Due to its biological properties, human amniotic membrane (hAM) is widely studied in the field of tissue engineering and regenerative medicine. hAM is already very attractive for wound healing and it may be helpful as a support for bone regeneration. However, few studies assessed its potential for guided bone regeneration (GBR). The purpose of the present study was to assess the potential of the hAM as a membrane for GBR. In vitro, cell viability in fresh and cryopreserved hAM was assessed. In vivo, we evaluated the impact of fresh versus cryopreserved hAM, using both the epithelial or the mesenchymal layer facing the defect, on bone regeneration in a critical calvarial bone defect in mice. Then, the efficacy of cryopreserved hAM associated with a bone substitute was compared to a collagen membrane currently used for GBR. In vitro, no statistical difference was observed between the conditions concerning cell viability. Without graft material, cryopreserved hAM induced more bone formation when the mesenchymal layer covered the defect compared to the defect left empty. When associated with a bone substitute, such improved bone repair was not observed. These preliminary results suggest that cryopreserved hAM has a limited potential for GBR.
Strontium (Sr) has shown effectiveness for stimulating bone remodeling. Nevertheless, the exact therapeutic values are not established yet. Authors hypothesized that local application of Sr-enriched ceramics would enhance bone remodeling in constant osteoporosis of rabbits’ femoral neck bone. Seven different bone conditions were analyzed: ten healthy rabbits composed a control group, while other twenty underwent ovariectomy and were divided into three groups. Bone defect was filled with hydroxyapatite 30% (HAP) and tricalcium phosphate 70% (TCP) granules in 7 rabbits, 5% of Sr-enriched HAP/TCP granules in 7, but sham defect was left unfilled in 6 rabbits. Bone samples were obtained from operated and non-operated legs 12 weeks after surgery and analyzed by histomorphometry and immunohistochemistry (IMH). Mean trabecular bone area in control group was 0.393 mm2, in HAP/TCP – 0.226 mm2, in HAP/TCP/Sr – 0.234 mm2 and after sham surgery – 0.242 mm2. IMH revealed that HAP/TCP/Sr induced most noticeable increase of nuclear factor kappa beta 105 (NFkB 105), osteoprotegerin (OPG), osteocalcin (OC), bone morphogenetic protein 2/4 (BMP 2/4), collagen type 1α (COL-1α), interleukin 1 (IL-1) with comparison to intact leg; NFkB 105 and OPG rather than pure HAP/TCP or sham bone. We concluded that Sr-enriched biomaterials induce higher potential to improve bone regeneration than pure bioceramics in constant osteoporosis of femoral neck bone. Further studies on bigger osteoporotic animals using Sr-substituted orthopedic implants for femoral neck fixation should be performed to confirm valuable role in local treatment of osteoporotic femoral neck fractures in humans.
Osteoinductive capacity of demineralized bone matrix (DBM) is sometimes insufficient or shows high variability between different batches of DBM. Here, we tried to improve its osteoinductive activity by alkali-urea or trypsin treatment but this strategy was unsuccessful. Then, we tested the enrichment of DBM with a bone protein extract (BPE) containing osteogenic growth factors comparing two sources: cortical bone powder and DBM. The osteoinductive capacity (alkaline phosphatase activity) of the obtained BPEs was evaluated in vitro in C2C12 cells. Specific protein levels present in the different BPE was determined by enzyme-linked immunosorbent assay or by a multiplex assay. BPE from cortical bone powder showed a lack of osteoinductive effect, in agreement with the low content on osteoinductive factors. In contrast, BPE from DBM showed osteoinductive activity but also high variability among donors. Thus, we decided to enrich DBM with BPE obtained from a pool of DBM from different donors. Following this strategy, we achieved increased osteoinductive activity and lower variability among donors. In conclusion, the use of a BPE obtained from a pool of demineralized bone to enrich DBM could be used to increase its osteoinductive effect and normalize the differences between donors.
The development of in-vitro techniques to characterise the behaviour of cells in biomedical scaffolds is a rapidly developing field. However, until now it has not been possible to visualise, directly in 3D, the extent of cell migration using a desktop X-ray microCT. This paper describes a new technique based on cell labelling with a radio opacifier (barium sulphate), which permits cell tracking without the need for destructive sample preparation. The ability to track cells is highlighted via a comparison of cell migration through demonstrator lyophilised collagen scaffolds with contrasting pore size and interconnectivity. The results demonstrate the ease with which the technique can be used to characterise the effects of scaffold architecture on cell infiltration.
