GMP-compliant sponge-like dressing containing MSC lyo-secretome: Proteomic network of healing in a murine wound model.

Chronic wounds account for 3% of total healthcare expenditure of developed countries; thus, innovative therapies, including Mesenchymal Stem Cells (MSCs) end their exosomes are increasingly considered, even if the activity depends on the whole secretome, made of both soluble proteins and extracellular vesicles. In this work, we prove for the first time the in vivo activity of the whole secretome formulated in a sponge-like alginate wound dressing to obtain the controlled release of bioactive substances. The product has been prepared in a public GMP-compliant facility by a scalable process; based on the murine model, treated wounds healed faster than controls without complications or infections. The treatment induced a higher acute inflammatory process in a short time and sustained the proliferative phase by accelerating fibroblast migration, granulation tissue formation, neovascularization and collagen deposition. The efficacy was substantially supported by the agreement between histological and proteomic findings. In addition to functional modules related to proteolysis, complement and coagulation cascades, protein folding and ECM remodeling, in treated skin, emerged the role of specific wound healing related proteins, including Tenascin (Tnc), Decorin (Dcn) and Epidermal growth factor receptor (EGFR). Of note, Decorin and Tenascin were also components of secretome, and network analysis suggests a potential role in regulating EGFR. Although further experiments will be necessary to characterize better the molecular keys induced by treatment, overall, our results confirm the whole secretome efficacy as novel "cell-free therapy". Also, sponge-like topical dressing containing the whole secretome, GMP- compliant and "ready-off-the-shelf", may represent a relevant point to facilitate its translation into the clinic.

Development and evaluation of silver sulfadiazine loaded microsponge based gel for partial thickness (second degree) burn wounds.

Silver sulfadiazine has been frequently used as an antibacterial agent for topical treatment of partial thickness burn wounds. In this study, we present the preparation of silver sulfadiazine microsponges by w/o/w emulsion solvent evaporation method. Formulation variables were optimized by using 32 factorial design. The optimized microsponges were characterized by FTIR, DSC, PXRD, particle size analysis, SEM analysis and mercury intrusion porosimetry studies. Viscosity, texture analysis and ex vivo drug deposition study of optimized microsponge loaded gel were also evaluated. The safety of the optimized gel was assessed by MTT assay using epidermal keratinocyte (HaCaT) and mouse embryonic fibroblast (NIH-3T3) cell lines. In vitro antibacterial studies were carried out to compare the antibacterial inhibitory efficiency of the optimized gel against the commercial product. The efficacy of the optimized gel was evaluated by the partial thickness (second degree) burn wound model in mice. Optimized microsponge loaded gel enhanced the drug retaining capacity in the skin layers, by 3 fold higher to that of a commercial product. The antibacterial inhibitory efficiency of optimized gel was similar to the commercial product against the Staphylococcus aureus and Pseudomonas aeruginosa. Optimized gel showed reduced frequency of application, no skin irritation, low cytotoxicity on dermal cell lines and enhanced wound contraction.

Pharmacokinetics of gelatin sponge microparticles in a rabbit VX2 liver tumor model of hepatic arterial chemoembolization.

The objective of this study is to investigate pharmacokinetics of gelatin sponge microparticles (GSMs) combined with epirubicin in a rabbit VX2 liver tumor model of hepatic arterial chemoembolization (TACE). Eighteen successful models of VX2 in New Zealand white rabbits was established, which were divided into three groups randomly: HAI group (n = 6), the epirubicin solution (epirubicin 10 mg mixed with saline 10 ml into the hepatic artery); GSMs-TACE group (n = 6), GSMs (20 mg) mixed with epirubicin solution (1 mg/ml); c-TACE group (n = 6), epirubicin (10 mg) mixed with lipiodol (10 ml). Each rabbit was administrated epirubicin at dose adjusted for a 1 mg/kg. Samples were collected from femoral vein at 5, 10, 20, 30, 40, 60, 90, and 120 min after therapy after 120 min; rabbit was killed, and tumor and peritumoral normal liver tissue was cised. Epirubicin concentrations in plasma and tumor were measured. The epirubicin concentration in plasma was significantly lower in GSMs-TACE group than in HAI group. C max in there groups after administration was 28.77 ± 7.15 µg/ml in c-TACE group, 83.84 ± 32.28 µg/ml in GSMs-TACE group, and 238.46 ± 23.44 µg/ml in HAI group at 5 min, respectively. The epirubicin concentration in tumor tissue was 53.06 ± 16.9 µg/g in c-TACE group, 44.49 ± 16.80 µg/g in the GSMs-TACE group, and 18.32 ± 8.30 µg/g in HAI group, respectively. Epirubicin concentration of GSMs-TACE group was significantly higher than that of HAI group (P < 0.05). The area under the curve (AUC) at 0-120 min in c-TACE, GSMs-TACE, and HAI groups were 1,815 ± 889.88, 3,416 ± 799.90, and 11,899 ± 2,717.17 µg min/ml, respectively. The AUC was lower in GSMs-TACE group than in HAI group (P < 0.05). Compared with HAI, GSMs-TACE has higher epirubicin concentrations in tumor and lower concentrations in plasma. The results show that GSMs-TACE has a feature of slow drug release-it may be one of the mechanisms of GSMs-TACE for HCC.

