Hybrid scaffolds based on PLGA and silk for bone tissue engineering

Porous silk scaffolds, which are considered to be natural polymers, cannot be used alone because they have a long degradation rate, which makes it difficult for them to be replaced by the surrounding tissue. Scaffolds composed of synthetic polymers, such as PLGA, have a short degradation rate, lack hydrophilicity and their release of toxic by‐products makes them difficult to use. The present investigations aimed to study hybrid scaffolds fabricated from PLGA, silk and hydroxyapatite nanoparticles (Hap NPs) for optimized bone tissue engineering. The results from variable‐pressure field emission scanning electron microscopy (VP–FE–SEM), equipped with EDS, confirmed that the fabricated scaffolds had a porous architecture, and the location of each component present in the scaffolds was examined. Contact angle measurements confirmed that the introduction of silk and HAp NPs helped to change the hydrophobic nature of PLGA to hydrophilic, which is the main constraint for PLGA used as a biomaterial. Thermo‐gravimetric analysis (TGA) and FT–IR spectroscopy confirmed thermal decomposition and different vibrations caused in functional groups of compounds used to fabricate the scaffolds, which reflected improvement in their mechanical properties. After culturing osteoblasts for 1, 7 and 14 days in the presence of scaffolds, their viability was checked by MTT assay. The fluorescent microscopy results revealed that the introduction of silk and HAp NPs had a favourable impact on the infiltration of osteoblasts. In vivo experiments were conducted by implanting scaffolds in rat calvariae for 4 weeks. Histological examinations and micro‐CT scans from these experiments revealed beneficial attributes offered by silk fibroin and HAp NPs to PLGA‐based scaffolds for bone induction. Copyright © 2015 John Wiley & Sons, Ltd.     

Characterization of isoquinolin alkaloids from <Emphasis Type="Italic">Fumaria agraria</Emphasis> and evaluation of their antiproliferative activity against human breast cancer cell lines

Fumaria agraria (Fumariaceae) is an annual plant used traditionally in Algeria for various medicinal purposes. Alkaloids extracts from areal parts were subjected to solvent fractionation and GCMS analysis. Rates of alkaloids recorded were respectively of 700 mg/100 g of Dry Weight of total alkaloids “TA”, 400 mg/100 g DWof neutral fraction “NF”, 40 mg/100 g DW of acid fraction “AF” and 250 mg/100 g DWof the basic fraction “BF”. Protopine was found to be the most potent alkaloid of the total extract (41.55%). Fractionation showed that Protopine was found essentially in the BF fraction (60.7%). Antiproliferative activities of total alkaloids extract and BF fraction were assessed on MCF-7 and MDAMB- 231 breast cancer and MCF10A mammary normal cell lines, by trypan blue exclusion essay. Both TA extract and BF fraction showed potent antiproliferative activities against MCF-7 breast cancer cells in varying degrees with respective IC₅₀ of 47.8 ± 1.8 µg/ml and 17.6 ± 0.4 µM, without cell death induction but no impact was observed on normal MCF-10A cell growth. MCF7 Cell line was most sensitive (IC₅₀ of 17,6 ± 0,4µM) to BF fraction effect than MDAMB-231 one (IC₅₀ of 36,4 ± 0,6µM). Isoquinolein alkaloids of Fumaria agraria and specially the fraction which contain Protopine showed potent antiproliferative activity against breast cancer cells.     

Role of electrospun fibre diameter and corresponding specific surface area (SSA) on cell attachment

