healthy and sufficient alveolar bone. Bone deficiencies may be the result of extraction traumas, periodontal disease and infection. In these cases, placement of titanium implants is contraindicated until a vertical bone augmentation is obtained. This goal is achieved using bone graft materials, which should simulate extracellular matrix (ECM), in order to promote osteoblast proliferation and fill the void, maintaining the space without collapsing until the new bone is formed. In this work, we design, develop and characterize a novel, moldable chitosan-pectin hydrogel reinforced by biphasic calcium phosphate particles with size in the range of 100-300µm. The polysaccharide nature of the hydrogel mimics the ECM of natural bone, and the ceramic particles promote high osteoblast proliferation, assessed by Scanning Electron Microscopy analysis. Swelling properties allow significant adsorption of water solution (up to 200% of solution content) so that the bone defect space can be filled by the material in an in vivo scenario. The incorporation of ceramic particles makes the material stable at different pH and increases the compressive elastic modulus, toughness and ultimate tensile strength. Furthermore, cell studies with SAOS-2 human osteoblastic cell line show high cell proliferation and adhesion already after 72h, and the presence of ceramic particles increases the expression of alkaline phosphatase activity after 1week. These results suggest a great potential of the developed moldable biomaterials for the regeneration of the alveolar bone. STATEMENT OF SIGNIFICANCE: The positive fate of a surgical procedure involving the insertion of a titanium screw still depends on the quality and quantity of alveolar bone which is present in the extraction site. Available materials are basically hard scaffold materials with un-predictable behavior in different condition and difficult shaping properties. In this work we developed a novel pectin-chitosan hydrogel reinforced with ceramic particles. Polysaccharides simulate the extracellular matrix of natural bone and the extensive in vitro cells culture study allows to assess that the incorporation of the ceramic particles promote a pro-osteogenic response. Shape control, easy adaption of the extraction site, predictable behavior in different environment condition, swelling properties and an anti-inflammatory response are the significant characteristics of the developed biomaterial.

Hydrogel control of water uptake by pectins during in vitro pollen hydration of Eucalyptus globulus.

PREMISE OF THE STUDY: Upon pollination, dehydrated pollen grains take water out of the stigma surface, an event that constitutes the first functional checkpoint of sexual reproduction in higher plants. Little is known about possible functional connections between rehydration speed and further steps of fertilization. Here we addressed the mechanisms of water uptake control by dehydrated pollen grains. Because dehydrated cells have no energy-driven active mechanism such as membrane-based osmoregulation for controlling water movement, we tested the hypothesis that another mechanism might exist, namely, the use of hydrogel-behaving molecules. METHODS: We developed an imaging protocol to visualize and quantify the rate of water entry into pollen grains of Eucalyptus globulus and tested the influence of different treatments linked to hydrogel-behaving molecules. We complemented these analyses by immunostaining pectins in the pollen grain with monoclonal antibodies JIM5 and JIM7. KEY RESULTS: Water entry seemed to proceed exclusively through the germination apertures of the pollen grain, and the changes observed in different hydration media are compatible with hydrogel behavior. When JIM5 and JIM7 were used to characterize pectins on the germination apertures during hydration, pectin localization and esterification changed during hydration and were affected by the hydration solutions. These results suggest that chemical modification of the pectins may change their hydrogel behavior, thus modifying the hydration speed. CONCLUSIONS: The hydrogel behavior of pectins and pectin localization on apertures strongly suggest that pectins act like "valves" for water entry, enabling a regulated process of water uptake into the dehydrated pollen grain. We propose that this regulation evolved in terms of achieving the correct self-organization of molecules and cellular components to resume metabolism and pollen tube growth, especially in species that are subject to demanding environmental water stress.

Synergistic enhancement of ectopic bone formation by supplementation of freshly isolated marrow cells with purified MSC in collagen-chitosan hydrogel microbeads.