Calibrated bioresorbable microspheres: a preliminary study on the level of occlusion and arterial distribution in a rabbit kidney model.

PURPOSE: To assess the level of occlusion and arterial distribution of calibrated bioresorbable microspheres (BRMS-I and BRMS-II) compared with tris-acryl gelatin microspheres (TGMS) after renal embolization. MATERIALS AND METHODS: Six rabbits underwent renal embolization with 100-300 µm BRMS-I and TGMS; three rabbits received partial occlusion (group 1, n = 3), and three rabbits received total occlusion (group 2, n = 3). Four other rabbits received 100-300 µm BRMS-II (with higher cross-linking density than BRMS-I) in the left kidneys reaching total occlusion (group 3, n = 4). Coronal sections of the kidneys were histologically analyzed. Ease of injection, microsphere deformation, vessel sizes, and arterial distribution were assessed. RESULTS: The injection of BRMS-I, BRMS-II, and TGMS through microcatheters went smoothly without any clogging. In group 1, BRMS identification was easier than TGMS. In group 2, both BRMS-I and TGMS were observed in all three arterial levels (interlobar, arcuate, and interlobular arteries) without a significant difference (P = .84). BRMS-I were not significantly different from TGMS in the mean diameter of vessels occluded (197 µm ± 23 vs 158 µm ± 21, P = .25) or the microsphere deformation (8.85% ± 0.53% vs 11.80% ± 0.64%, P = .071). In group 3, the arterial distribution of BRMS-II was significantly different from BRMS-I and TGMS (P < .0001). CONCLUSIONS: In occluding arteries, 100-300 µm BRMS-I were not significantly different from 100-300 µm TGMS. Arterial distribution of BRMS can be influenced by their cross-linking density.

Synthetic hydrogel scaffold is an effective vehicle for delivery of INFUSE (rhBMP2) to critical-sized calvaria bone defects in rats.

Medtronic's INFUSE Bone Graft provides surgeons with a potent tool for stimulating bone formation. Current delivery vehicles that rely on Absorbable Collagen Sponges (ACS) require excessive quantities of the active ingredient in INFUSE, recombinant human Bone Morphogenic Protein-2 (rhBMP2), to achieve physiologically relevant concentrations of the growth factor, driving up the cost of the product and increasing the likelihood of undesirable side effects in neighboring tissues. We demonstrate that a simple light-mediated, thiol-ene chemistry can be used to create an effective polymer delivery vehicle for rhBMP2, eliminating the use of xenographic materials and reducing the dose of rhBMP2 required to achieve therapeutic effects. Comprised entirely of synthetic components, this system entraps rhBMP2 within a biocompatible hydrogel scaffold that is degraded by naturally occurring remodeling enzymes, clearing the way for new tissue formation. When tested side-by-side with ACS in a critical-sized bone defect model in rats, this polymeric delivery system significantly increased bone formation over ACS controls.

Release of vancomycin from multilayer coated absorbent gelatin sponges.

Wounds have the potential to become infected during any surgical procedure. Gelatin sponges that are commonly used to absorb blood during invasive surgeries would benefit tremendously if they released antibiotics. In this work, we have examined coating a commercial gelatin sponge with degradable polymer multilayer films containing vancomycin. The effect of the film on sponge absorption capabilities and the effect of the sponge on drug release kinetics were both examined. Application of vancomycin containing layer-by-layer assembled films to this highly porous substrate greatly increased drug loading up to approximately 880% compared to a flat substrate. Vancomycin drug release was extended out to 6 days compared to 2 days for film coated flat substrates. Additionally, the absorbent properties of the gelatin sponge were actually enhanced by up to 170% due to the presence of the vancomycin film coating. A comparison of film coated sponges with sponges soaked directly in vancomycin demonstrated the ability of the multilayer films to control drug release. Film released vancomycin was also found to remain highly therapeutic with unchanged antimicrobial properties compared to the neat drug, demonstrated by quantifying vancomycin activity against Staphylococcus aureus in vitro.

A comparison of three transarterial lipiodol-based formulations for hepatocellular carcinoma: in vivo biodistribution study in humans.