In order to develop scaffolds for tissue regeneration applications, it is important to develop an understanding of the kinetics of cell attachment as a function of scaffold geometry. In the present study, we investigated how the specific surface area of electrospun scaffolds affected cell attachment and spreading. Number of cells attached to the scaffold was measured by the relative intensity of a metabolic dye (MTS) and cell spreading was analysed for individual cells by measuring the area of projected F‐actin cytoskeleton. We varied the fibre diameter to obtain a specific surface area distribution in the range 2.24–18.79 µ m^?1. In addition, we had one case where the scaffolds had beads in them and therefore had non‐uniform fibres. For each of these different geometries, we varied the cell‐seeding density (0.5–1 × 10⁵) and the serum concentration (0–12%) over the first 8 h in an electrospun polycaprolactone NIH 3T3 fibroblast system. Cells on beaded scaffolds showed the lowest attachment and almost no F‐actin spreading in all experiments indicating uniform fibre diameter is essential for electrospun scaffolds. For the uniform fibre scaffolds, cell attachment was a function of scaffold specific surface area (SSA) (18.79–2.24 µ m^?1) and followed two distinct trends: when scaffold SSA was < 7.13 µ m^?1, cell adhesion rate remained largely unchanged; however, for SSA > 7.13 µ m^?1 there was a significant increase in cellular attachment rate with increasing SSA. This indicated that nanofibrous scaffolds increased cellular adhesion compared to microfibrous scaffolds. This phenomenon is true for serum concentrations of 7.5% and higher. For 5% and lower serum concentration, cell attachment is low and higher SSA fails to make a significant improvement in cell attachment. When cell attachment was investigated at a single‐cell level by measuring the projected actin area, a similar trend was noted where the effect of higher SSA led to higher projected area for cells at 8 h. These results indicate that uniform electrospun scaffolds with SSA provide a faster cell attachment compared to lower SSA and beaded scaffolds. These results indicate that continuous electrospun nanofibrous scaffolds may be a good substrate for rapid tissue regeneration. Copyright © 2009 John Wiley & Sons, Ltd.     

Fibrous biodegradable l‐alanine‐based scaffolds for vascular tissue engineering

In vascular tissue engineering, three‐dimensional (3D) biodegradable scaffolds play an important role in guiding seeded cells to produce matrix components by providing both mechanical and biological cues. The objective of this work was to fabricate fibrous biodegradable scaffolds from novel poly(ester amide)s (PEAs) derived from l ‐alanine by electrospinning, and to study the degradation profiles and its suitability for vascular tissue‐engineering applications. In view of this, l ‐alanine‐derived PEAs (dissolved in chloroform) were electrospun together with 18–30% w/w polycaprolactone (PCL) to improve spinnability. A minimum of 18% was required to effectively electrospin the solution while the upper value was set in order to limit the influence of PCL on the electrospun PEA fibres. Electrospun fibre mats with average fibre diameters of ~0.4 µm were obtained. Both fibre diameter and porosity increased with increasing PEA content and solution concentration. The degradation of a PEA fibre mat over a period of 28 days indicated that mass loss kinetics was linear, and no change in molecular weight was found, suggesting a surface erosion mechanism. Human coronary artery smooth muscle cells (HCASMCs) cultured for 7 days on the fibre mats showed significantly higher viability (p < 0.0001), suggesting that PEA scaffolds provided a better microenvironment for seeded cells compared with control PCL fibre mats of similar fibre diameter and porosity. Furthermore, elastin expression on the PEA fibre mats was significantly higher than the pure PEA discs and pure PCL fibre mat controls (p < 0.0001). These novel biodegradable PEA fibrous scaffolds could be strong candidates for vascular tissue‐engineering applications. Copyright © 2012 John Wiley & Sons, Ltd.     

Annulus fibrosus tissue engineering using lamellar silk scaffolds

Degeneration of the intervertebral disc (IVD) represents a significant muscular skeletal disease. Recently, scaffolds composed of synthetic, natural and hybrid biomaterials have been investigated as options to restore the IVD; however, they lack the hallmark lamellar morphological features of annulus fibrosus (AF) tissue. The goal of regenerating the disc is to achieve anatomical morphology as well as restoration of mechanical and biological function. In this study, two types of scaffold morphology formed from silk fibroin were investigated towards the goal of AF tissue restoration. The first design mimics the lamellar features of the IVD that are associated with the AF region. The second is a porous spongy scaffold that serves as a control. Toroidal scaffolds were formed from the lamellar and porous silk material systems to generate structures with an outer diameter of 8 mm, inner diameter of 3.5 mm and a height of 3 mm. The inter‐lamellar spacing in the lamellar scaffold was 150–250 µm and the average pore sizes in the porous scaffolds were 100–250 µm. The scaffolds were seeded with porcine AF cells and, after growth over defined time frames in vitro, histology, biochemical assays, mechanical testing and gene expression indicated that the lamellar scaffold generated results that were more favourable in terms of ECM expression and tissue function than the porous scaffold for AF tissue. Further, the seeded porcine AF cells supported the native shape of AF tissue in the lamellar silk scaffolds. The lamellar silk scaffolds were effective in the formation of AF‐like tissue in vitro. Copyright © 2012 John Wiley & Sons, Ltd.     

