Yahya Açil

Bis-phossy jaws - high and low risk factors for bisphosphonate-induced osteonecrosis of the jaw.

INTRODUCTION Bisphosphonates (BPs) have transformed our ability to treat certain malignancies, osteoporosis and hypercalcaemia. This class of drug is assumed to be well tolerated by most. There are some important caveats to this assumption, however, one of the significances being the risk of osteonecrosis of the jaw (ONJ). MATERIAL AND METHODS This multi-centre retrospective study examined the role of different BPs on the development of ONJ, its clinical presentation and the efficacy of various treatment modalities, comparing these findings with the available literature. RESULTS A total of 78 patients from 17 centres were identified with ONJ. A majority of patients identified with ONJ had used Pamidronate or Zoledronate (93.6%) intravenously. 94.9% of patients had received BP in the course of treatment for malignancies and a majority had also received prior chemotherapy or exogenous steroids. 82.1% of patients had received BP for more than 1 year. The mean time from the introduction of BP to the development of ONJ in 24 patients from our department was 31.8 months. CONCLUSIONS The most common intraoral manifestation was exposed necrotic jawbone. Tooth extractions and oral surgical intervention appear to place patients on BP therapy at risk of ONJ, especially after intravenous BP treatments. ONJ proved in this study to be remarkably refractory to treatment, with radical resection being the only curative approach. We recommend that all patients receive necessary dental treatment prior to commencing BP therapy.

Culture of cells gained from temporomandibular joint cartilage on non-absorbable scaffolds

The objective of this study was to investigate the adhesion, spreading and extracellular matrix synthesis of temporomandibular joint (TMJ) derived cells on non-absorbable scaffold materials to ultimately provide a durable stress-absorbent framework within tissue-engineered disc transplants. Scaffolds were prepared by polyamide monofilaments, expanded polytetrafluoroethylene (ePTFE) monofilaments, polyglycolic acid monofilaments (control) or natural bone mineral blocks (control). These scaffolds were incubated for 2, 4 and 8 weeks under common culture conditions with cells (human and porcine) harvested from the TMJ-disc or the articular eminence. The specimens were examined by scanning electron microscopy and transmission electron microscopy. The type of collagen synthesized was analyzed by SDS-PAGE. The cells were strongly adherent to all of the materials. Independent of their origin the cells became confluent on all scaffolds within four weeks. They filled recesses loosely and covered the constructs by an envelope of dense stratified cell layers. Moreover, the cells expressed collagen type II, which is specific for chondrocytes. Thus, it could be demonstrated, that ePTFE, polyamide, polyglycolic acid and natural bone mineral have an excellent compatibility in a three-dimensional cell culture system. ePTFE and polyamide scaffolds may be well suited for the development of tissue-engineered stress-resistant articular disc transplants.

Platelet-rich Plasma and Platelet-rich fibrin in human cell culture

OBJECTIVES The clinical use of platelet-rich plasma (PRP) for preprosthetic surgery has been a matter of controversy until now. Only recently, a new blood preparation has been developed which results in platelet-rich fibrin (PRF). The objective of the present investigation was to examine the growth factor release from PRP and PRF in vitro. STUDY DESIGN Whole blood samples from healthy participants (n = 10) were drawn to generate PRP and PRF. Human osteoblasts (O), human fibroblasts (F), and human osteoblast-derived osteosarcoma cells (Saos-2) were used for the cell culture. Cells of each cell line were cultivated, and PRP- or PRF-preparations added for ten days. The drawn medium was pooled and the quantities of growth factors (platelet-derived growth factor isomers AB and BB, insulin-like growth factor I, and transforming growth factor (TGF) isomers beta1 and beta2) analyzed by enzyme-linked immunosorbent assay. RESULTS In osteoblast and Saos-2 cultures, cytokine concentrations were significantly higher for PRP than for PRF (P < .05). In fibroblast cultures, results were the same with the exception of TGF-beta2 (P < .05). CONCLUSIONS This study demonstrates that PRP application in cell cultures leads to higher levels of growth factors than PRF application.

Effect of oligofructose or dietary calcium on repeated calcium and phosphorus balances, bone mineralization and trabecular structure in ovariectomized rats*.

