Incorporation of a sequential BMP-2/BMP-7 delivery system into chitosan-based scaffolds for bone tissue engineering

The aim of this study was to develop a 3-D construct carrying an inherent sequential growth factor delivery system. Poly(lactic acid-co-glycolic acid) (PLGA) nanocapsules loaded with bone morphogenetic protein BMP-2 and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) nanocapsules loaded with BMP-7 made the early release of BMP-2 and longer term release of BMP-7 possible. 3-D fiber mesh scaffolds were prepared from chitosan and from chitosan–PEO by wet spinning. Chitosan of 4% concentration in 2% acetic acid (CHI4–HAc2) and chitosan (4%) and PEO (2%) in 5% acetic acid (CHI4– PEO2–HAc5) yielded scaffolds with smooth and rough fiber surfaces, respectively. These scaffolds were seeded with rat bone marrow mesenchymal stem cells (MSCs). When there were no nanoparticles the initial differentiation rate was higher on (CHI4–HAc2) scaffolds but by three weeks both the scaffolds had similar alkaline phosphatase (ALP) levels. The cell numbers were also comparable by the end of the third week. Incorporation of nanoparticles into the scaffolds was achieved by two different methods: incorporation within the scaffold fibers (NP–IN) and on the fibers (NP–ON). It was shown that incorporation on the CHI4–HAc2 fibers (NP–ON) prevented the burst release observed with the free nanoparticles, but this did not influence the total amount released in 25 days. However NP–IN for the same fibers revealed a much slower rate of release; ca. 70% released at the end of incubation period. The effect of single, simultaneous and sequential delivery of BMP-2 and BMP-7 from the CHI4–HAc2 scaffolds was studied in vitro using samples prepared with both incorporation methods. The effect of delivered agents was higher with the NP–ON samples. Delivery of BMP-2 alone suppressed cell proliferation while providing higher ALP activity compared to BMP-7. Simultaneous delivery was not particularly effective on cell numbers and ALP activity. The sequential delivery of BMP-2 and BMP-7, on the other hand, led to the highest ALP activity per cell (while suppressing proliferation) indicating the synergistic effect of using both growth factors holds promise for the production of tissue engineered bone.This project was conducted within the scope of the EU FP6 NoE Project Expertissues (NMP3-CT-2004-500283). We acknowledge the support to PY through the same project in the form of an integrated PhD grant. We also would like to acknowledge the support from Scientific and Technical Research Council of Turkey (TUBITAK) through project METUNANOBIOMAT (TBAG 105T508)

3D plotted PCL scaffolds for stem cell based bone tissue engineering

The ability to control the architecture and strength of a bone tissue engineering scaffold is critical to achieve a harmony between the scaffold and the host tissue. Rapid prototyping (RP) technique is applied to tissue engineering to satisfy this need and to create a scaffold directly from the scanned and digitized image of the defect site. Design and construction of complex structures with different shapes and sizes, at micro and macro scale, with fully interconnected pore structure and appropriate mechanical properties are possible by using RP techniques. In this study, RP was used for the production of poly(e-caprolactone) (PCL) scaffolds. Scaffolds with four different architectures were produced by using different configurations of the fibers (basic, basic-offset, crossed and crossed-offset) within the architecture of the scaffold. The structure of the prepared scaffolds were examined by scanning electron microscopy (SEM), porosity and its distribution were analyzed by micro-computed tomography (m-CT), stiffness and modulus values were determined by dynamic mechanical analysis (DMA). It was observed that the scaffolds had very ordered structures with mean porosities about 60%, and having storage modulus values about 1!107 Pa. These structures were then seeded with rat bone marrow origin mesenchymal stem cells (MSCs) in order to investigate the effect of scaffold structure on the cell behavior; the proliferation and differentiation of the cells on the scaffolds were studied. It was observed that cell proliferation was higher on offset scaffolds (262000 vs 235000 for basic, 287000 vs 222000 for crossed structure) and stainings for actin filaments of the cells reveal successful attachment and spreading at the surfaces of the fibers. Alkaline phosphatase (ALP) activity results were higher for the samples with lower cell proliferation, as expected. Highest MSC differentiation was observed for crossed scaffolds indicating the influence of scaffold structure on cellular activities