Differentiation of human endometrial stem cells into urothelial cells on a three‐dimensional nanofibrous silk–collagen scaffold: an autologous cell resource for reconstruction of the urinary bladder wall

Reconstruction of the bladder wall via in vitro differentiated stem cells on an appropriate scaffold could be used in such conditions as cancer and neurogenic urinary bladder. This study aimed to examine the potential of human endometrial stem cells (EnSCs) to form urinary bladder epithelial cells (urothelium) on nanofibrous silk–collagen scaffolds, for construction of the urinary bladder wall. After passage 4, EnSCs were induced by keratinocyte growth factor (KGF) and epidermal growth factor (EGF) and seeded on electrospun collagen‐V, silk and silk–collagen nanofibres. Later we tested urothelium‐specific genes and proteins (uroplakin‐Ia, uroplakin‐Ib, uroplakin‐II, uroplakin‐III and cytokeratin 20) by immunocytochemistry, RT–PCR and western blot analyses. Scanning electron microscopy (SEM) and histology were used to detect cell–matrix interactions. DMEM/F12 supplemented by KGF and EGF induced EnSCs to express urothelial cell‐specific genes and proteins. Either collagen, silk or silk–collagen scaffolds promoted cell proliferation. The nanofibrous silk–collagen scaffolds provided a three‐dimensional (3D) structure to maximize cell‐matrix penetration and increase differentiation of the EnSCs. Human EnSCs seeded on 3D nanofibrous silk–collagen scaffolds and differentiated to urothelial cells provide a suitable source for potential use in bladder wall reconstruction in women. Copyright © 2013 John Wiley & Sons, Ltd.     

Automated washing of human progenitor cells: evaluation of apoptosis and cell necrosis

Background: High‐dose chemotherapy followed by reinfusion of autologous stem cells harvested from peripheral blood has been increasingly applied for a variety of disorders. The critical importance of cell dose in the clinical outcome, after transplant, has motivated the need to develop techniques aimed at reducing cell losses and increasing reproducibility. Objectives: The aim of this study is to evaluate the efficacy of the Sepax S‐100 device to process thawed HPC‐A products in comparison with manual procedure. Methods/Materials: We have analysed viability, total nucleated cells (TNC), haematopoietic progenitors and CD34+ cells recovery. Results: The TNC and CD34+ cells recovery in the automatic procedure was of 91·9% (73–100; SD ± 12·60) and 86·7% (69–100; SD ± 10·21), respectively. Instead the recovery of TNC and CD34+ cells using the manual method was of 84·7% (47–100; SD ± 22·9) and 80·29% (23–100; SD ± 25·96). The results, obtained from the assessment of viability of CD34+ both 7‐AAD)+ and AnnV+ showed a high percentage of necrosis and apoptosis in this cell subset by using the manual procedure in respect to the Sepax automated system. Conclusion: Overall, our data suggest that the automated washing procedure is safe and suitable for processing of thawed HPC‐A products and can be daily used in clinical routine.     