While antibiotic-eluting polymethylmethacrylate space maintainers have shown efficacy in the treatment of bacterial periprosthetic joint infection and osteomyelitis, antifungal-eluting space maintainers are associated with greater limitations for treatment of fungal musculoskeletal infections including limited elution concentration and duration. In this study, we have designed a porous econazole-eluting space maintainer capable of greater inhibition of fungal growth than traditional solid space maintainers. The eluted econazole demonstrated bioactivity in a concentration-dependent manner against the most common species responsible for fungal periprosthetic joint infection as well as staphylococci. Lastly, these porous space maintainers retain compressive mechanical properties appropriate to maintain space before definitive repair of the joint or bony defect.
The target of the recent study is to achieve a significant inexpensive and eco-friendly way for getting ZTA/HA composites, based on the nano-HA derived from the eggshell biogenic source. Combining simultaneously the porous structure; which is considered as a bone formation key, with developed mechanical properties and adequate biocompatibility, is another purpose of this study. Furthermore, the impact of ZTA addition from 10–30 mass-%, fabricated by uniaxial pressing and sintering at 1200–1300?°C for 2?h, on the physical and mechanical properties, microstructure and phase composition of ZTA/HA composite bodies was investigated. The results demonstrated that the increasing of ZTA content increases the bodies’ apparent porosity and decreases the bulk density due to the decomposition of HA into β-TCP. Where the formation of β-TCP possessed the predominant impact on the mechanical properties of the sintered ZTA/HA composites. ICP, SEM, EDX and thin film XRD results of composites containing 20 mass-% ZTA affirmed the excellent bioactivity of the bodies.
Phosphate-based glasses have been attracting attention due to their possible medical applications arising from unique dissolution characteristics in the human body leading to the possibility of new tissue regeneration. In this study, the leaching kinetics of a series of melt-quenched Sr-doped phosphate glasses are presented. Regardless of the presence of Sr, all the glasses have an initial linear and sustained release of the ions followed by a plateau. To guarantee proper nutritional support to the growing tissue during regeneration and to mimic the 3-dimensional architecture of tissues, organic scaffold systems have been developed. However, their poor mechanical strength has limited their application. To overcome this problem, cross-linkers can be used although this then limits the solubility of the materials. To succeed in dealing with such a limitation, in this paper, by freeze-drying, the aforementioned soluble melt-quenched phosphate glasses were combined as powders with collagen fibres from bovine achilles tendon to make degradable scaffolds. The scaffolds were characterized by SEM, EDX and BET. Changes to the dissolution behaviour of the glasses arising from the presence of collagen interacting with the ions leached were reported. Furthermore, the ability of the scaffolds to induce hydroxyapatite (HA) formation was evaluated: one the elaborated scaffold could grow an HA-like layer after a week in SBF. Based on the results obtained, a possible application in restorative dentistry is proposed for one or more materials.
Anticoagulation therapy plays a vital role in the prevention of blood clot formation during hemodialysis and hemofiltration, especially for critical care patients. Here, we synthesized a novel argatroban (Arg)-modified polysulfone (PSf) membrane for anticoagulation. Arg was grafted onto the PSF membrane via chemical modification to increase membrane hydrophilicity. Protein adsorption, coagulation, as well as activation of platelets and complement systems were greatly reduced on the Arg-modified PSf membrane. Thus, the recalcification time and the activated partial thrombin time (APTT) were increased after the modification. In comparison with the pristine PSf membrane, the Arg-modified PSf membrane showed better hemocompatibility and anticoagulation properties, indicating its potential for applications in hemodialysis and hemofiltration.
Modification of the PSf membrane has been investigated in attempts to further enhance the anticoagulation properties of the hemodialysis membranes, including a heparin-modified PSf membrane. However, heparin can inhibit plasma-free thrombin, and cause the occurrence of heparin-induced thrombocytopenia (HIT), which increases the risk of bleeding during dialysis in critical care patients. To address this problem, we modified PSf membrane with as a novel direct thrombin inhibitors, argatroban (Arg). It can reversibly bind to thrombin, inhibiting not only the plasma-free thrombin in the blood, but also clot-bound thrombin.