This study aimed to evaluate and compare the biodistribution properties of three transarterial Lipiodol-based therapeutic regimens in human hepatocellular carcinoma (HCC). In this prospective study with 13 patients randomly allocated to one of three study groups, each of the patients received transcatheter intra-arterial administration into a solitary HCC with one of three different Lipiodol-based formulations: Lipiodol-ethanol mixture (LEM; Group A), Lipiodol alone (Group B), and Lipiodol and gelatin pledgets (Group C). With the use of radioactive iodine-131-labeled Lipiodol, each group was assessed for (1) pattern of Lipiodol accumulation in the lungs within the first 2 weeks as evaluated by single-photon emission computed tomography and (2) decomposition of Lipiodol formulation within the first 2 weeks as evaluated by radioactivity detected in peripheral blood and urine. The degree of Lipiodol retention in the tumor within the first 4 weeks was evaluated with CT. No statistically significant difference in Lipiodol accumulation in the lungs was detected among the three groups. However, the peak accumulation in the lungs was delayed 3 days for Group A compared to Groups B and C. The degree of Lipiodol retention within the tumor in Group A was significantly greater than that in Groups B and C on day 14 (p = 0.014) and day 28 (p = 0.013). This study showed that LEM is associated with a greater embolic effect in intrahepatic HCC at 4 weeks, and a comparable degree of lung shunting and decomposition rates, compared with ethanol-free Lipiodol formulations.

Nano-ferrosponges for controlled drug release.

Magnetic sponge-like hydrogels (ferrosponges) were fabricated by using an in-situ synthesis of magnetic nanoparticles (MNPs) in the presence of various concentrations of gelatin. The resulting ferrosponges show an interconnected nanopore structure which serves as a reservoir to accommodate therapeutic drugs and the nanoporous networks demonstrate magnetic sensitive behavior under the application of magnetic field. The ferrosponges showed high swelling ratios, together with excellent elasticity and hydrophilicity, allow them to response rapidly to an external magnetic stimulation for fast and repeatable swelling-deswelling (or expansion-contractile) operations. The ferrosponges with lower gelatin concentration exhibited good performance on magnetification. Furthermore, drug release from the ferrosponges is relatively magnetic-sensitive and is dominated by its magnetism and associated interaction between the magnetic nanoparticle and the gelatin matrix under an external magnetic field. Higher MNPs concentration in the ferrosponges exhibited higher degree of magnetic sensitive which is due to stronger interparticle forces. By taking these peculiar magnetic sensitive behaviors of the ferrosponges, a novel drug delivery system can be designed for medical uses.

Controlling bone morphogenetic protein diffusion and bone morphogenetic protein-stimulated bone growth using fibrin glue.

STUDY DESIGN: An in vitro and in vivo study. OBJECTIVE: To evaluate the ability of fibrin glue to limit diffusion of recombinant human bone morphogenetic protein (rhBMP)-2 and its ability to protect spinal nerves from rhBMP-2 stimulated bone growth. SUMMARY OF BACKGROUND DATA: Studies have shown bone morphogenetic protein (rhBMP-2) stimulated bone growth can encroach on the spinal canal and nerves, causing neural compression. More recently, rhBMP-2 use in the cervical spine has been associated with life-threatening swelling. Fibrin glue has been used as a biologic carrier but has not been evaluated for its ability to limit rhBMP-2. METHODS: In phase 1 of the study, rhBMP-2 soaked absorbable collagen sponges (ACS) were encapsulated in fibrin glue and immediately incubated in physiologic lactated ringers solution at 38 degrees C. Samples of solution were tested for rhBMP-2 concentration. In phase 2 of the study, rats were surgically treated with laminectomy and placement of rhBMP-2/ACS versus laminectomy and placement of fibrin glue before placement of rhBMP-2/ACS. After 8 weeks, animals were euthanized and imaged using micro-computerized tomography. RESULTS: The diffusion study showed a significant limitation in rhBMP-2 diffusion when encapsulated in fibrin glue. The laminectomy study revealed blockage of bone formation by fibrin glue and protection of the spinal canal. CONCLUSIONS: Fibrin glue can limit the diffusion of rhBMP-2, and, thus, it can be used to help protect the spinal canal and nerve roots from rhBMP-2 stimulated bone growth.

Bio-sorption of acidic gelatine hydro-gels implanted in the back tissues of Fisher's rats.

Recent advance in tissue engineering therapy requires new scaffold materials. Acidic gelatine powders (10 wt%) were, thus, dissolved in water, were or were not cross-linked, and freeze-dried. After sterilization, prepared small sponges were implanted in 7-week-old Fisher's rats' subcutaneous tissues for up to 2 weeks. Sponges absorbed body fluid and changed into hydro-gels in vivo. Non-cross-linked hydro-gels were absorbed within 3 days, while cross-linked hydro-gels were eliminated after 7 days' implantation. Histological observations revealed that the common captivation process was mild while granulocytes and macrophages were encountered. Because acidic gelatine sponges can accommodate various basic growth factors, it can be speculated that prepared sponges might be used as short-time hydro-gel scaffolds and growth-factor carriers.