Nano/microparticle incorporated chitosan fibers as tissue engineering scaffolds

[Excerpt] The aim of this study was to develop a bone tissue engineering scaffold with an inherent bone morphogenetic proteins BMP-2 and BMP-7 sequential delivery system. BMPs were encapsulated in poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and poly(lactic acid-co-glycolic acid) (PLGA) nano/microparticules which are then introduced to a chitosan matrix by two methods: embedding in the chitosan fibers and then forming the scaffold or by forming the chitosan scaffold and then introducing the nano/microparticules. [...]info:eu-repo/semantics/publishedVersio

Effect of scaffold architecture and BMP-2/BMP-7 delivery on in vitro bone regeneration

The aim of this study was to develop 3-D tissue engineered constructs that mimic the in vivo conditions through a self-contained growth factor delivery system. A set of nanoparticles providing the release of BMP-2 initially followed by the release of BMP-7 were incorporated in poly(ε-caprolactone) scaffolds with different 3-D architectures produced by 3-D plotting and wet spinning. The release patterns were: each growth factor alone, simultaneous, and sequential. The orientation of the fibers did not have a significant effect on the kinetics of release of the model protein BSA; but affected proliferation of bone marrow mesenchymal stem cells. Cell proliferation on random scaffolds was significantly higher compared to the oriented ones. Delivery of BMP-2 alone suppressed MSC proliferation and increased the ALP activity to a higher level than that with BMP-7 delivery. Proliferation rate was suppressed the most by the sequential delivery of the two growth factors from the random scaffold on which the ALP activity was the highest. Results indicated the distinct effect of scaffold architecture and the mode of growth factor delivery on the proliferation and osteogenic differentiation of MSCs, enabling us to design multifunctional scaffolds capable of controlling bone healing.This project was conducted within the scope of the EU FP6 NoE Project Expertissues (NMP3-CT-2004-500283). We acknowledge the support to PY through the same project in the form of an integrated PhD grant. We also would like to acknowledge the support from Scientific and Technical Research Council of Turkey (TUBITAK) through project METUNANOBIOMAT (TBAG 105T508)

Effects of Ethylene oxide resterilisation and In- Vitro degradation on mechanical properties of partially absorbable composite hernia meshes

Background: Prosthetic mesh repair for abdominal wall hernias is widely used because of its technical simplicity and low hernia recurrence rates. The most commonly used material is pure polypropylene mesh, however newer  composite materials are recommended by some centers because of their advantages. However, these meshes are more expensive than pure polypropylene meshes. Resterilisation of a pure polypropylene mesh has been  shown to be quite safe, and many centers prefer slicing a large mesh into smaller pieces that suitable for hernia type or defect size. Nevertheless there is no data about the safety after resterilisation of the composite meshes.Objective: To search the effects of resterilisation and In vitro degradation in phosphate buffered saline solution on the physical structure and the mechanical properties of partially absorbable lightweigth meshes.Design: Laboratory-based research.Subjects: Two composite meshes were used in the study: One mesh is consisted of monofilament polypropylene and monofilament polyglecaprone -a copolymer of glycolide and epsilon (ε)-caprolactone- (Ultrapro®, 28 g/m2, Ethicon, Hamburg, Germany), and the other one consisted of multifilament polypropylene and multifilament polyglactine (Vypro II®, 30 g/m2, Ethicon, Hamburg, Germany). Two large meshes were cut into rectangular specimens sized 50x20 mm for mechanical testing and 20x20 mm for In vitro degradation experiments. Meshes were divided into control group with no resterilisation and gas resterilisation. Ethylene oxide gas sterilisation was performed at 55°C for 4.5 hours. In vitro degradation in 0.01 M phosphate buffered saline (PBS, pH 7.4) solution at 37 ± 1°C for 8  weeks was applied to one subgroup in each mesh group. Tensiometric measurements and scanning electron microscopyic evaluations were completed for control and resterilisation specimens.Results: Regardless of resterilisation, when meshes were exposed to In vitro degradation, all mechanical parameters decreased significantly. Highest reduction in mechanical properties was observed for Ultrapro due to the degradation of absorbable polyglecaprone and polyglactin parts of these meshes. It was observed that resterilisation by ethylene oxide did not have significant difference on the degradation characteristics and almost similar physical structures  were observed for resterilised and non-resterilised meshes. For Vypro II meshes, no significant mechanical  difference was observed between resterilised and non-resterilised meshes after degradation while resterilised Ultrapro meshes exhibited stronger characteristics than non-resterilised counterparts, after degradation