Ti-based implants sometimes fail to integrate with surrounding bone tissue due to insufficiency of new bone formation and surface bonding. To overcome this problem, this research focused on establishing a sustained bone growth factor delivery system by applying anodized TiO2 nanotube arrays and PLGA film on the titanium implant surface. TiO2 nanotube arrays were made by anodic oxidation method, and were then filled with rhBMP2 by vacuum freeze-drying. Next, PLGA was deposition on the surface of this material. The designed system was characterized, pharmacokinetic release rate of rhBMP2 was determined. Adhesion, proliferation, and differentiation activity of osteoblasts cultured on the new surfaces and traditional titanium surfaced were compared. SEM showed that a surface of TiO2 nanotube arrays were successfully generated. PLGA membranes of 50?nm, 250?nm, 800?nm thickness were successfully deposited on the surfaces of TiO2 nanotube layers by using 1%, 3%, 10% PLGA solutions. PLGA film of 250?nm thickness showed ideally controlled release of rhBMP2, lasting for 4 weeks. Furthermore, 250?nm thickness PLGA film improved osteoblast adhesion, proliferation, and levels of alkaline phosphatase. In conclusion, the PLGA film / TiO2 nanotube growth factor delivery system can effectively sustain the release of rhBMP-2, and promote proliferation and differentiation of MC3T3-E1 osteoblasts.
Electrospun membranes have the potential to act as an effective barrier for wounds from the external environment to prevent pathogens. In addition, materials with good antibacterial properties can effectively fight off the invading pathogens. In this paper, we report the development of a novel electrospun polyvinyl alcohol (PVA) membrane containing biosynthesized silver nanoparticle (bAg) for wound dressing applications. Plant extract from a medicinal plant Mimosa pudica was utilized for the synthesis of bAg. Synthesized bAg were characterized by Ultraviolet-Visible (UV) Spectroscopy and Fourier Transform Infrared Spectroscopy (FTIR). The morphology of bAg was obtained from Transmission Electron Microscopy (TEM) and found that they were spherical in morphology with average particle size 7.63?±?1.2?nm. bAg nanoparticles incorporated PVA membranes were characterized using several physicochemical techniques such as Scanning Electron Microscopy (SEM), Energy Dispersive X-Ray Spectroscopy (EDS) and X-Ray Diffraction (XRD) analysis. Experimental results confirmed the successful incorporation of bAg in PVA fibers. PVA nanofiber membranes incorporated with bAg showed good mechanical strength, excellent exudate uptake capacity, antibacterial activity, blood compatibility and cytocompatibility.
The amnion muscle combined graft (AMCG) conduits showed good clinical results in peripheral nerves gap repair. It combines the human amniotic membrane with autologous skeletal muscle fibres. These results seem attributable to the biological characteristics of human amniotic membrane: Pluripotency, anti-inflammatory and low immunogenicity.We here evaluate the final outcome of nerve regeneration morphologically and functionally, across the AMCG compared to nerve autograft. Fourteen Wistar rats were divided into two groups: In Group A, including 6 rats, the left forelimb was treated performing a 1.5?cm length gap on median nerve that was then reconstructed with a reverse autograft. In Group B, including 8 rats, the gap was reconstructed with AMCG. Functional results were evaluated at 30, 60 and 90 days performing grasping tests. Morphological and stereological analyses were performed at T90 using high-resolution light microscopy and design-based stereology. The AMCG conduits revealed nerve fibres regeneration and functional recovery. Functional recovery was observed in both groups with AMCG conduits group showing lower values and a regeneration of median nerves with more myelinated fibres with the same axon size, but thinner myelin than the autograft group. Though the autograft remains the gold standard to restore wide nerve gaps, the AMCG conduit has proved to be effective in enabling nerve regeneration through a critical rat’s nerve gap of 15?mm. These findings empirically support the great clinical results obtained using AMCG conduit to restore traumatic nerve’s gap from 3 to 6?cm of mixed forearm nerves.
Osteoclasts are large multinucleated giant cells that actively resorb bone during the physiological bone turnover (BTO), which is the continuous cycle of bone resorption (by osteoclasts) followed by new bone formation (by osteoblasts). Osteoclasts secrete chemotactic signals to recruit cells for regeneration of vasculature and bone. We hypothesize that a biomaterial that attracts osteoclasts and re-establishes BTO will induce a better healing response than currently used bone graft materials. While the majority of bone regeneration efforts have focused on maximizing bone deposition, the novelty in this approach is the focus on stimulating osteoclastic resorption as the starter for BTO and its concurrent new vascularized bone formation. A biodegradable tyrosine-derived polycarbonate, E1001(1k), was chosen as the polymer base due to its ability to support bone regeneration in vivo. The polymer was functionalized with a RGD peptide or collagen I, or blended with β-tricalcium phosphate. Osteoclast attachment and early stages of active resorption were observed on all substrates. The transparency of E1001(1k) in combination with high resolution confocal imaging enabled visualization of morphological features of osteoclast activation such as the formation of the “actin ring” and the “ruffled border”, which previously required destructive forms of imaging such as transmission electron microscopy. The significance of these results is twofold: (1) E1001(1k) is suitable for osteoclast attachment and supports osteoclast maturation, making it a base polymer that can be further modified to optimize stimulation of BTO and (2) the transparency of this polymer makes it a suitable analytical tool for studying osteoclast behavior.