The preparation and characterisation of drug-loaded alginate and chitosan sponges.

Sponges composed of sodium alginate and chitosan were prepared via a freeze drying process in order to assess the utility of mixed sponges as potential wound dressings or matrices for tissue engineering. Sponge preparation involved dissolving both polymers (either individually or mixed) in 1% acetic acid and freeze-drying the corresponding solutions. The mechanical properties of the sponges were assessed using texture analysis and the microstructure examined using scanning electron microscopy. The dissolution of a model drug (paracetamol) from the sponges was assessed as a function of polysaccharide composition. It was noted that the sponges had a flexible yet strong texture, as assessed macroscopically. Measurement of the resistance to compression ('hardness') indicated that the chitosan sponges were the 'hardest' while the alginate sponges showed the least resistance to compression, with all sponges showing a high degree of recovery. In contrast, the breaking force (tensile force) of the sponges were greatest for the single component systems, while the elongation prior to breaking was similar for each material. SEM studies indicated that the mixed systems had a less well-defined microstructure than the single component sponges. This was ascribed to the two polysaccharides interacting in aqueous solution via coulombic forces, leading to a more randomly ordered network being formed on freezing. Dissolution studies indicated that systems containing chitosan alone showed the slowest release profile, with the mixed systems showing a relatively rapid dissolution profile. The use of chitosan and alginates together, therefore, appears to allow the formulator to manipulate both the mechanical properties and the drug release properties of the sponges.

The effect on the middle-ear cavity of an absorbable gelatine sponge alone and with corticosteroids.

The objectives of this study were to establish whether there is an obvious difference between intact mucosa and abraded mucosa of the middle-ear cavity in respect to the potential side effects from the application of absorbable gelatine sponge (Gelfoam) and to investigate if Gelfoam combined with corticosteroid ointment (cortimycine, sterile 1% hydrocortisone acetate) can reduce the occurrence of these effects. Twenty Albino rats were used in the study. These animals were divided into four groups, with ten ears in each group. In group A, the middle-ear mucosa was kept intact, and Gelfoam was inserted into the middle-ear cavity. In group B, the middle-ear mucosa was abraded, and Gelfoam was inserted. In group C, Gelfoam with corticosteroid was implanted over the intact mucosa, and in group D, the mucosa was abraded prior to the insertion of Gelfoam with corticosteroid. The changes were evaluated 8 weeks postoperatively. In group A, there was a minimal increase in fibroblastic activity, vascular proliferation with mild to moderate fibrosis and all but two tympanic membranes were perfectly normal. However, in group B, we encountered a significant increase in fibroblastic activity, vascular proliferation and fibrosis, and we observed that all tympanic membranes were moderately to severely thickened. These histopathologic changes related to Gelfoam were noted to be decreased in group C and especially in group D. As previously reported in the literature, Gelfoam was found to promote the formation of connective tissue in the middle-ear cavity regardless of the status of the mucosa. The unwanted effects of this material may be decreased if it is combined with corticosteroids in the middle-ear cavity.

Effect of composition on the physicochemical properties and active substance release from gelatin-alginate sponge.

The aim the study was physicochemically characterize and develop ability of the active substance (cefradine) from the implantable porous carriers. The drug delivery systems consisting of the gelatine and alginic acid sodium salt and glycerol (GL) or peanut oil (AO). Gelatin-alginate sponge was prepared by foamed components and next freeze-dried this foam. The composition of the sponges affected on the sorption ability and on the stability to proteolytic enzymes. Owing to porous structure obtained specific profile of active substance release.

Tissue implantation test in rabbits with gelatine sponges.

The purpose of this implantation study was to evaluate possible toxic effects and quality and quantity of resorption of two different resorbable gelatine sponges when implanted into the paravertebral muscle of the rabbit for a period of 7, 14 or 21 days and to evaluate bioequivalence of two types of test material. Test materials (Gelaspon) and a commercially available reference material were compared in three groups with 6 rabbits each. Tissue reaction to the implants and sponge degradation were scored on a graded scale and the sponges were compared for evaluation. No treatment-related mortality or body weight changes were observed. Clinical signs, indicative of local effects, were mainly seen for reference material. Tissue reaction after 7 days was comparable, after 14 and 21 days a significant increase in granulomatous reaction was seen for reference material. Sponge degradation was complete for test materials after 14 and 21 days without inflammatory tissue reaction, but small sponge rests of reference material were still present, accompanied by an increased inflammatory response. Results show test materials to fulfil the clinical requirements for implant material concerning fast resorption without inflammatory reactions and its superiority to reference material. Bioequivalence of used types of test material could be confirmed.