Three‐dimensional tissue culture model of human breast cancer for the evaluation of multidrug resistance

Multidrug resistance (MDR) is one of the major obstacles to improving outcomes of chemotherapy in tumour patients. However, progress has been slow to overcome this phenomenon due to the limitations of current cell/tissue models in recapitulating MDR behaviour of tumour cells in vitro. To address this issue, a more pathologically relevant, three‐dimensional (3D) culture of human breast cancer cells was developed by seeding the adriamycin‐resistant cells MCF‐7R in silk‐collagen scaffolds. The cultures of the parental cell line MCF‐7 served as controls. Distinct growth profiles of MCF‐7R and MCF‐7 cells were observed when they were cultured in the scaffolds in comparison with those in the monolayer culture, including cell proliferation, cellular aggregate formation, and expression of drug resistance‐related genes/proteins. Moreover, the 3D cultures of these cell lines especially the cultures of MCF‐7R exhibited a significantly enhanced drug resistance evidenced by their increased IC₅₀ values to the anticancer drugs and improved drug efflux capability. An altered cell cycle distribution and improved percentage of breast cancer stem cell (BCSC)‐like cells was also found in the present study. This might play an important role in promoting the drug‐resistance production in those 3D cultures. Thus, we established improved 3D cultures of MDR human breast cancer. It would provide a robust tissue model for use to evaluate the efficacy of anticancer drugs, explore mechanisms of MDR, and enrich BCSCs in vitro.     

Poly(amino acid)‐based fibrous scaffolds modified with surface‐pendant peptides for cartilage tissue engineering

In this study, fibrous scaffolds based on poly(γ‐benzyl‐l ‐glutamate) (PBLG) were investigated in terms of the chondrogenic differentiation potential of human tooth germ stem cells (HTGSCs). Through the solution‐assisted bonding of the fibres, fully connected scaffolds with pore sizes in the range 20–400 µm were prepared. Biomimetic modification of the PBLG scaffolds was achieved by a two‐step reaction procedure: first, aminolysis of the PBLG fibres’ surface layers was performed, which resulted in an increase in the hydrophilicity of the fibrous scaffolds after the introduction of N⁵‐hydroxyethyl‐l ‐glutamine units; and second, modification with the short peptide sequence azidopentanoyl–GGGRGDSGGGY–NH₂, using the 'click' reaction on the previously modified scaffold with 2‐propynyl side‐chains, was performed. Radio‐assay of the ¹²⁵I‐labelled peptide was used to evaluate the RGD density in the fibrous scaffolds (which varied in the range 10^–3–10 pm /cm²). All the PBLG scaffolds, especially with density 90 ± 20 fm /cm² and 200 ± 100 fm /cm² RGD, were found to be potentially suitable for growth and chondrogenic differentiation of HTGSCs. Copyright © 2015 John Wiley & Sons, Ltd.     

Development of endothelium‐denuded human umbilical veins as living scaffolds for tissue‐engineered small‐calibre vascular grafts

Tissue‐engineered small‐calibre vessel grafts may help to alleviate the lack of graft material for coronary and peripheral bypass grafting in an increasing number of patients. This study explored the use of endothelium‐denuded human umbilical veins (HUVs) as scaffolds for vascular tissue engineering in a perfusion bioreactor. Vessel diameter (1.2 ± 0.4 mm), wall thickness (0.38 ± 0.09 mm), uniaxial ultimate failure stress (8029 ± 1714 kPa) and burst pressure (48.4 ± 20.2 kPa, range 28.4–83.9 kPa) were determined in native samples. The effects of endothelium removal from HUVs by enzymatic digestion, hypotonic lysis and dehydration were assessed. Dehydration did not significantly affect contractile function, tetrazolium dye reduction, mechanical strength and vessel structure, whereas the other methods failed in at least one of these parameters. Denudation by dehydration retained laminin, fibronectin, collagen and elastic fibres. Denuded HUVs were seeded in a perfusion bioreactor with either allogeneic HUVs endothelial cells or with saphenous vein endothelial cells harvested from patients with coronary artery disease. Seeding in a perfusion bioreactor resulted in a confluent monolayer of endothelial cells from both sources, as judged by histology and scanning electron microscopy. Seeded cells contained von Willebrand factor and CD31. In conclusion, denuded HUVs should be considered an alternative to decellularized blood vessels, as the process keeps the smooth muscle layer intact and functional, retains proteins relevant for biomechanic properties and for cell attachment and provides a suitable scaffold for seeding an autologous and flow‐resistant endothelium. Copyright © 2012 John Wiley & Sons, Ltd.     