PURPOSE: Bone marrow-derived mesenchymal stem cells (MSC) can differentiate osteogenic lineages, but their tissue regeneration ability is inconsistent. The bone marrow mononuclear cell (BMMC) fraction of adult bone marrow contains a variety of progenitor cells that may potentiate tissue regeneration. This study examined the utility of BMMC, both alone and in combination with purified MSC, as a cell source for bone regeneration. METHODS: Fresh BMMC, culture-expanded MSC, and a combination of BMMC and MSC were encapsulated in collagen-chitosan hydrogel microbeads for pre-culture and minimally invasive delivery. Microbeads were cultured in growth medium for 3 days, and then in either growth or osteogenic medium for 17 days prior to subcutaneous injection in the rat dorsum. RESULTS: MSC remained viable in microbeads over 17 days in pre-culture, while some of the BMMC fraction were nonviable. After 5 weeks of implantation, microCT and histology showed that supplementation of BMMC with MSC produced a strong synergistic effect on the volume of ectopic bone formation, compared to either cell source alone. Microbeads containing only fresh BMMC or only cultured MSC maintained in osteogenic medium resulted in more bone formation than their counterparts cultured in growth medium. Histological staining showed evidence of residual microbead matrix in undifferentiated samples and indications of more advanced tissue remodeling in differentiated samples. CONCLUSIONS: These data suggest that components of the BMMC fraction can act synergistically with predifferentiated MSC to potentiate ectopic bone formation. The microbead system may have utility in delivering desired cell populations in bone regeneration applications.

Design and formulation of a topical hydrogel integrating lemongrass-loaded nanosponges with an enhanced antifungal effect: in vitro/in vivo evaluation.

Lemongrass oil (LGO) is a volatile oil extracted from the leaves of Cymbopogon citratus that has become one of the most important natural oils in the pharmaceutical industry because of its diverse pharmacologic and clinical effects. However, LGO suffers from low aqueous solubility, which could lead to a reduced effect. Moreover, the instability of its major active constituent, citral, could lead to volatilization, reaction with other formulation ingredients, and consequently, skin irritation. To surmount these problems, this research aims to formulate lemongrass-loaded ethyl cellulose nanosponges with a topical hydrogel with an enhanced antifungal effect and decreased irritation. The minimal inhibitory concentration and minimal fungicidal concentration of LGO against Candida albicans strain ATC 100231, determined using the broth macrodilution method, were found to be 2 and 8 µL/mL, respectively. The emulsion solvent evaporation technique was used for the preparation of the nanosponges. The nanosponge dispersions were then integrated into carbopol hydrogels (0.4%). Nine formulations were prepared based on a 32 full factorial design employing the ethyl cellulose:polyvinyl alcohol ratio and stirring rate as independent variables. The prepared formulations were evaluated for particle size, citral content, and in vitro release. Results revealed that all the nanosponge dispersions were nanosized, with satisfactory citral content and sustained release profiles. Statistical analysis revealed that both ethyl cellulose:polyvinyl alcohol ratio and stirring rate have significant effects on particle size and percentage released after 6 hours; however, the effect of the stirring rate was more prominent on both responses. The selected hydrogel formulation, F9, was subjected to surface morphological investigations, using scanning and transmission electron microscopy, where results showed that the nanosponges possess a spherical uniform shape with a spongy structure, the integrity of which was not affected by integration into the hydrogel. Furthermore, the selected formulation, F9, was tested for skin irritation and antifungal activity against C. albicans, where results confirmed the nonirritancy and the effective antifungal activity of the prepared hydrogel.

An autologous platelet-rich plasma hydrogel compound restores left ventricular structure, function and ameliorates adverse remodeling in a minimally invasive large animal myocardial restoration model: a translational approach: Vu and Pal "Myocardial Repair: PRP, Hydrogel and Supplements".

