Development of local vancomycin delivery system from fibrin gel to prevent Staphylococcus aureus biofilms graft infection

Prosthetic vascular graft infection is one of the most severe vascular surgery complications. Fibrin gel (FG) hasmany useful characteristics as biocompatibility, biodegradation, adhesion, and haemostasis to develop the localantibiotic delivery system. In this study, human plasma was collected from peripheral blood that was used tocreate fibrin gel by supplement ion Ca2?. Antibiotic-containing fibrin gel was then evaluated in some characteristics such as surface structure, biodegradation, antibiotic delivery, cytotoxicity, and bacterial biofilmprevention in vitro and in vivo. The results showed that fibrin gel was excellent material for the extendeddelivery of antibiotics. Most importantly, antibiotic-containing fibrin gel was not toxic for human fibroblastcells in vitro and inhibited bacterial biofilm growth in vitro and in vivo. This research is the first step indeveloping an antibiotic delivery system for effective graft infection treatments.

Effect of Soft Fibrin Gels onTransdifferentiation Behaviour in Activated Hepatic Stellate Cell / 中山大学学报(医学科学版)

[Objective]To determine whether activated hepatic stellate cell(HSC)become quiescent phenotype if cultured in a soft extracellular matrix(ECM).[Methods]HSC-T6 cells which stably expressed myofibroblast(MFB) phenotype,were cultured in the 2D and 3D in-vitro matrix culture models that were constructed by 1 mg/mL fibrin gel. The proliferation activity of HSC-T6 was determined by cell counting kit-8(CCK8)assay at different time points(24 h, 48 h,72 h,96 h). Immunofluorescence and gelatin zymography analysis were performed to detect the expression of α-SMA,MMP-2,and MMP-9. Moreover,the cellular surface morphology and deformability was detected by AFM.[Results]After cultured in the fibrin gel,the cellular proliferation activity,expression of α-SMA,MMP-2 and MMP-9,and cellular deformability of activated HSC-T6 was obviously down-regulated in contrast to the control group (P<0.05),and there were also differences in the inhibition of cellular proliferation activity,the assembly of α-SMA stress fiber and the effect of cellular deformability between 2D and 3D fibrin gel cultured(P<0.05).[Conclusions]Soft fibrin gel inhibited the transdifferentiation of activated HSC-T6,but could not make it revert to α-SMA negative quies-cent phenotype.

Cartilage Engineering Using Fibrin Gel and Chondrocyte Cell Sheets / 中山大学学报(医学科学版)

Objective]To study the feasibility of Cartilage engineering using fibrin gel and chondrocyte cell sheets.[Methods]rabbit auricular chondrocytes were isolated and cultured to form cell sheets in flasks. The cell sheets were harvested using cell scrapers,and cut into fragments. The two precursor solutions of Fibrin gel were used to suspend the cell sheet fragments and isolated chondrocytes,and then added into the wells of a 48-well plate to form Gelatinous chondroid disc constructs. After in vitro culture, the constructs were implanted into nude mice. After 8 weeks,the constructs were harvested,and the specimens were evaluated using grossly observing, histological and immunohistochemical observation. [Results]Mature cartilage discs were obtained. The histomorphology of the explanted discs appeared non-uniform cartilaginous tissue comprise of regenerated cartilage islands with different size and irregular shape. Immunohistochemistry staining demonstrated that type II collagen highly expressed in the ECM of the cartilage islands. In 1 of the 8 discs,partial ossification was observed.[Conclusion]Fibrin gel is a favourable carrier. Artificial cartilage with stereochemical structure was constructed via combining the fibrin gel and chondrocyte cell sheets.

Arcabouço de PRP-gel associado a células tronco mesenquimais: uso em lesões condrais em modelo experimental equino / PRP-gel scaffold associated with mesenchymal stem cells: use in experimental chondral defect of equine models

O plasma rico em plaquetas (PRP) é conhecido por apresentar propriedades anabólicas, anti-inflamatórias e capacidade de gelificação. Atualmente o PRP é considerado eficaz na reparação da cartilagem, sendo sua capacidade de formação de gel indicada para o preenchimento de defeitos condrais. O objetivo desse estudo foi analisar o uso do PRP ativado, no formato de arcabouço, como suporte para o implante de células tronco mesenquimais (CTM), no preenchimento e tratamento de lesões condrais induzidas em equinos. Doze equinos foram submetidos a uma cirurgia artroscópica no tempo zero do experimento (T0), onde foi induzida uma lesão condral de 15 mm de diâmetro na tróclea medial femoral dos membros pélvicos direito. As 12 articulações foram divididas em dois grupos distintos com seis articulações cada (GA e GB). As articulações do GA foram submetidas ao tratamento com o implante de CTM em gel de PRP. As articulações de GB foram o grupo controle do experimento. As CTMs foram extraídas do tecido adiposo e o PRP em gel foi obtido por protocolo de dupla centrifugação seguido da adição de trombina liofilizada. Após cinco meses (T150) foi realizada nova artroscopia para avaliação macroscópica do local, coleta de amostras do tecido de reparação para análises de microscopia eletrônica, sendo realizadas imagens ressonância magnética e tomografia computadorizada no local do implante no GA. Observamos que o gel de PRP associado às CTM demonstrou ser adequado no tratamento de defeitos condrais experimentais dos equinos. GA evidenciou um melhor aspecto macroscópico e microscópico do tecido de reparação, sendo que GB mostrou maior desorganização das fibras colágenas. Nas imagens de ressonância magnética e tomografia computadorizada apenas foi relevante o local da lesão condral. O arcabouço de gel de PRP demonstrou ser apropriado no suporte do tratamento com as CTMs, sendo de fácil aplicação e efetivo, demonstrando resultados promissores na reparação de lesões condrais induzidas.(AU)