Cerebral haemodynamics and carbon dioxide reactivity during sepsis syndrome

Background

Most patients with sepsis develop potentially irreversible cerebral dysfunctions. It is yet not clear whether cerebral haemodynamics are altered in these sepsis patients at all, and to what extent. We hypothesized that cerebral haemodynamics and carbon dioxide reactivity would be impaired in patients with sepsis syndrome and pathological electroencephalogram patterns.

Methods

After approval of the institutional ethics committee, 10 mechanically ventilated patients with sepsis syndrome and pathological electroencephalogram patterns underwent measurements of cerebral blood flow and jugular venous oxygen saturation before and after reduction of the arterial carbon dioxide partial pressure by 0.93 ± 0.7 kPa iu by ypervent ilation. The cerebral capillary closing pressure was determined from transcranial Doppler measurements of the arterial blood flow of the middle cerebral artery and the arterial pressure curve. A t test for matched pairs was used for statistical analysis (P < 0.05).

Results

During stable mean arterial pressure and cardiac index, reduction of the arterial carbon dioxide partial pressure led to a significant increase of the capillary closing pressure from 25 ± 11 mmHg to 39 ± 15 mmHg (P < 0.001), with a consecutive decrease of blood flow velocity in the middle cerebral artery of 21.8 ± 4.8%/kPa (P < 0.001), of cerebral blood flow from 64 ± 29 ml/100 g/min to 39 ± 15 ml/100 g/min (P < 0.001) and of jugular venous oxygen saturation from 75 ± 8% to 67 ± 14% (P < 0.01).

Conclusion

In contrast to other experimental and clinical data, we observed no pathological findings in the investigated parameters of cerebral perfusion and oxygenation.     

Viability and neuronal differentiation of neural stem cells encapsulated in silk fibroin hydrogel functionalized with an IKVAV peptide

Three‐dimensional (3D) porous scaffolds combined with therapeutic stem cells play vital roles in tissue engineering. The adult brain has very limited regeneration ability after injuries such as trauma and stroke. In this study, injectable 3D silk fibroin‐based hydrogel scaffolds with encapsulated neural stem cells were developed, aiming at supporting brain regeneration. To improve the function of the hydrogel towards neural stem cells, silk fibroin was modified by an IKVAV peptide through covalent binding. Both unmodified and modified silk fibroin hydrogels were obtained, through sonication, with mechanical stiffness comparable to that of brain tissue. Human neural stem cells were encapsulated in both hydrogels and the effects of IKVAV peptide conjugation on cell viability and neural differentiation were assessed. The silk fibroin hydrogel modified by IKVAV peptide showed increased cell viability and an enhanced neuronal differentiation capability, which contributed to understanding the effects of IKVAV peptide on the behaviour of neural stem cells. For these reasons, IKVAV‐modified silk fibroin is a promising material for brain tissue engineering. Copyright © 2015 John Wiley & Sons, Ltd.     

Evaluation of decellularized tilapia skin as a tissue engineering scaffold

Decellularized bovine and porcine tissues have been used as scaffolds to support tissue regeneration but inherit religious restrictions and risks of disease transmission to humans. Decellularized marine tissues are seen as attractive alternatives due to their similarity to mammalian tissues, reduced biological risks, and less religious restrictions. The aim of this study was to derive an acellular scaffold from the skin of tilapia and evaluate its suitability as a tissue engineering scaffold. Tilapia skin was treated with a series of chemical and enzymatic treatments to remove cellular materials. The decellularized tilapia skin (DTS) was then characterized and evaluated in vitro and in vivo to assess its biological compatibility. The results indicated that the decellularization process removed 99.6% of the DNA content from tilapia skin. The resultant DTS was shown to possess a high denaturation temperature of 68.1 ± 1.0°C and a high Young's modulus of 56.2 ± 14.4 MPa. The properties of DTS were also compared against those of crosslinked electrospun tilapia collagen membrane, another form of tilapia‐derived collagen scaffold. In vitro studies revealed that both DTS and crosslinked electrospun tilapia collagen promoted cellular metabolic activity, differentiation, and mineralization of murine preosteogenic MC3T3‐E1 cells. The rat calvarial defect model was used to evaluate the in vivo performance of the scaffolds, and both scaffolds did not induce hyperacute rejections. Furthermore, they enhanced bone regeneration in the critical defect compared with the sham control. This study suggests that tilapia‐derived scaffolds have great potential in tissue engineering applications.     