AIMS: Cell-based myocardial restoration has not penetrated broad clinical practice yet due to poor cell retention and survival rates. In this study, we attempt a translational, large-scale restorative but minimally invasive approach in the pig, aiming at both structurally stabilizing the left ventricular (LV) wall and enhancing function following ischemic injury. METHODS AND RESULTS: A myocardial infarction (MI) was created by permanent ligation of left circumflex coronary artery through a small lateral thoracotomy. Thirty-six Yorkshire pigs were randomized to receive transthoracic intramyocardial injection into both infarct and border zone areas with different compounds: 1) Hyaluronic acid-based hydrogel; 2) autologous platelet-rich plasma (PRP); 3) ascorbic acid-enriched hydrogel (50 mg/L), combined with IV ibuprofen (25 mg/kg) and allopurinol (25 mg/kg) (cocktail group); 4) PRP and cocktail (full-compound); or 5) saline (control). The latter two groups received daily oral ibuprofen (25 mg/kg) for 7 days and allopurinol (25 mg/kg) for 30 days, postoperatively. Hemodynamic and echocardiographic studies were carried out at baseline, immediately after infarction and at end-point. Eight weeks after MI, the full-compound group had better LV fractional area change, ejection fraction and smaller LV dimensions than the control group. Also, dp/dtmax was significantly higher in the full-compound group when the heart rate increased from 100 bpm to 160bpm in stress tests. Blood vessel density was higher in the full-compound group, compared to the other treatment groups. CONCLUSIONS: A combination of PRP, anti-oxidant and anti-inflammatory factors with intramyocardial injection of hydrogel has the potential to structurally and functionally improve the injured heart muscle while attenuating adverse cardiac remodeling after acute myocardial infarction.

A novel poly(γ-glutamic acid)/silk-sericin hydrogel for wound dressing: Synthesis, characterization and biological evaluation.

A novel multifunctional poly(γ-glutamic acid)/silk sericin (γ-PGA/SS) hydrogel has been developed and used as wound dressing. The physical and chemical properties of the γ-PGA/SS gels were systemically investigated. Furthermore, these γ-PGA/SS gels have been found to promote the L929 fibroblast cells proliferate, and in the in vivo study, significant stimulatory effects were also observed on granulation and capillary formation on day 9 in H-2-treated wounds, indicating that this new complex hydrogel could maintain a moist healing environment, protect the wound from bacterial infection, absorb excess exudates, and promote cell proliferation to reconstruct damaged tissue. Considering the simple preparation process and excellent biological property, this γ-PGA/SS hydrogel might have a wide range of applications in biomedical and clinical areas.

Glucan HBP-A increase type II collagen expression of chondrocytes in vitro and tissue engineered cartilage in vivo.

OBJECTIVE: Although chondroprotective activities have been documented for polysaccharides, the potential target of different polysaccharide may differ. The study was aimed to explore the effect of glucan HBP-A in chondrocyte monolayer culture and chondrocytes-alginate hydrogel constructs in vivo, especially on the expression of type II collagen. METHODS: Chondrocytes isolated from rabbit articular cartilage were cultured and verified by immunocytochemical staining of type II collagen. Chondrocyte viability was assessed after being treated with HBP-A in different concentrations. Morphological status of chondrocytes-alginate hydrogel constructs in vitro was observed by scanning electron microscope (SEM). The constructs were treated with HBP-A and then injected to nude mice subcutaneously. Six weeks after transplantation, the specimens were observed through transmission electron microscopy (TEM). The mRNA expressions of disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTs-5), aggrecan and type II collagen in both monolayer culture and constructs were determined by real time polymerase chain reaction (PCR). The expression of type II collagen and matrix metalloproteinases-3 (MMP-3) in chondrocyte monolayer culture was also tested through Western blot and enzyme linked immunosorbent assay (ELISA), respectively. RESULTS: MMP-3 secretion and ADAMTs-5 mRNA expression in vitro were inhibited by HBP-A at 0.3 mg/mL concentration. In morphological study, there were significant appearance of collagen in those constructs treated by HBP-A. Accordingly, in both chondrocyte monolayer culture and chondrocytes-alginate hydrogel constructs, the expression of type II collagen was increased significantly in HBP-A group when compared with control group (P<0.001). CONCLUSIONS: The study documented that the potential pharmacological target of glucan HBP-A in chondrocytes monolayer culture and tissue engineered cartilage in vivo may be concerned with the inhibition of catabolic enzymes MMP-3, ADAMTs-5, and increasing of type II collagen expression.

Improved conduction and increased cell retention in healed MI using mesenchymal stem cells suspended in alginate hydrogel.

