Feza Korkusuz

An investigation of the chemical synthesis and high-temperature sintering behaviour of calcium hydroxyapatite (HA) and tricalcium phosphate (TCP) bioceramics

The experimental conditions for the synthesis of sub-micrometre,spherical particles of calcium hydroxyapatite[Ca10(PO4)6(OH)2] (HA) andtricalcium phosphate [Ca3(PO4)2] (TCP) areinvestigated through chemical coprecipitation from the aqueous solutions ofcalcium nitrate and di-ammonium hydrogen phosphate salts. The precipitationprocess employed was also found to be suitable for the production ofsub-micrometre HA/TCP composite powders in situ. The synthesized pureHA and TCP powders were found to be stable even at 1300°C in air forprolonged heating times. Bioceramic sample characterization was achieved bypowder X-ray diffraction (XRD), scanning electron microscopy (SEM), energydispersive X-ray spectroscopy (EDX), and density and surface area measurements.Crystallographic analyses of HA powders were performed by the Rietveld method onthe powder XRD data.

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.

Sulbactam‐cefoperazone polyhydroxybutyrate‐co‐ hydroxyvalerate (PHBV) local antibiotic delivery system: In vivo effectiveness and biocompatibility in the treatment of implant‐related experimental osteomyelitis

In this study, a novel antibiotic carrier system for use in the treatment of implant-related and chronic osteomyelitis was developed. Sulbactam-cefoperazone was introduced to rods of polyhydroxybutyrate-co-hydroxyvalerate (22 mol % HV, w/w), a member of a family of microbial-origin polymer that is biodegradable, biocompatible, and osteoconductive due to its piezoelectric property. The antibiotic-loaded carrier was implanted into the infection site that was induced by Staphylococcus aureus inoculation into the rabbit tibia. The effectiveness of this was assessed macroscopically, radiographically, bacteriologically, and histopathologically. Findings of infection subsided on day 15 and almost complete remission was observed on day 30. The control side that contained antibiotic-free rods, however, worsened. These findings prompted us to conclude that the novel biodegradable antibiotic carrier developed in the present study seems to be a promising candidate for use in the treatment of severe bone infection.

Bone generation on PHBV matrices: an in vitro study

Bone formation was investigated in vitro by culturing rat marrow stromal osteoblasts in biodegradable, macroporous poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV) matrices over a period of 60 days. Foams 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 (300-500 microm) were used. PHBV foams were treated with rf-oxygen plasma (100 W 10 min) to modify their surface chemistry and hydrophilicity with the aim of increasing the reattachment of osteoblasts. Osteoblasts were isolated from rat bone marrow and seeded onto PHBV foams. The cell density on and in the foams was determined with MTS assay. MTS results showed that osteoblasts proliferated on PHBV. Twenty-one days after seeding of incubation, growth of osteoblasts on matrices and initiation of mineralization were observed by confocal laser scanning microscopy. Increasing ALP and osteocalcin secretion during 60 days confirmed the osteoblastic phenotype of the derived stromal cells. SEM, histological evaluations and confocal laser scanning microscopy showed that osteoblasts could grow inside the matrices and lead to mineralization. Cells exhibited spindle-like morphology and had a diameter of 10-30 microm. Based on these, it could confidently be stated that PHBV seems to be a promising polymeric matrix material for bone tissue engineering.

A novel osteochondral implant

A novel implant for the use as an osteochondral graft was designed. This implant was prepared by stepwise formation of calcium phosphate crystals within the matrix of a lyophilised collagen sponge. Chondrocytes were then grown on this material to create the osteochondral implant. The implant was characterized with light microscopy, scanning electron microscopy (SEM), electron diffraction crystallography (EDX), and IR. It was observed with IR that the implant had a peak, that was not found so distinctly in its components, at 1400 cm(-1), implying a strong interaction of the two main ingredients of the implant, calcium phosphate and collagen. This strong interaction was also shown in the graft degradation test while the untreated collagen sponge degraded rapidly (in one day) the mineral loaded implant was able to maintain its integrity for two weeks. In the chondrocyte culture medium degradation of the implant was shown by a decrease of the calcium content and calcium to phosphorous ratio. Also, EDX revealed the presence of sulfur one and two weeks after incubation, an element not found among the components of the implant, possibly due to the development of an extracellular matrix. SEM showed that the form of the crystals of calcium phosphate differed depending on whether they were prepared on the template, collagen, or in the absence of a template. The chondrocytes appeared to be growing in number on the implant and their shapes were morphologically normal. The chondrocyte loaded collagen-calcium phosphate composite could thus be considered a potential tissue engineered osteochondral implant.