We investigated the effects of dietary oligofructose and Ca on bone structure in ovariectomized rats, using microradiography and histomorphometry. Ninety-six animals were allocated to seven experimental groups: G1, sham-operated; G2-G7, ovariectomized. Semi-purified diets containing 5 g Ca/kg (recommended content) without oligofructose (G1, G2) or with 25, 50 or 100 g oligofructose/kg (G3, G4, G5) or 10 g Ca/kg (high content) without oligofructose (G6) or with 50 g oligofructose/kg (G7) were fed for 16 weeks. At the recommended level of Ca, high oligofructose (G5) increased femur mineral levels in ovariectomized rats, while medium oligofructose did so at high Ca. Increasing Ca in the absence of oligofructose did not increase femur mineral content. Trabecular bone area (%) analysed in the tibia was 10.3 (sem 1.2) (G1), 7.7 (sem 0.6) (G2), 9.3 (sem 0.7) (G3), 9.4 (sem 1.0) (G4), 9.5 (sem 0.7) (G5), 10.2 (sem 0.8) (G6), and 12.6 (sem 0.8) (G7). At the recommended level of Ca, 25 g oligofructose/kg prevented loss of trabecular area due to increased trabecular thickness, while 50 or 100 g oligofructose/kg increased trabecular perimeter. At high Ca, oligofructose prevented loss of bone area due to increased trabecular number but similar thickness (G7 v. G6). When Ca was raised in the presence of oligofructose (G7), trabecular area and cortical thickness were highest, while loss of trabecular connectivity was lowest of all groups. At the same time, lumbar vertebra Ca was higher; 44.0 (sem 0.8) (G7) compared with 41.6 (sem 0.8) (G2), 41.4 (sem 0.7) (G4), and 40.5 (sem 1.0) mg (G6). We conclude that ovariectomy-induced loss of bone structure in the tibia was prevented but with different trabecular architecture, depending on whether dietary Ca was increased, oligofructose was incorporated, or both. Oligofructose was most effective when dietary Ca was high.

Platelet‐rich fibrin membranes as scaffolds for periosteal tissue engineering

OBJECTIVES Platelet-rich fibrin (PRF)-based membranes have been used for covering alveolar ridge augmentation side in several in vivo studies. Few in vitro studies on PRF and no studies using human periosteal cells for tissue engineering have been published. The aim is a comparison of PRF with the commonly used collagen membrane Bio-Gide as scaffolds for periosteal tissue engineering. MATERIAL AND METHODS Human periosteal cells were seeded on membrane pieces (collagen [Bio-Gide] and PRF) at a density of 10(4) cells/well. Cell vitality was assessed by fluorescein diacetate (FDA) and propidium iodide (PI) staining, biocompatibility with the lactate dehydrogenase (LDH) test and proliferation level with the MTT, WST and BrdU tests and scanning electron microscopy (SEM). RESULTS PRF membranes showed slightly inferior biocompatibility, as shown by the LDH test. The metabolic activity measured by the MTT and WST tests was higher for PRF than for collagen (BioGide). The proliferation level as measured by the BrdU test (quantitative) and SEM examinations (qualitative) revealed higher values for PRF. CONCLUSION PRF appears to be superior to collagen (Bio-Gide) as a scaffold for human periosteal cell proliferation. PRF membranes are suitable for in vitro cultivation of periosteal cells for bone tissue engineering.

Three-dimensional cultivation of human osteoblast-like cells on highly porous natural bone mineral.

In this study, we investigated the growth and extracellular matrix synthesis of human osteoblast-like cells on highly porous natural bone mineral. Human bone cells were isolated from trabecular bone during routine iliac crest biopsies. Under conventional culture conditions, trabecular bone cells were able to assume the organization of a three-dimensional structure on a porous natural bone mineral (Bio-Oss(R) Block). Scanning electron microscopy examination after 6 weeks revealed multiple cell layers on the trabecular block. Transmission electron microscopy examination after 6 weeks revealed the accumulation of mature collagen fibrils in the intracellular and extracellular spaces, and showed multilayered, rough endoplasmic reticulum as well as mitochondria-rich cells surrounded by dense extracellular matrix. These morphological observations suggest that the cell layer may resemble the natural three-dimensional structure. Biochemical analysis revealed that the hydroxylysylpyridinoline, lysylpyridinoline, and hydroxyproline content of the cell layer increased in a time-dependent manner, whereas in monolayer culture without natural bone mineral, no measurable amounts of hydroxylysylpyridinoline or lysylpyridinoline, and a barely measurable amount of hydroxyproline, were noted. Mature collagen extracted by ethylenediaminetetraacetic acid-demineralization from the cell layer on natural bone mineral showed an identical electrophoretic pattern to that observed in human bone, as evaluated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The present study demonstrated an excellent biocompatibility of the highly porous natural bone mineral in a three-dimensional bone cell culture system, and thus its potential for tissue-engineered growth of human bone.

A comparison of biocompatibility and osseointegration of ceramic and titanium implants: an in vivo and in vitro study

This study compared the biocompatibility in vitro and the osseointegration in vivo of zirconium and titanium implants regarding implant surfaces and the bone-implant contacts. The different implant surfaces and the biocompatibility of zirconium versus titanium implants were determined by vitality and cytotoxic tests in vitro. The contact of the osteoblasts to the implant surface was determined by scanning electron microscopy (SEM). The in vivo study for osseointegration was performed in domestic pigs over 4 and 12 weeks. In each animal, 4 zirconium and 4 titanium implants (WhiteSky, BlueSky, Bredent, Germany) were inserted in the os frontale and analysed by histomorphometry. Cytotoxicity and SEM showed good biocompatibility in relation to the investigated implant materials. Histological results showed direct bone-implant contact of the implant surfaces. The zirconium implants showed a slight delay in osseointegration in terms of bone-implant contact as measured by histomorphometry (after 4 weeks, zirconium (59.3 ± 4.6%) versus titanium (64.1 ± 3.9%); after 12 weeks, zirconium (67.1 ± 2.3%) versus titanium (73.6 ± 3.2%). A statistically significant difference between the two groups was not observed. The results indicated similar biocompatibility and osseointegration for zirconium compared to titanium implants.

