Vasif Hasirci

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.

Sequential growth factor delivery from complexed microspheres for bone tissue engineering

Aim of the study was to design a 3D tissue-engineering scaffold capable of sequentially delivering two bone morphogenetic proteins (BMP). The novel delivery system consisted of microspheres of polyelectrolyte complexes of poly(4-vinyl pyridine) (P(4)VN) and alginic acid loaded with the growth factors BMP-2 and BMP-7 which themselves were loaded into the scaffolds constructed of PLGA. Microspheres carrying the growth factors were prepared using polyelectrolyte solutions with different concentrations (4-10%) to control the growth factor release rate. Release kinetics was studied using albumin as the model drug and the populations that release their contents very early and very late in the release study were selected to carry BMP-2 and BMP-7, respectively. Foam porosity changed when the microspheres were loaded. Bone marrow derived stem cells (BMSC) from rats were seeded into these foams. Alkaline phosphatase (ALP) activities were found to be lowest and cell proliferation was highest at all time points with foams carrying both the microsphere populations, regardless of BMP presence. With the present doses used neither BMP-2 nor BMP-7 delivery had any direct effect on proliferation, however, they enhanced osteogenic differentiation. Co-administration of BMP enhanced osteogenic differentiation to a higher degree than with their single administration.

Macroporous poly(3-hydroxybutyrate-co-3-hydroxyvalerate) matrices for bone tissue engineering

Macroporous poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV) matrices were prepared after solvent evaporation and solute leaching. PHBV solutions with different concentrations were prepared in chloroform: dichloromethane (1:2, v/v). In order to create a matrix with high porosity and uniform pore sizes, sieved sucrose crystals (75-300 or 300-500 microm) were used. PHBV foams were treated with rf-oxygen plasma to modify their surface chemistry and hydrophilicity with the aim of increasing the reattachment of osteoblasts. Surface characteristics, pore sizes and their distribution on PHBV surface were studied by scanning electron microscopy (SEM) and Scion Image Analysis Program. Void volume, pore sizes and density of foams were found to be significantly affected by foam preparation conditions. Stability of PHBV foams in aqueous media was studied. Their weight and density were unchanged for a period of 120 days and then a significant decrease was observed for the rest of the study (60 days). Osteoblasts were seeded onto the foams and their proliferation inside the matrices was also determined by SEM. After 29 and 60 days of incubation, growth of osteoblasts on matrices was observed.

Biomaterials in orthopedics

Biocompatibility and the biomaterial/tissue interface: hard tissue-biomaterial interactions, Petek Korkusuz, Feza Korkusuz material characteristics and biocompatibility of low ridigity titanium alloys for biomedical applications, Mitsuo Niinomi,Tomokazu Hattori and Shigeo Niwa the corrosion and biocompatibility of orthopaedic implants, Nadim James Hallab, Robert M. Urban and Joshua J. Jacobs technologies for the surface modification of biomaterials, Aron B. Anderson, Anthony W. Dallmier,Stephen J. Chudzik, Lise W. Duran, Patrick E. Guire, Robert W. Hergenrother, Muhammad A. Lodhi, Amy E. Noval, Ronald F. Ofstead and Klaus R. Wormuth. Bioabsorbable biomaterials for bone repair: rational design of absorbable polymers for orthopaedicrepair, James B. Beil, Jorge Heller and Kirk P. Adriano synthesis and evaluation of a poly(propylene glycol-co-fumaric acid) bone graft extender, Stephen A. Doherty, David D. Hile, Donald L. Wise, Kai-Uwe Lewandrowski and Debra J. Trantolo self-reinforced bioabsorbable devices for osteofixation of craniofacial bones, Nureddin Ashammakhi, Timo Waris, Willy Serlo and Pertti Tormala osseous grafting materials for periodontal defects, David D. Hile, Stephen A. Doherty, Stephen T. Sonis, DonaldL. Wise, Kai-Uwe Lewandrowski and Debra J. Trantolo guided diaphysis regeneration, C. Olson, S.D. Wagner and Thomas D. McGee bioresorbable skeletal fixation systems in craniofacial surgery, Mutaz B. Habal. Nondegradable materials in orthopaedics:osseointegration principles in orthopedics - basic research and clinical applications, Lars Carlsson, Warren Macdonald, C. Magnus Jacobsson and Tomas Albrektsson recent developments in bone cements, Kemal Serbetci and Nesrin Hasirci three-dimensionallyengineered hydroxyapatite ceramics with interconnected pores as a bone substitute, Akira Myoui, Noriyuki Tamai, Masataka Nishikawa, Nobuhito Araki, Takanobu Nakase, Shosuke Akita and Hideki Yoshikawa the histological and immunhistochemical aspects of theinterfacial membranes of cemented total hip and knee arthroplastics, Najat Al-Saffar and Jochanan H. Boss ceramic spine prosthesis, Noboru Hosono, Hironobu Sakaura, Tetsuo Ohwada, Kazuo Yonenobu and Hideki Yoshikawa safety aspects of alumina andzirconia ceramics in hip surgery, Gunther Heimke plasma-sprayed hydroxyapatite-coated and plasma-sprayed titanium-coated implants in vivo and clinics, Y. Yang, Kazuhisa Bessho and Joo L. Ong calcium phosphate ceramics in Japan, Masataka Nishikawa andHajime Ohgushi aspects to consider in the clinical applications of NiTi and NiTiCu shape memory alloys, F. J. Gil and J.A. Planell.