INTRODUCTION: Mesenchymal stem cells (MSCs) have been associated with reduced arrhythmias; however, the mechanism of this action is unknown. In addition, limited retention and survival of MSCs can significantly reduce efficacy. We hypothesized that MSCs can improve impulse conduction and that alginate hydrogel will enhance retention of MSCs in a model of healed myocardial infarction (MI). METHODS AND RESULTS: Four weeks after temporary occlusion of the left anterior descending artery (LAD), pigs (n = 13) underwent a sternotomy to access the infarct and then were divided into two studies. In study 1, designed to investigate impulse conduction, animals were administered, by border zone injection, 9-15 million MSCs (n = 7) or phosphate-buffered saline (PBS) (control MI, n = 5). Electrogram width measured in the border zone 2 weeks after injections was significantly decreased with MSCs (-30 ± 8 ms, p < 0.008) but not in shams (4 ± 10 ms, p = NS). Optical mapping from border zone tissue demonstrated that conduction velocity was higher in regions with MSCs (0.49 ± 0.03 m/s) compared to regions without MSCs (0.39 ± 0.03 m/s, p < 0.03). In study 2, designed to investigate MSC retention, animals were administered an equal number of MSCs suspended in either alginate (2 or 1 % w/v) or PBS (n = 6/group) by border zone injection. Greater MSC retention and survival were observed with 2% alginate compared to PBS or 1% alginate. Confocal immunofluorescence demonstrated that MSCs survive and are associated with expression of connexin-43 (Cx43) for either PBS (control), 1%, or 2% alginate. CONCLUSIONS: For the first time, we are able to directly associate MSCs with improved impulse conduction and increased retention and survival using an alginate scaffold in a clinically relevant model of healed MI.

Hydrogel-based 3D model of patient-derived prostate xenograft tumors suitable for drug screening.

The lack of effective therapies for bone metastatic prostate cancer (PCa) underscores the need for accurate models of the disease to enable the discovery of new therapeutic targets and to test drug sensitivities of individual tumors. To this end, the patient-derived xenograft (PDX) PCa model using immunocompromised mice was established to model the disease with greater fidelity than is possible with currently employed cell lines grown on tissue culture plastic. However, poorly adherent PDX tumor cells exhibit low viability in standard culture, making it difficult to manipulate these cells for subsequent controlled mechanistic studies. To overcome this challenge, we encapsulated PDX tumor cells within a three-dimensional hyaluronan-based hydrogel and demonstrated that the hydrogel maintains PDX cell viability with continued native androgen receptor expression. Furthermore, a differential sensitivity to docetaxel, a chemotherapeutic drug, was observed as compared to a traditional PCa cell line. These findings underscore the potential impact of this novel 3D PDX PCa model as a diagnostic platform for rapid drug evaluation and ultimately push personalized medicine toward clinical reality.

Designing unconventional Fmoc-peptide-based biomaterials: structure and related properties.

We have recently employed L-amino acids in the lipase-catalyzed biofabrication of a class of self-assembling Fmoc-peptides that form 3-dimensional nanofiber scaffolds. Here we report that using d-amino acids, the homochiral self-assembling peptide Fmoc-D-Phe3 (Fmoc-F*F*F*) also forms a 3-dimensional nanofiber scaffold that is substantially distinguishable from its L-peptide and heterochiral peptide (F*FF and FF*F*) counterparts on the basis of their physico-chemical properties. Such chiral peptides self-assemble into ordered nanofibers with well defined fibrillar motifs. Circular dichroism and atomic force microscopy have been employed to study in depth such fibrillar peptide structures. Dexamethasone release kinetics from PLGA and CS-PLGA nanoparticles entrapped within the peptidic hydrogel matrix encourage its use for applications in drug controlled release.

Blockade of the cerebral aqueduct in rats provides evidence of antagonistic leptin responses in the forebrain and hindbrain.