Thermal and mechanical properties of hydroxyapatite impregnated acrylic bone cements

Abstract Improvement of mechanical, thermal and biological properties of acrylic-based bone cements by adding various chemicals is a novel research area. Among these additives, hydroxyapatite (HA) is proven to be a biocompatible and osteoconductive material and it strongly integrates with bone. In this study, HA-containing acrylic bone cements were prepared and thermal and mechanical properties of the resultant cements were examined. In order to achieve a proper and homogeneous distribution of HA particles in the polymer matrix, very low viscosity cement compositions were prepared by mixing poly(methylmethacrylate) particles with two different molecular weights. Addition of HA into the cement increased the viscosity while making workability easier. With this novel formulation, polymerization temperature decreased from 111 to 87 °C, compressive strength increased from 96 to 122 MPa and sufficient compressive fatigue strength of 77 MPa at 106 cycles was provided. HA-containing acrylic bone cements demonstrated higher mechanical strength than the reference cement. The screening tests by in vivo applications achieved biological compatibility. It can be concluded that HA-containing acrylic bone cement may effectively be used in the clinical field in the future.

Poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) based tissue engineering matrices.

In this study, the aim was to produce tissue-engineered bone using osteoblasts and a novel matrix material, poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV). In order to prepare a porous PHBV matrix with uniform pore size, sucrose crystals were loaded in the foam and then leached leaving pores behind. The surface of the PHBV matrix was treated with rf-oxygen plasma to increase the surface hydrophilicity. SEM examination of the PHBV matrices was carried out. Stability of PHBV foams in aqueous media was studied. The pH decrease is an indication of the degradation extent. The weight and density were unchanged for a period of 120 days but then a significant decrease was observed for the rest of the study. Osteoblast cells were then isolated from rat bone marrow and seeded onto PHBV matrices. The metabolization and proliferation on the foams was determined with MTS assay which showed that osteoblasts proliferated on PHBV. It was also found that cells proliferated better on large pore size foams (300–500 μm) than on the small pore size foams (75–300 μm). Production of ALP was measured spectrophotometrically. The present study demonstrated that PHBV matrices are suitable substrates for osteoblast proliferation and differentiation.

In vitro antibiotic release from poly(3-hydroxybutyrate-co-3-hydroxyvalerate) rods

Provision and maintenance of adequate concentrations of antibiotics at infection sites is very important in treating highly resistant infections. For diseases like implant related osteomyelitis (IRO) it is best to provide this locally via implanted drug formulations, as systemic administration of the antibiotic may not be effective due to damaged vasculature. In this study, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) rods containing 7, 14 and 22% (mol) 3-hydroxyvalerate were loaded with sulbactam:cefoperazone or gentamicin #174;, and their antibiotic release behaviours were studied under in vitro conditions in physiological phosphate buffer at room temperature. The release patterns were representative of release from monolithic devices where a rapid early release phase is followed by a slower and prolonged release. With PHBV 22 rods, the latter phase continued for #168 2 months. This duration is critical because a proper antibiotic therapy of IRO requires the minimal effective concentration for at least 6 weeks. After in vitro release, voids with sharp edges were detected on the rods, indicating that the drug crystals dissolved but the polymer did not undergo erosion within this test period. Changing the polymer:drug ratio from 2:1 to 20:1 substantially decreased the drug release rate. A change of polymer type, however, did not lead to any detectable changes in the release patterns. Gentamicin #174; release also followed a similar pattern, except that the concentration of the drug in the release medium exhibited a decrease after long release periods, indicating degradation (or decomposition) of the antibiotic in the release medium.

In vivo response to biodegradable controlled antibiotic release systems

In this study, the major goal was to evaluate in vitro and in vivo findings by macroscopy, radiology, and histology to determine the effectiveness of therapy of experimental implant-related osteomyelitis with antibiotic carrier rods constructed of microbial polyesters. The polymers used were poly(3-hydroxybutyrate-co-4-hydroxyvalerate) [P(3-HB-co-4-HB)] and poly(3-hydroxybutyrate-co-3-hydroxy- valerate) [P(3-HB-co-3-HV)]. Both the Sulperazone and the Duocid-P(3-HB-co-4-HB) rods with a drug to polymer ratio of 1:1 (w/w) were effective in treating the bone infection that was experimentally initiated by inoculation of a hemolytic strain of Staphylococcus aureus (coagulase positive; phage type 52/52b) together with metal implants into the medullary area of rabbit tibia. Macroscopical data revealed that the effectiveness of therapy was apparent at week 6 for all categories tested. Radiological findings with Duocid- and Sulperazone-loaded P(3-HB-co-4-HB) rods improved significantly when judged by changes in periosteal elevation, widening of bone shaft, new bone formation, and soft-tissue deformation after 6 weeks of implantation. Histologically the signs of infection were found to subside by weeks 3 and 6. Inflammatory cells were replaced with bone-forming cells upon treatment with Sulperazone-P(3-HB-co-4-HB) and Duocid-P(3-HB-co-4-HB). Osteoblastic activity was prominent. Intramedullary inflammation, although still present, started to be replaced by fibrous or bony tissue. Histological findings presented the subsidence of infection. In summary, the antibiotic-loaded biopolymeric rods appeared to have potential as a new controlled-release system for the treatment of implant related osteomyelitis and chronic osteomyelitis.