In vivo application of biodegradable controlled antibiotic release systems for the treatment of implant-related osteomyelitis

In this study the construction and in vivo testing of antibiotic-loaded polyhydroxyalkanoate rods were planned for use in the treatment of implant-related osteomyelitis. The rods were constructed of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate), carrying 50% (w/w) Sulperazone or Duocid. They were implanted in rabbit tibia in which implant-related osteomyelitis (IRO) had been induced with Staphylococcus aureus. The effectiveness of the antibiotics in the treatment of IRO was determined. The establishment of IRO with bacterial inoculation was complete after 3 weeks with 100% infection rate in all groups. There was no contamination or super-infection. Both antibiotics were found to be highly effective against the bacteria. Following the application of Sulperazone-P(3-HB-co-4-HB) rods, no infective agents could be isolated from the infection site within the 6-week test period, indicating complete treatment of the infection. Macroscopical evaluation at follow-up revealed no drainage, minimal swelling and increase in local warmth, most probably due to the surgery rather than to a reaction towards the implant. The overall scores for radiological findings by the end of 6 weeks were 0.8/5 for the antibiotic-loaded rod implanted in the right limb, and 1.1/5 for the antibiotic-free rod implanted in the left limb. There was no statistical difference between the antibiotic-loaded and antibiotic-free polymeric rods. In vivo drug release was almost complete within the first week. One interesting observation, however, was that the therapy was still very effective even when the release rate was very high. In the SEM of in vitro tested rods, the polymeric component was unchanged in 2 weeks while the drug leached out, leaving voids behind. In vivo, however, the morphology of the implant was significantly modified within 6 weeks post-implantation. Since a substantial degree of the in vivo drug release was complete within 1 week, we believe that dissolution of the drug must be the predominant mechanism through which the drug release is controlled.

Development of a calcium phosphate-gelatin composite as a bone substitute and its use in drug release.

This study was carried out to develop a calcium phosphate-gelatin composite implant that would mimic the structure and function of bone for use in filling voids or gaps and to release bioactive compounds like drugs, growth hormones into the implant site to assist healing. XDS analysis of the synthesized calcium phosphate revealed a calcium to phosphorus molar ratio of ca. 2.30, implying a less erodible material than hydroxyapatite (1.67). Release of the antibiotic gentamicin from the implant was with a burst, whether in situ or in vivo, followed by an almost constant release for about three months. It was found that the release rate could be decreased by increasing the density of the gelatin membrane. Upon implantation into rabbit tibia the release duration was substantially shortened (to about 4 weeks) with respect to the in situ tests basically due to the degradation of gelatin. In vivo studies with rabbits confirmed this degradation. The composite was perfectly biocompatible as shown by the histological studies. It, thus, has a great potential as a bone substitute material.

Retinal pigment epithelium cell culture on surface modified poly(hydroxybutyrate-co-hydroxyvalerate) thin films.

There is currently no effective treatment for the retinal disorders caused by retinal pigment epithelium (RPE) degeneration. Transplantation of allografts is the main strategy towards correction of this malady. Tissue engineering could offer hope and involve the use of biodegradable polymeric templates to replace diseased or lost RPE. In this study PHBV8 film was chosen as a temporary substrate for growing retinal pigment epithelium cells as an organized monolayer before their subretinal transplantation. The surface of the PHBV8 film was rendered hydrophilic by oxygen plasma treatment to increase the reattachment of D407 cells on the film surface. Power and duration was changed, from 50 W, 10 min to 100 W, 20 min during plasma treatment. The effect of these two parameters on surface hydrophilicity, morphology, topography, surface composition of PHBV8 thin films was studied using AFM, SEM, and phase contrast microscopy. The effect of changes in surface characteristics on cell reattachment, spreading and cell growth rate was investigated. It was found that as the treatment level was increased the surface hydrophilicity increased and roughness was decreased probably due to ablation. The PHBV8 film treated with 100 W 10 min was found to be the most suitable for 24 h reattachment of D407 cells. The cells were also grown to confluency as an organized monolayer suggesting PHBV8 film as a potential temporary substrate for subretinal transplantation to replace diseased or damaged retinal pigment epithelium.