Previously, we reported that low-dose leptin infusions into the fourth ventricle produced a small but significant increase in body fat. These data contrast with reports that injections of higher doses of leptin into the fourth ventricle inhibit food intake and weight gain. In this study, we tested whether exogenous leptin in the fourth ventricle opposed or contributed to weight loss caused by third ventricle leptin infusion by blocking diffusion of CSF from the third to the fourth ventricle. Male Sprague-Dawley rats received third ventricle infusions of PBS or 0.3 µg leptin/24 h from miniosmotic pumps. After 4 days, rats received a 3-µl cerebral aqueduct injection of saline or of thermogelling nanoparticles (hydrogel) that solidified at body temperature. Third ventricle leptin infusion inhibited food intake and caused weight loss. Blocking the aqueduct exaggerated the effect of leptin on food intake and weight loss but had no effect on the weight of PBS-infused rats. Leptin reduced both body fat and lean body mass but did not change energy expenditure. Blocking the aqueduct decreased expenditure of rats infused with PBS or leptin. Infusion of leptin into the third ventricle increased phosphorylated STAT3 in the VMHDM of the hypothalamus and the medial NTS in the hindbrain. Blocking the aqueduct did not change hypothalamic p-STAT3 but decreased p-STAT3 in the medial NTS. These results support previous observations that low-level activation of hindbrain leptin receptors has the potential to blunt the catabolic effects of leptin in the third ventricle.

Vascular smooth muscle cell optimization of vasculogenesis within naturally derived, biodegradable, hybrid hydrogel scaffolds.

BACKGROUND: As vascularization represents the rate-limiting step in permanent incorporation of hydrogel-based tissue-regeneration templates, the authors sought to identify the material chemistry that would optimize endothelial cell adhesion and invasion into custom hydrogel constructs. The authors further investigated induction of endothelial tubule formation by growth factor supplementation and paracrine stimulation. METHODS: Hydrogel scaffolds consisting of combinations of alginate, collagen type I, and chitosan were seeded with human umbilical vein endothelial cells and maintained under standard conditions for 14 days. Cell density and invasion were then evaluated. Tubule formation was evaluated following basic fibroblast growth factor addition or co-culture with human aortic smooth muscle cells. RESULTS: Human umbilical vein endothelial cells demonstrated greatest cell-surface density and invasion volumes with alginate and collagen (10:1 weight/weight) scaffolds (p < 0.05). Supplementation with basic fibroblast growth factor increased surface density but neither invasion nor tubule formation. A significant increase in tubule content/organization was observed with increasing human aortic smooth muscle cell-to-human umbilical vein endothelial cell ratio co-culture. CONCLUSIONS: Alginate and collagen 10:1 scaffolds allow for maximal cellularization compared with other combinations studied. Growth factor supplementation did not affect human umbilical vein endothelial cell invasion or morphology. Paracrine signaling by means of co-culture with human umbilical vein endothelial cells stimulated endothelial tubule formation and vascular protonetwork organization. These findings serve to guide future endeavors toward fabrication of prevascularized tissue constructs.

Extração de bromelina dos resíduos de abacaxi ( Ananas comosus ) por sistemas de duas fases aquosas e sua aplicação em hidrogel polimérico / Extraction of bromelain from pineapple waste ( Ananas comosus ) by aqueous two-phase systems and its application in polymeric hydrogels

Bromelina é um nome coletivo para enzimas proteolíticas encontradas no talo, fruto e folhas do abacaxi (Ananas comosus Merr). A bromelina possui propriedades anti-inflamatórias, de debridamento, entre outras. Para a produção da bromelina deve-se, preferencialmente, usar resíduos do abacaxi, visto que os produtos do fruto têm aplicação comercial. Este trabalho teve como objetivo a extração de bromelina a partir de cascas de abacaxi através de sistema de duas fases aquosas (SDFA), e sua aplicação em hidrogel polimérico. Foram realizados estudos de estabilidade da bromelina comercial, em que se observou maior estabilidade em pH 5,0 com menor perda da atividade relativa em todas as temperaturas estudadas (20, 30, 40 e 50°C). O estudo da extração da bromelina em SDFA formado por polietileno glicol (PEG) e ácido poliacrílico (PAA) (com auxílio da análise de variância de parâmetros como rendimento, fator de purificação e coeficiente de partição) proporcionou rendimento de 335% e fator de purificação de 25,8. Os hidrogéis poliméricos à base de PEG estudados apresentaram-se flexíveis, com pouca elasticidade e taxa de absorção superior a 1000%. Hidrogel carreado de bromelina pelo método de turgescência proporcionou a maior liberação da enzima, assim como a maior atividade (80% da bromelina liberada em 24 h e 278 ± 89 U/mL)