Collagen scaffolds with in situ‐grown calcium phosphate for osteogenic differentiation of Wharton's jelly and menstrual blood stem cells

The aim of this research was to investigate the osteogenic differentiation potential of non‐invasively obtained human stem cells on collagen nanocomposite scaffolds with in situ‐grown calcium phosphate crystals. The foams had 70% porosity and pore sizes varying in the range 50–200 µm. The elastic modulus and compressive strength of the calcium phosphate containing collagen scaffolds were determined to be 234.5 kPa and 127.1 kPa, respectively, prior to in vitro studies. Mesenchymal stem cells (MSCs) obtained from Wharton's jelly and menstrual blood were seeded on the collagen scaffolds and proliferation and osteogenic differentiation capacities of these cells from two different sources were compared. The cells on the composite scaffold showed the highest alkaline phosphatase activity compared to the controls, cells on tissue culture polystyrene and cells on collagen scaffolds without in situ‐formed calcium phosphate. MSCs isolated from both Wharton's jelly and menstrual blood showed a significant level of osteogenic activity, but those from Wharton's jelly performed better. In this study it was shown that collagen nanocomposite scaffolds seeded with cells obtained non‐invasively from human tissues could represent a potential construct to be used in bone tissue engineering. Copyright © 2012 John Wiley & Sons, Ltd.     

Cytomodulin‐functionalized porous PLGA particulate scaffolds respond better to cell migration,actin production and wound healing in rodent model

In the present study, porous PLGA microparticulate scaffolds (PMS_P), surface‐hydrolysed scaffolds (PMS_Hyd) and cytomodulin‐coupled scaffolds (PMS_CM) were prepared and characterized. After coupling the particles with cytomodulin, the size was reduced from 334 µm (span 0.53) to 278 µm due to hydrolysis, and contact angle also decreased from 70.87 ± 8.56 to 31.43 ± 7.43, indicating an increase in hydrophilicity. Surface roughness and pore density increased, along with an increase in surface area from 9.59 ± 0.36 to 16.82 ± 0.064 m²/g after attaching the biomolecule CM onto the PLGA particles. In vitro cell culture experiments on human dermal fibroblasts (HDFs) were performed for 21 days, in which MTT assay indicated two‐fold higher cell proliferation on PMS_Hyd than on PMS_CM; however, cell distribution, cell spreading and actin production were significantly higher on PMS_CM than on other scaffolds. Migration of cells from PMS_CM to a 2D plate was gradual but the migrated cells attained early confluence, indicating the preservation of normal cellular functions. In a full‐thickness wound mouse model, PMS_CM exhibited 80% wound closure within 2 weeks. Further, at the end of week 3, the inflammatory cell count in the PMS_CM group was reduced to one‐third of the control group, while in PMS_P and PMS_Hyd the extent of inflammation was much higher and more severe. In the case of PMS_CM, abundant fibroblast proliferation, early formation of the scar tissue, eschar formation and inward movement of the wound margins (a zipper‐like movement) towards the deeper layers of the skin suggested advanced wound healing. Cytomodulin‐coupled scaffolds ensured better cell spreading and migration and thus enabled rapid wound healing (see Supporting information, Figure S1). Copyright © 2012 John Wiley & Sons, Ltd.     