The platelet-rich plasma (PRP) is characterized by its anabolic, anti-inflammatory and gelling capability. Nowadays, the PRP is considered effective in the repair of cartilage defects, and its gelling capability is proper to filling chondral defects. So, the aim of this study was to investigate the use of activated PRP as a fibrin gel scaffold, such as support for the use with mesenchymal stem cells (MSC), on the treatment of experimentally chondral articular defects. Twelve horses were subjected to an arthroscopic surgery at time zero of the experiment (T0). A chondral defect of 15 mm diameter was created on the medial femoral trochlea and these 12 joints were divided into two groups each with six joints in each group (GA and GB). The joints of the GA were treated with implantation of MSC and PRP-gel. GB joints were the control group. MSCs were cultivated from adipose tissue and PRP-gel was obtained by double centrifugation protocol followed by addition of lyophilized thrombin. After five months (T150) was performed new arthroscopy for macroscopic evaluation of the defect local, collect samples of tissue repair for electron microscopy assessment and also was implemented a magnetic resonance images and computed tomography on GA. It was observed that the PRP-gel associated with CTMs showed a suitable treatment of experimental chondral defects in horses. GA showed a better macroscopic and microscopic appearance of the tissue repair. GB showed smaller number of chondrocytes and increased collagen fibers disorganization. At the magnetic resonance and computed tomography imaging only the local of chondral defect was viewed. The PRP-gel scaffold was satisfactory to use and support MSCs implantation. It showed an easy handling and it was effective, showing a promising results in the repair of induced chondral defects.(AU)

Neuronal differentiation of PC12 and embryonic stem cells in two- and three-dimensional in vitro culture.

The quality of neuronal differentiation and reduction in apoptosis that occurred in two-dimensional (2D) and three-dimensional (3D) culture conditions is compared. PC12 and embryonic stem cells are two commonly utilized cell lines for the study of neuronal regeneration. These cells were induced to neuronally differentiate by adding NGF and retinoic acid respectively. Total neurite length and expression of neuronal markers (MAP-2 and β3-tubulin) was assessed by morphometry and immunocytochemistry. Also, TUNEL assay was used to detect apoptosis. Upon exposure to a differentiation media in the 3D fibrin gel, PC12 and embryonic stem cells stopped dividing, had increased adhesion to the substratum, extended neurite processes and expressed neuronal markers. The same results, however, were not observed with the 2D culture. Also, the apoptosis index performed by TUNEL assay demonstrated a reduction in the degree of apoptosis in the 3D culture compared to 2D culture. Fibrin matrix supports growth and neuronal differentiation of PC12 and embryonic stem cells. In addition, the 3D culture enhanced cellular resistance to apoptosis when compared to the 2D culture. It appears as if a 3D culture system may offer a better technique for future neuronal tissue engineering investigations.

Complex of growth differentiation factor-5 and fibrin gel for repair of lumbar disc injury in rabbits / 中华创伤杂志

Objective To investigate the result of local injection of growth differentiation factor-5 (GDF-5) and fibrin gel for treatment of lumbar disc injury in rabbits.Methods Lumbar puncture with a 20-gauge needle was performed at L3/4,L4/5,and L5/6 discs of 40 New Zealand white rabbits.After needle puncture,L3/4 discs were injected with GDF-5 and fibrin gel (compound group) ; L4/5 discs fibrin gel (fibrin gel group) ; L5/6 discs nothing (blank control group).Two weeks later,intervertebral disc degeneration in each group was observed via radiography,MRI and nucleus proteoglycan content detection and histological examination.Results At postoperative 2-,4-,8-,and 12-weeks,X-ray films revealed a gradual decrease in disc height index (DHI) among the three groups,but the decreasing velocity was lower in compound group than in other two groups (P ＜ 0.05).On MRI,the signal of intervebral discs among the three groups diminished progressively with time,but a relatively lower decreasing was observed in compound group (P ＜ 0.05).At postoperative 4-week,proteoglycan content of the nucleus pulposus was (6.3-± 0.4) in compound group,higher than (5.9-0.4) in blank control group and (5.8-± 0.3)in fibrin gel group (P ＜0.05).At postoperative 2-week,histological evaluation showed (5.28 ±0.41)points in compound group,lower than (7.54 ± 0.53) points in blank control group and (7.21 ± 0.44)points in fibrin gel group (P ＜ 0.05).Conclusion Local injection of GDF-5 and fibrin gel facilitates the restoration of the injured discs and delays further disc degeneration.