Bromelain is a collective name for the proteolytic enzymes found in the stem, fruit and leaves of pineapple (Ananas comosus Merr.). Bromelain possesses anti-inflammatory properties, debridement, among others. For bromelain production one should preferably use the waste materials, whereas pineapple fruit products have commercial application. This study aimed to extract bromelain from pineapple peels using aqueous two-phase system (ATPS), and its application in polymeric hydrogels. Stability studies of commercial bromelain were performed, which found greater stability at pH 5.0 with minor loss of relative activity at all temperatures studied. The study of bromelain extraction in ATPS composed by polyethylene glycol (PEG) and poly acrylic acid (PAA) (with assistance of variance analysis of parameters such as yield, purification factor and partition coefficient) showed yield 335% and purification factor of 25.8. The PEG-based hydrogels studied presented flexibility, low elasticity and swelling ratio higher than 1000%. Hydrogel containing bromelain, loading by embedding (solvent sorption) method, yielded the highest enzyme release, as well as the highest activity (80% bromelain released over 24 h and 278 ± 89 U / mL)

Benzoyl peroxide formulated polycarbophil/carbopol 934P hydrogel with selective antimicrobial activity, potentially beneficial for treatment and prevention of bacterial vaginosis.

The human vagina is colonized by a variety of indigenous microflora; in healthy individuals the predominant bacterial genus is Lactobacillus while those with bacterial vaginosis (BV) carry a variety of anaerobic representatives of the phylum Actinobacteria. In this study, we evaluated the antimicrobial activity of benzoyl peroxide (BPO) encapsulated in a hydrogel against Gardnerella vaginalis, one of the causative agents of BV, as well as indicating its safety for healthy human lactobacilli. Herein, it is shown that in well diffusion assays G. vaginalis is inhibited at 0.01% hydrogel-encapsulated BPO and that the tested Lactobacillus spp. can tolerate concentrations of BPO up to 2.5%. In direct contact assays (cells grown in a liquid culture containing hydrogel with 1% BPO or BPO particles), we demonstrated that hydrogels loaded with 1% BPO caused 6-log reduction of G. vaginalis. Conversely, three of the tested Lactobacillus spp. were not inhibited while L. acidophilus growth was slightly delayed. The rheological properties of the hydrogel formulation were probed using oscillation frequency sweep, oscillation shear stress sweep, and shear rate sweep. This shows the gel to be suitable for vaginal application and that the encapsulation of BPO did not alter rheological properties.

Controlling the adhesion and differentiation of mesenchymal stem cells using hyaluronic acid-based, doubly crosslinked networks.

We have created hyaluronic acid (HA)-based, cell-adhesive hydrogels that direct the initial attachment and the subsequent differentiation of human mesenchymal stem cells (MSCs) into pre-osteoblasts without osteogenic supplements. HA-based hydrogel particles (HGPs) with an average diameter of 5-6 µm containing an estimated 2.2 wt% gelatin (gHGPs) were synthesized by covalent immobilization of gelatin to HA HGPs prepared via an inverse emulsion polymerization technique. Separately, a photocrosslinkable HA macromer (HAGMA) was synthesized by chemical modification of HA with glycidyl methacrylate (GMA). Doubly crosslinked networks (DXNs) were engineered by embedding gHGPs in a secondary network established by HAGMA at a particle concentration of 2.5 wt%. The resultant composite gels, designated as HA-gHGP, have an average compressive modulus of 21 kPa, and are non-toxic to the cultured MSCs. MSCs readily attached to these gels, exhibiting an early stage of stress fiber assembly 3 h post seeding. By day 7, stellate-shaped cells with extended filopodia were found on HA-gHGP gels. Moreover, cells had migrated deep into the matrix, forming a three dimensional, branched and interconnected cell community. Conversely, MSCs on the control gels lacking gelatin moieties formed isolated spheroids with rounded cell morphology. After 28 days of culture on HA-gHGP, Type I collagen production and mineral deposition were detected in the absence of osteogenic supplements, suggesting induction of osteogenic differentiation. In contrast, cells on the control gels expressed markers for adipogenesis. Overall, the HA-gHGP composite matrix has great promise for directing the osteogenic differentiation of MSCs by providing an adaptable environment through the spatial presentation of cell-adhesive modules.