Effect of propofol and thiopentone on regional blood flow in brain and peripheral tissues during normoxia and hypoxia in the dog

Summary. The effect of propofol and thiopentone on cerebral (CBF), myocardial (MBF), muscular, and arterial hepatic blood flow was assessed with radiolabeled microspheres in 12 chronically instrumented dogs, six given propofol and six thiopentone. Tissue blood flows were measured in the awake animal, after 30 min of normoxic anaesthesia (room air), and after 30 min of hypoxic anaesthesia using a mixture of 10% O₂ and 3% CO₂ in nitrogen. The decrease in CBF from awake to normoxic anaesthesia was similar with propofol and thiopentone (propofol: 77 ±8 to 38±3 ml min^-1 100 g^-1, P<0.01; thiopentone: 66±3 to 33±2 ml min^-1 100 g^-1, P<0.01). During hypoxia, CBF rose moderately in the two groups (respectively + 19% and +28%, P<0.05). The MBF increased in propofol and thiopentone groups after 30 min of anaesthesia with air (propofol: 97 ±23 to 137 ± 15 ml min^-1 100 g^-1; thiopentone: 82 ±7 to 141 ± 10 ml min^-1 100 g^-1) and increased still more during hypoxia. The increase in MBF was related to an increase in heart rate and blood pressure. The quadriceps blood flow decreased during anaesthesia in normoxia and in hypoxia. The diaphragmatic blood flow increased with thiopentone under hypoxia. The hepatic arterial blood flow was unchanged. It is concluded that the effects of propofol on regional blood flows are very similar to those of thiopentone.     

Improving pore interconnectivity in polymeric scaffolds for tissue engineering

A new scaffold fabrication technique aiming to enhance pore interconnectivity for tissue engineering has been developed. Medical grade poly(lactic acid) was utilized to generate scaffolds by a solvent‐evaporating/particulate‐leaching technique, using a new dual‐porogen system. Water‐soluble sodium chloride particles were used to control macro‐pore size in the range 106–255 µm, while organic naphthalene was utilized as a porogen to increase pore interconnections. The three‐dimensional (3D) morphology of the scaffolds manufactured with and without naphthalene was examined by optical coherence tomography and scanning electron microscopy. The mechanical properties of the scaffolds were characterized by compression tests. MG63 osteoblast cells were seeded in the scaffolds to study the cell attachment and viability evaluated by confocal microscopy. It was revealed that introducing naphthalene as the second porogen in the solvent‐evaporating/particulate‐leaching process resulted in improvement of the pore interconnectivity. Cells grew in both scaffolds fabricated with and without naphthalene. They exhibited strong green fluorescence when using a live/dead fluorescent dye kit, indicating that the naphthalene in the scaffold process did not affect cell viability. Copyright © 2009 John Wiley & Sons, Ltd.     

Effect of SanOrg123781A, a synthetic hexadecasaccharide, on clot-bound thrombin and factor Xa in vitro and in vivo

Summary. Factor (F)Xa and thrombin bound to the clot during its formation contribute to the propensity of thrombi to activate the coagulation system. The aim of this work was to study the inhibition of clot‐bound FXa and clot‐bound thrombin by SanOrg123781A, a synthetic hexadecasaccharide that enhances the inhibition of thrombin and FXa by antithrombin (AT). SanOrg123781A, designed to exhibit low non‐specific binding to proteins other than AT, was compared with heparin. In buffer, heparin and SanOrg123781A inhibited FXa and thrombin at similar concentrations [concentration inhibiting 50% (IC₅₀) of Xa and IIa activity were, respectively: heparin 120 ± 7 and 3 ± 1 ng mL^?1; SanOrg123781A 77 ± 5 and 4 ± 1 ng mL^?1]. In human plasma, the activity of both compounds was reduced, although the activity of heparin was much more affected than that of SanOrg123781A (IC₅₀ values for inhibition of FXa and FIIa activity were, respectively: heparin 100 ± 5 and 800 ± 40 ng mL^?1; SanOrg123781A 10 ± 5 and 30 ± 3 ng mL^?1). We demonstrated, in agreement with our previous results, that the procoagulant activity of the clot is essentially due to clot‐bound FXa and to some extent to clot‐bound thrombin. We showed that heparin and SanOrg123781A were able to inhibit fragment F1+2 generation induced by clot‐bound FXa with IC₅₀ values of 2 ± 0.5 µg mL^?1 and 0.6 ± 0.2 µg mL^?1, respectively. Both compounds also inhibited clot‐bound thrombin activity, the IC₅₀ values of heparin and SanOrg123781A being 1 ± 0.01 µg mL^?1 and 0.1 ± 0.1 µg mL^?1, respectively. Moreover, both heparin and SanOrg123781A significantly inhibited fibrinopeptide A generated by the action of clot‐bound thrombin on fibrinogen but also by free thrombin generated from prothrombin by clot‐bound FXa with IC₅₀ values of 4 ± 0.6 and 1 ± 0.1 µg mL^?1, respectively. As with clot‐bound enzymatic activities, SanOrg123781A was three times more active than heparin in vivo on fibrinogen accretion onto a pre‐existing thrombus and as activators of recombinant tissue‐type plasminogen activator‐induced thrombolysis. In conclusion, due to the specific activities of SanOrg123781A, this compound is much more active than heparin in the presence of plasma proteins, on clot‐bound enzymes and in in vivo models of thrombosis/thrombolysis.     