Contact guidance enhances the quality of a tissue engineered corneal stroma

Corneal stroma is a very complex structure, composed of 200 lamellae of oriented collagen fibers. This highly complex nature of cornea is known to be important for its transparency and mechanical integrity. Thus, an artificial cornea design has to take into account this complex structure. In this study, behavior of human corneal keratocytes on collagen films patterned with parallel channels was investigated. Keratocytes proliferated well on films and reached confluency after 7 days in the incubation medium. Nearly all of the cells responded to the patterns and were aligned in contrast to the cells on unpatterned surfaces. Collagen type I and keratan sulfate secreted by keratocytes on patterned films appeared to be aligned in the direction of the patterns. The films showed an intermediate degradation over the course of a month. On the whole, transparency of the films increased with degradation and decreased by the presence of the cells. The decrease was, however, low and transparency level was maintained on the patterned films while on the unpatterned films a sharp decrease in transparency was followed by an improvement. This was due to the more organized distribution of cells and the oriented secretion of extracellular matrix molecules on patterned collagen films. Thus, these results suggest that application of contact guidance in cornea tissue engineering may facilitate the remodeling process, hence decrease the rehabilitation period. © 2007 Wiley Periodicals, Inc

Biodegradable nanomats produced by electrospinning : expanding multifunctionality and potential for tissue engineering

With increasing interest in nanotechnology, development of nanofibers (n-fibers) by using the technique of electrospinning is gaining new momentum. Among important potential applications of n-fiber-based structures, scaffolds for tissue-engineering represent an advancing front. Nanoscaffolds (n-scaffolds) are closer to natural extracellular matrix (ECM) and its nanoscale fibrous structure. Although the technique of electrospinning is relatively old, various improvements have been made in the last decades to explore the spinning of submicron fibers from biodegradable polymers and to develop also multifunctional drug-releasing and bioactive scaffolds. Various factors can affect the properties of resulting nanostructures that can be classified into three main categories, namely: (1) Substrate related, (2) Apparatus related, and (3) Environment related factors. Developed n-scaffolds were tested for their cytocompatibility using different cell models and were seeded with cells for to develop tissue engineering constructs. Most importantly, studies have looked at the potential of using n-scaffolds for the development of blood vessels. There is a large area ahead for further applications and development of the field. For instance, multifunctional scaffolds that can be used as controlled delivery system do have a potential and have yet to be investigated for engineering of various tissues. So far, in vivo data on n-scaffolds are scarce, but in future reports are expected to emerge. With the convergence of the fields of nanotechnology, drug release and tissue engineering, new solutions could be found for the current limitations of tissue engineering scaffolds, which may enhance their functionality upon in vivo implantation. In this paper electrospinning process, factors affecting it, used polymers, developed n-scaffolds and their characterization are reviewed with focus on application in tissue engineering

Effects of Ethylene Oxide Resterilization and In-vitro Degradation on Mechanical Properties of Partially Absorbable Composite Hernia Meshes*