Electro-conductive nanocomposites have several applications in biomedical field. Development of a biocompatible electro-conductive polymeric materials is therefore of prime importance. In this study, electro-conductive nanofibrous mats of PLGA/CNT were fabricated through different methods including blend electrospinning, simultaneous PLGA electrospinning and CNT electrospraying and ultrasound-induced adsorption of CNTs on the electrospun PLGA nanofibers. The morphology and diameter of fibers were characterized by SEM and TEM, showing the lowest average diameters of 477?±?136?nm for PLGA/MWCNT blend nanocomposites. MWCNT-sprayed PLGA specimens showed significant lower water contact angle (83°), electrical resistance (3.0?×?104?Ω) and higher mechanical properties (UTS: 5.50?±?0.46?MPa) compared to the untreated PLGA scaffolds. Also, results of PC12 cell study demonstrated highest viability percentage on the MWCNT-sprayed PLGA nanofibers. We propose that the conductive nanocomposites have capability to use as tool for the neural regeneration and biosensors.
Osteogenicity and osteointegration of materials is one of the key elements of the success of bone implants. Poly(methyl methacrylate) (PMMA) is the basic compound of bone cement and has been widely investigated for other orthopedic applications, but its poor osteointegration and the subsequent loosening of implant material limits its widespread use as bone implants. Micropillar features on substrate surfaces were recently reported to modulate cell behavior through alteration of cell morphology and promotion of osteogenesis. Utilization of this pillar-decorated topography may be an effective approach to enhance osteogenicity of polymeric surfaces. The aim of this study was to investigate the effect of cell morphology on the micropillar features on attachment, proliferation, and osteogenic activity of human osteoblast-like cells. A series of solvent cast PMMA films decorated with 8?µm high square prism micropillars with pillar width and interpillar distances of 4, 8 and 16?µm were prepared from photolithographic templates, and primary human osteoblast-like cells (hOB) isolated from bone fragments were cultured on them. Micropillars increased cell attachment and early proliferation rate compared to unpatterned surfaces, and triggered distinct morphological changes in cell body and nucleus. Surfaces with pillar dimensions and gap width of 4?µm presented the best osteogenic activity. Expression of osteogenic marker genes was upregulated by micropillars, and cells formed bone nodule-like aggregates rich in bone matrix proteins and calcium phosphate. These results indicated that micropillar features enhance osteogenic activity on PMMA films, possibly by triggering morphological changes that promote the osteogenic phenotype of the cells.
Oral administration of drugs presents important limitations, which are frequently not granted the importance that they really have. For instance, hepatic metabolism means an important drug loss, while some patients have their ability to swell highly compromised (i.e. unconsciousness, cancer…). Sublingual placement of an accurate Pharmaceutical Dosage Form is an attractive alternative. This work explores the use of the β-chitosan membranes, from marine industry residues, composed with marine sediments for dual sublingual drug delivery. As proof of concept, the membranes were loaded with a hydrophilic (gentamicin) and a hydrophobic (dexamethasone) drug. The physico-chemical and morphological characterization indicated the successful incorporated of diatomaceous earth within the chitosan membranes. Drug delivery studies showed the potential of all formulations for the immediate release of hydrophilic drugs, while diatomaceous earth improved the loading and release of the hydrophobic drug. These results highlight the interest of the herein developed membranes for dual drug delivery.
3D fibrous scaffolds have received much recent attention in regenerative medicine. Use of fibrous scaffolds has shown promising results in tissue engineering and wound healing. Here we report the development and properties of a novel fibrous scaffold that is useful for promoting wound healing. A scaffold made of salmon fibrinogen and chitosan is produced by electrospinning, resulting in a biocompatible material mimicking the structure of the native extracellular matrix (ECM) with suitable biochemical and mechanical properties. The scaffold is produced without the need for enzymes, in particular thrombin, but is fully compatible with their addition if needed. Human dermal fibroblasts cultured on this scaffold showed progressive proliferation for 14 days. Split-thickness experimental skin wounds treated and untreated were compared in a 10-day follow-up period. Wound healing was more effective using the fibrinogen-chitosan scaffold than in untreated wounds. This scaffold could be applicable in various medical purposes including surgery, tissue regeneration, burns, traumatic injuries, and 3D cell culture platforms.