In vivo biocompatibility assessment of poly (ether imide) electrospun scaffolds

Poly(ether imide) (PEI), which can be chemically functionalized with biologically active ligands, has emerged as a potential biomaterial for medical implants. Electrospun PEI scaffolds have shown advantageous properties, such as enhanced endothelial cell adherence, proliferation and low platelet adhesion in in vitro experiments. In this study, the in vivo behaviour of electrospun PEI scaffolds and PEI films was examined in a murine subcutaneous implantation model. Electrospun PEI scaffolds and films were surgically implanted subcutaneously in the dorsae of mice. The surrounding subcutaneous tissue response was examined via histopathological examination at 7 and 28 days after implantation. No serious adverse events were observed for both types of PEI implants. The presence of macrophages or foreign body giant cells in the vicinity of the implants and the formation of a fibrous capsule indicated a normal foreign body reaction towards PEI films and scaffolds. Capsule thickness and inflammatory infiltration cells significantly decreased for PEI scaffolds during days 7–28 while remaining unchanged for PEI films. The infiltration of cells into the implant was observed for PEI scaffolds 7 days after implantation and remained stable until 28 days of implantation. Additionally some, but not all, PEI scaffold implants induced the formation of functional blood vessels in the vicinity of the implants. Conclusively, this study demonstrates the in vivo biocompatibility of PEI implants, with favourable properties of electrospun PEI scaffolds regarding tissue integration and wound healing. Copyright © 2015 John Wiley & Sons, Ltd.     

Ultrasound‐guided plasma rich in growth factors injections and scaffolds hasten motor nerve functional recovery in an ovine model of nerve crush injury

In the present study we evaluated the motor recovery process of peripheral nerve injury (PNI), based on electrophysiological and histomorphometric criteria, after treatment with plasma rich in growth factors (PRGF) injections and scaffolds in an ovine model. Three groups of sheep underwent a nerve crush lesion: the first group (n = 3) was left to recover spontaneously (SR); the second group was administered saline injections (SI; n = 5) and a third group (n = 6) received PRGF injections and scaffolds immediately after the crush injury. At post‐intervention week 8, 70% of sheep in the PRGF group were CMAP‐positive, with no electrophysiological response in the rest of the groups. Histomorphometric analysis 12 weeks after the surgical intervention revealed that the average axonal density of the SR (1184 ± 864 axons/µm²) and SI (3109 ± 2450 axons/µm²) groups was significantly inferior to the control (8427 ± 2433 axons/µm²) and also inferior to the PRGF group (5276 ± 4148 axons/µm²), showing no significant differences between the control and PRGF groups. The axonal size of the SR and SI groups was significantly smaller compared with the control group (18 ± 4 µm²), whereas the axonal size of the PRGF group (6 ± 5 µm²) did not show statistical differences from the control. Morphometry of the target muscles indicated that the PRGF group had the lowest percentage volume reduction 12 weeks after the crush injury. The PRGF group had larger muscle fibre areas than the SI and SR groups, although the differences did not reach statistical significance. Overall, these data suggest that the PRGF injections and scaffolds hastened functional axon recovery and dampened atrophy of the target muscles in an ovine model. Copyright © 2015 John Wiley & Sons, Ltd.