Preparation and optimization of fibrin-gel-coated vancomycin alginate beads / 中华创伤杂志

Objective To prepare and optimize fibrin-gel-coated vaneomycin alginate beads (FG-Vanco-AB)and investigate their possible use in treatment of osteomyelitis or prevention of infection.Methods Vancomycin alginate beads were produced by dropping vancomycin and alginate mixed liquor into calcium chloride solution.Beads including high vancomycin content were prepared and chosen by optimizing different concentrations of vancomycin solution and alginate solution.These beads were coated with fibrin gel formed by different concentrations of fibrin and the same concentration thrombin.The optimized beads were selected based on available release time,when vancomycin in medium could kill Staphylococcus aureus(ATCC25923). Results Higher content of vancomycin in bead resulted in increase of vancomycin concentration and alginate concentration in mixed liquid.The highest vancomycin content beads were prepared by 16%alginate and 50 mr/ml vancomycin,up to(27.36±0.90)%.The further results showed that vancomycin concentrations from beads coated with fibrin at 75 mg/ml and thrombin at 400 IU/ml could kill Staphylococcus aureus and remained above the breakpoint sensitivity for 19 days.Conclusion The available release time is prolonged,and the possibility of clinical use is conspicuously increased after vancomycin beads are optimized by adjusting the rate of mixed component and fibrin gel coat.

Elementary study on construction of engineered blood vessel using chitosan-fibrin gel as scaffold / 中国康复理论与实践

@#ObjectiveTo investigate the feasibility of construction of engineered blood vessel using chitosan tube and fibrin gel as scaffold.MethodsVascular endothelial cells and smooth muscle cells were harvested from aortas of a rat, respectively. After expansion in vitro, vascular endothelial cells were seeded onto the inner surface of chitosan tube and smooth muscle cells mixed with fibrin gel seeded onto outer surface of the scaffold to construct engineered blood vessels. Inverted microscope, immunohistochemical staining and scanning electronic microscope were used to evaluate the construct.ResultsVascular endothelial cells formed monolayer and covered the inner surface of chitosan tube. Smooth muscle cells survived in the fibrin gel and grew in a 3-dimensional manner. ConclusionChitosan-fibrin gel may be potentially used as scaffold of engineered blood vessels.

Hepatocyte Transplantation Using Fibrin Gel / 대한이식학회지

PURPOSE: Whole liver transplantation, an effective therapy for many inherited and acquired hepatic disorders, has limitations including donor shortage and fatal surgical complications. Hepatocyte transplantation, which is simpler and less expensive than whole liver transplantation, allows the use of living related donors, permits the use of a single donor organ for multiple recipients, and makes possible the cryopreservation of hepatocytes for future use. However, choosing a proper scaffold for hepatocyte transplantation hampers wide use of hepatocyte transplantation. We performed hepatocyte transplantation using fibrin gel, as a cell transplantation scaffold and evaluated their effectiveness. METHODS: Female, five week old FVB mice, were prepared for donors, and two male, five week old nude mice, were used as recipients. Liver cells were isolated from FVB donors. The cell viability exceeded 95% as assessed by the trypan blue exclusion method. For three nude mice, 5x10(6) cells resuspended in 500microliter of fibrinogen were mixed with 500microliter thrombin, and were injected into the peritoneal cavity of each mouse. One nude mouse was transplanted with 5x10(6) cells resuspended in 500 microliter medium, which served as a negative control. Specimens were retrieved at one week, and histological and immunohistochemical analyses wereperformed. RESULTS: In the negative control, all transplanted hepatocytes disappeared at one week. In mice transplanted both fibrin gel and hepatocytes, conglomerates containing hepatocytes were observed on the intestinal mesentery. The hepatocytes were identified by H & E staining and immunohistochemistry using anti-hepatocyte antibody. Functional activity was evaluated with PAS staining. CONCLUSION: In this preliminary study, stable hepatocyte engraftment was achieved in hepatocyte transplantation with fibrin gel, but not in hepatocyte transplantation without scaffold. More studies on comparison between fibrin gel and injectable scaffolds would be necessary. Improvement on both initial vascularization and proliferation of transplanted hepatocytes is a target of our future work.