The advantages of synthetic bone graft substitutes over autogenous bone grafts include abundant graft volume, lack of complications related to the graft harvesting, and shorter operation and recovery times for the patient. We studied a new synthetic supercritical CO2 –processed porous composite scaffold of β-tricalcium phosphate and poly(L-lactide-co-caprolactone) copolymer as a bone graft substitute in a rabbit calvarial defect. Bilateral 12?mm diameter critical size calvarial defects were successfully created in 18 rabbits. The right defect was filled with a scaffold moistened with bone marrow aspirate, and the other was an empty control. The material was assessed for applicability during surgery. The follow-up times were 4, 12, and 24 weeks. Radiographic and micro-CT studies and histopathological analysis were used to evaluate new bone formation, tissue ingrowth, and biocompatibility. The scaffold was easy to shape and handle during the surgery, and the bone-scaffold contact was tight when visually evaluated after the implantation. The material showed good biocompatibility and its porosity enabled rapid invasion of vasculature and full thickness mesenchymal tissue ingrowth already at four weeks. By 24 weeks, full thickness bone ingrowth within the scaffold and along the dura was generally seen. In contrast, the empty defect had only a thin layer of new bone at 24 weeks. The radiodensity of the material was similar to the density of the intact bone. In conclusion, the new porous scaffold material, composed of microgranular β-TCP bound into the polymer matrix, proved to be a promising osteoconductive bone graft substitute with excellent handling properties.
The effects of the addition of Sr on the microstructures, mechanical properties and bio-corrosion properties of Mg-2Zn alloys were investigated. Examination of the microstructures indicates that Sr addition promoted grain refinement and the formation of secondary phases. The results for the mechanical and corrosion property analyses show that the Mg-2Zn-0.2Sr alloy exhibited the best mechanical properties and bio-corrosion resistance. Notably, fine grain structure can promote strength and ductility, while the excessive secondary phases Mg17Sr2 resulted in the degradation of mechanical performance. Moreover, corrosion resistance could be improved by reducing the impurity concentrations and forming a denser corrosion product layer, while the secondary phases accelerated the corrosion process by forming micro-galvanic couples with the Mg matrix. It is considered that the addition of Sr (0.2%) represents the main contributing factor to these improved properties. The Mg-2Zn-0.2Sr alloy provides excellent strength and corrosion resistance for biomedical applications.
Heart valve diseases remain common in industrialized countries. Bioprosthetic heart valves, introduced as free of anticoagulation therapy alternatives to mechanical substitutes. Still they suffer from long term failure due to calcification. Different treatment methods introduced to inhibit calcification, have so far been limited in success. Glycosaminoglycans (GAGs) possess properties including high negative charge, anticoagulation and anti-inflammatory activity that make them a potential solution for calcification problem. In this study, heparin hydrogel was prepared and characterized both chemically and mechanically. After that, heparin hydrogel embedded bovine pericardial tissues, fixed with glutaraldehyde, were produced and tested for their mechanical behavior and anticalcifcation potential in vitro using the constant composition model. In the calcification experiments, tissues were divided into three groups: a) Controls without treatment, b) Hydrogel treated tissues and c) Tissues with raw heparin dissolved in the calcification solution. The results showed that embedding of tissue with hydrogel had no stiffening effect on its mechanical behavior. Calcification assessment showed a significant efficacy on inhibition of calcium phosphate deposition of hydrogel treated (second group) in comparison to untreated tissues (control, first group). Calcification inhibition potential was very similar in both the second and raw heparin (third group). Histological data confirmed the obtained results, suggesting that heparin treatment is a promising anticalcification agent.
Extrahepatic transplantation of islets of Langerhans could aid in better survival of islets after transplantation. When islets are transfused into the liver 60-70% of them are lost immediately after transplantation. An important factor for a successful extrahepatic transplantation is a well-vascularized tissue surrounding the implant. There are many strategies known for enhancing vessel formation such as adding cells with endothelial potential, the combination with angiogenic factors and / or applying surface topography at the exposed surface of the device. Previously we developed porous, micropatterned membranes which can be applied as a lid for an islet encapsulation device and we showed that the surface topography induces human umbilical vein endothelial cell (HUVEC) alignment and interconnection. This was achieved without the addition of hydrogels, often used in angiogenesis assays. In this work, we went one step further towards clinical implementation of the device by combining this micropatterned lid with Mesenchymal Stem Cells (MSCs) to facilitate prevascularization in vivo. As for HUVECs, the micropatterned membranes induced MSC alignment and organization in vitro, an important contributor to vessel formation, whereas in vivo (subcutaneous rat model) they contributed to improved implant prevascularization. In fact, the combination of MSCs seeded on the micropatterned membrane induced the highest vessel formation score in 80% of the sections.