Covalent immobilization of α-amylase onto pHEMA microspheres: preparation and application to fixed bed reactor

Abstract Microspheres of poly(2-hydroxyethyl methacrylate) with and without cross-linker were prepared by suspension polymerization. As the amount of cross-linker increased, the equilibrium water content, enzyme loading, immobilization efficiency and recovered activity were all adversely affected. Enzyme α-amylase was immobilized onto the microspheres after activation with epichlorohydrin. The K m value for the immobilized enzyme (0.90% w/v) was much greater than that of the free enzyme (0.53% w/v). It was found that the inactivation constant ( k i ) increased from 2.23 × 10 −8 min −1 at 20 °C to 1.45 × 10 −4 min −1 at 60 °C. Since the enzyme activity increased as the temperature increased, the temperature profile yielded a peak at 50 °C. For free enzyme this is at 45 °C. The residence time was proportional to the percentage hydrolysis until a residence time of 12 min was reached. Beyond this the activity increase could not match the increase in residence time. The pH profile yielded a broadening upon immobilization in addition to a small shift to higher pH (from 5.5 to 6.0). The continuous run at 30 °C, 1.0% w/v starch concentration and flow rate of 40 cm 3 h −1 led to only 20% loss in activity after a 120 h operation.

Sequential BMP-2/BMP-7 delivery from polyester nanocapsules

The aim of this study was to develop a nanosized, controlled growth factor release system to incorporate into tissue engineering scaffolds and thus activate the cells seeded in the scaffold. Nanocapsules of poly(lactic acid-co-glycolic acid) (PLGA) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) were loaded with the bone morphogenetic proteins BMP-2 and BMP-7, respectively, and with bovine serum albumin (BSA), the model protein. BSA-loading efficiency and release kinetics were used to determine the most appropriate nanocapsule pair to achieve the delivery of growth factors in a sequential manner, as occurs in natural processes. BSA-encapsulation efficiency was highest when the polymer concentration used in the preparation of PLGA and PHBV nanocapsules was 10% (w/v) (84.75% and 16.72%, respectively). Release of BSA was faster from PLGA than it was from PHBV. Based on the encapsulation efficiency and release data, 10% PLGA and 10% PHBV nanocapsules were chosen to provide the early BMP-2 and later BMP-7 release, respectively. Simultaneous, sequential delivery and individual release of the BMPs were studied for 7, 14, and 21 days, using rat bone marrow mesenchymal stem cells. Individual BMP-2 release suppressed cell proliferation while providing higher alkaline phosphatase activity with respect to BMP-7. The sequential delivery of BMP-2 and BMP-7 provided slightly lower proliferation than did simultaneous delivery, but the highest alkaline phosphatase activity of all indicated a synergistic effect on the osteogenic differentiation of mesenchymal stem cells caused by the use of the two growth factors in a sequential fashion.

Polyester based nerve guidance conduit design.

Nerve conduits containing highly aligned architecture that mimics native tissues are essential for efficient regeneration of nerve injuries. In this study, a biodegradable nerve conduit was constructed by converting a porous micropatterned film (PHBV-P(L-D,L)LA-PLGA) into a tube wrapping aligned electrospun fibers (PHBV-PLGA). The polymers were chosen so that the protective tube would erode slower than the fibrous core to achieve complete healing before the tube eroded. The pattern dimensions and the porosity (58.95 (%) with a maximum pore size of 4-5 microm) demonstrated that the micropatterned film would enable the migration, alignment and survival of native cells for proper regeneration. This film had sufficiently high mechanical properties (ultimate tensile strength: 3.13 MPa, Youngs Modulus: 0.08 MPa) to serve as a nerve guide. Electrospun fibers, the inner part of the tubular construct, were well aligned with a fiber diameter of ca. 1.5 microm. Fiber properties were especially influenced by polymer concentration. SEM showed that the fibers were aligned parallel to the groove axis of the micropatterned film within the tube as planned considering the nerve tissue architecture. This two component nerve conduit appears to have the right organization for testing in vitro and in vivo nerve tissue engineering studies.