The cytotoxic effects of three different bisphosphonates in-vitro on human gingival fibroblasts, osteoblasts and osteogenic sarcoma cells

INTRODUCTION Osteonecrosis of the jaw (ONJ) is an emerging condition in patients undergoing long-term administration of bisphosphonates (BP) for the treatment of osteoporosis and hypercalcaemia associated with malignancy, multiple myeloma, and metastatic breast and prostate cancers. This is a follow-up study, its purpose was to examine the effects in-vitro of intravenous zoledronic acid (ZOL) and pamidronate (PAM) and oral alendronate (FOS) on the human oral cavity using gingival fibroblasts and osteoblasts cells and, in addition, osteogenic sarcoma cells (SaOS-2-cells). MATERIALS AND METHODS Human gingival fibroblasts, osteoblasts and SaOS-2-cells were seeded on multiple 6-well plates at a density of 5 × 10(5)cells in a 4-week cell culture. Four different concentrations (1, 5, 10, 20 μM) of each BP (ZOL, PAM, FOS) and pyrophosphate were used in this study. RESULTS All BP decreased collagen production and lowered cell proliferation in-vitro. ZOL was the component with most inhibitory effect. CONCLUSION The findings in this study suggest that ZOL, PAM and FOS generally diminish cell proliferation and collagen production of human gingival fibroblasts, osteoblasts and SaOS-2-cells. The present follow-up study shows that not only ZOL and PAM but also FOS have a strong inhibitory effect on collagen production and cell survival in-vitro.

Effects of bone morphogenetic protein‐7 stimulation on osteoblasts cultured on different biomaterials

The objective of the present study was to investigate the effects of an in vitro stimulation of human osteoblasts by recombinant human bone morphogenetic protein‐7 (rhBMP‐7) on the collagen types and the quantity of the collagen cross‐links synthesized in a three‐dimensional culture on various biomaterials for bone replacement. Trabecular bone chips were harvested from human iliac crests, and cell cultures were established at standard conditions. One hundred and fifty nanograms per milliliter of rhBMP‐7 was added. For the second passage a cell scraper was used to bring the cells into suspension, and 100 μl osteoblasts (at a density of 3.3 × 105) were transferred onto nine blocks of either Bio‐Oss®, Tutoplast®, or PepGen p‐15™. Blocks incubated with cells that were not treated with rhBMP‐7 served as controls. Cell colonization of the biomaterials was observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) after a period of 2, 4, and 6 weeks. Throughout the experiment medium, supernatants were collected and collagen was characterized by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Finally, the collagen cross‐link residues hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) were quantified by HPLC. Within 4 weeks the cells became confluent on all of the studied biomaterials. All samples synthesized bone specific LP and collagen type I. However, in rhBMP‐7‐stimulated samples, the amount of HP and LP found was increased by 45% compared to non‐stimulated samples. Cell proliferation and collagen synthesis was similar on the different biomaterials, but was consistently reduced in specimen not stimulated with rhBMP‐7. In vitro stimulation of osteoblasts on Bio‐Oss, Tutoplast, or PepGen p‐15 with rhBMP‐7 and subsequent transplantation of the constructs might lead to an enhanced osseointegration of the biomaterials in vivo. J. Cell. Biochem. 86: 90–98, 2002.

Transmission electron microscopy comparison of methods for collecting in situ formed enamel pellicle

The in vivo formed salivary pellicle is composed of an outer globular and a densely structured basal layer. This study developed a method for selective recovering of these pellicle layers from the enamel surface. Two-hour in situ pellicles were formed by intraoral exposure of enamel specimens in two adults. Pellicle-covered enamel specimens were treated either mechanically (scraping with scaler, curette or razor blade, or rubbing with a sponge) or chemically (phosphate buffer, NaCl, NaOCl, CaCl2, NaSCN, urea, tetrahydrofurane, guanidine, SDS, HCl, or EDTA with or without additional ultrasonication). Specimens were processed for transmission electron microscopic analysis to detect pellicle residues remaining on the enamel surface after the different treatments. Most of the chemical treatments caused partial, incomplete removal of the globular layer. Complete removal of the globular layer without disruption of the basal layer was obtained by sponge rubbing or by CaCl2 combined with ultrasonication, whereas scraping caused partial disruption of the basal layer. Removal of the basal layer was observed after treatment with HCl, EDTA, or NaOCl combined with ultrasonication. Electrophoretical analysis of recovered pellicle fractions indicate that combination of sponge-rubbing followed by EDTA treatment can be recommended for stepwise removal of the globular and basal pellicle layers.