Rezumat Efectele resterilizãrii cu oxid de etilen aei degradãrii in-vitro asupra proprietãåilor mecanice ale plaselor chirurgicale din material mixt paråial absorbabile Principiul aei scopul lucrãrii: Protezarea cu plase chirurgicale a herniilor de perete abdominal este o tehnicã extensiv utilizatã datoritã simplitãåii sale aei ratelor scãzute de recurenåã aferente. Cele mai utilizate materiale sunt plasele din polipropilenã, însã noi materiale mixte sunt recomandate de unele centre datoritã avantajelor pe care le prezintã. Aceste plase sunt, însã, mai costisitoare decât cele realizate exclusiv din polipropilenã. Resterilizarea unei plase de polipropilenã purã s-a dovedit a fi o procedurã destul de sigurã, iar multe centre preferã sã taie o plasã de dimensiuni mari în mai multe plase mai mici, ce pot fi folosite pentru orice tip de hernie sau orice dimensiune a defectului. Cu toate acestea, nu existã date privind gradul de siguranåã al resterilizãrii plaselor din material mixt. Studiul curent a fost desfãaeurat în vederea evaluãrii efectelor resterilizãrii aei degradãrii in-vitro cu fosfat tamponat cu soluåie salinã asupra structurii fizice aei a proprietãåilor mecanice ale plaselor chirurgicale uaeoare paråial absorbabile. Materiale aei metode: Douã tipuri de plase din material mixt au fost utilizate în acest studiu: o plasã alcãtuitã din monofilamente de polipropilenã aei monofilamente de poliglecapronã -un copolimer din glicolid aei epsilon (ε)-caprolactonã (Ultrapro®, 28 g/m 2 , Ethicon, Hamburg, Germania), iar cealaltã din multifilamente de polipropilenã aei multifilamente de poliglactinã (Vypro II®, 30 g/m 2 , Ethicon, Hamburg, Germania). Douã plase de dimensiuni mari au fost tãiate în probe rectangulare de 50x20 mm pentru testarea mecanicã aei de 20x20 mm pentru experimentele de degradare in-vitro. Plasele au fost distribuite în grupuri de control, fãrã sterilizare, aei de resterilizare cu gaz. Sterilizarea gazoasã cu oxid de etilen a fost efectiatã la 55°C timp de 4,5 ore. Câte un subgrup din fiecare grup de plase a fost supus degradãrii in-vitro cu 0,01 M fosfat tamponat cu soluåie salinã (PBS, pH 7,4) la 37 ± 1°C timp de 8 sãptãmâni. Mãsurãtorile tensiometrice aei de microscopie electronicã au fost efectuate pentru probele de control aei cele resterilizate. Rezultate: Indiferent de resterilizare, la expunerea plaselor la degradarea in-vitro, toåi parametrii mecanici au scãzut semnificativ. Cea mai mare scãdere în termeni de proprietãåi mecanice a fost observatã la plasele Ultrapro, din cauza degradãrii componentelor de poliglecapronã aei poliglactinã absorbabile ale acestor plase. S-a observat cã resterilizarea cu oxid de etilen nu a determinat diferenåe semnificative în ce Cuvinte cheie: plasã chirurgicalã, hernie, plasã din material mixt, resterilizare, degradare, proprietãåi mecanice, tensiometrie, rezistenåã la tracåiune, polipropilenã, poliglecapronã, poliglactinã Abstract Background and Aim: Prosthetic mesh repair for abdominal wall hernias is widely used because of its technical simplicity and low hernia recurrence rates. The most commonly used material is pure polypropylene mesh, although newer composite materials are recommended by some centers due to their advantages. However, these meshes are more expensive than pure polypropylene meshes. Resterilization of a pure polypropylene mesh has been shown to be quite safe, and many centers prefer slicing a large mesh into smaller pieces, suitable for any hernia type or defect size. Nevertheless there is no data about the safety after resterilization of the composite meshes. The present study was carried out to investigate the effects of resterilization and in vitro degradation in phosphate buffered saline solution on the physical structure and the mechanical properties of partially absorbable lightweight meshes. Methods: Two composite meshes were used in the study: One mesh consists of monofilament polypropylene and monofilament polyglecaprone -a copolymer of glycolide and epsilon (ε) -caprolactone -(Ultrapro®, 28 g/m 2 , Ethicon, Hamburg, Germany), and the other one consisted of multifilament polypropylene and multifilament polyglactine (Vypro II®, 30 g/m 2 , Ethicon, Hamburg, Germany). Two large meshes were cut into rectangular specimens sized 50 x 20 mm for mechanical testing and 20 x 20 mm for in vitro degradation experiments. Meshes were divided into control group with no resterilization and gas resterilization. Ethylene oxide gas sterilization was performed at 55°C for 4.5 hours. In vitro degradation in 0.01 M phosphate buffered saline (PBS, pH 7.4) solution at 37 ± 1°C for 8 weeks was applied to one subgroup in each mesh group. Tensiometric measurements and scanning electron microscopic evaluations were completed for control and resterilization specimens. Results: Regardless of resterilization, when the meshes were exposed to in vitro degradation, all mechanical parameters decreased significantly. Highest reduction in mechanical properties was observed for Ultrapro due to the degradation of absorbable polyglecaprone and polyglactin parts of these meshes. It was observed that resterilization by ethylene oxide did not determine significant difference on the degradation characteristics and almost similar physical structures were observed for resterilized and non-resterilized meshes. For Vypro II meshes, no significant mechanical difference was observed between resterilized and non-resterilized meshes after degradation while resterilized Ultrapro meshes exhibited stronger characteristics than non-resterilized counterparts, after degradation. Conclusion: Resterilization with ethylene oxide did not affect the mechanical properties of partially absorbable composite meshes. No important surface changes were observed in scanning electron microscopy after resterilization