Søren Jepsen

Mandibular reconstruction with prefabricated vascularized bone grafts using recombinant human osteogenic protein-1: an experimental study in miniature pigs. Part II: Transplantation

Osteogenic Protein-1 (rhOP-1), also called bone morphogenetic protein-7 (BMP-7), is osteoinductive. The aim of this study was to present a new surgical technique: the prefabrication of a vascularized bone graft using rhOP-1 and its microsurgical transplantation. During 6 weeks, osteomuscular grafts were prefabricated in the latissimus dorsi muscle of five adult minipigs. Six hundred micrograms rhOP-1 on a carrier of xenogenic bone mineral in block form were used. The grafts were transplanted into defects of the mandibular angles performing a microsurgical anastomosis and using miniplates for fixation. Identical defects of the contralateral side were treated by direct application of 600 microg rhOP-1 and xenogenic bone mineral. A polychrome sequential labelling was applied. After transplantation the bone stayed viable, demonstrated by continuous apposition of fluorochromes (non-decalcified histologic sections) and bone scintigraphy. The reconstructive result was significantly superior in the prefabrication technique, assessed by histology and computerized tomography (CT). In conclusion, the method has a potential to become a clinical alternative for conventional vascularized bone grafts.

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.

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.

Mandibular reconstruction in miniature pigs with prefabricated vascularized bone grafts using recombinant human osteogenic protein-1: a preliminary study.

A new technique of prefabrication of vascularized bone grafts in the latissimus dorsi muscle of miniature pigs, using recombinant human osteogenic protein-1 (rhOP-1) and xenogenic bone mineral as a carrier, is presented. The grafts were used to reconstruct a mandible using microsurgical anastomosis of the thoracodorsal vessels of the flap with a branch of the external carotid artery and the external jugular vein. The technique provided enough bone to reconstruct a mandible. The bone marrow stayed viable after transplantation, demonstrated by fluorescence microscopy and scintigraphy. Compared with identical defects of the contralateral side, which were treated with directly applied rhOP-1/bone mineral, a significantly better result was obtained with the prefabrication technique. The prefabrication technique may provide a means to treat irradiated patients with bone grafts produced by osteoinductive proteins and has a potential to become a clinical alternative to conventional vascularized bone grafts.

Evaluation of selective caries removal by a fluorescence feedback-controlled Er:YAG laser in vitro

Aim: To establish a fluorescence threshold level that could guide a therapeutic Er:YAG laser through a caries lesion to determine a therapeutic endpoint of caries removal. Materials and Methods: A total of 65 extracted human teeth, 35 with dentine caries and 30 healthy, were used for this study. An Er:YAG laser system that emitted at a wavelength of 2.94 µm was used. The laser was equipped with a laser fluorescence feedback system, excitation wavelength 655 nm, to control the irradiation by the Er:YAG laser. The evaluated threshold levels of the fluorescence feedback system were 3, 7, 8, 10, 12, 15 and 20. After treatment the teeth were prepared for histological staining according to the method of Brown and Brenn for the identification of bacteria. The specimens were subjected to a quantitative evaluation of residual bacteria on the treated dentine surface. In addition, the internal fluorescence of dentine and potential fluorescence changes of dentine after laser irradiation were evaluated. Results: About 80% of the irradiated dentine surface showed residual bacteria with threshold levels of 20, 15, 12, and 10. Residual bacteria were not found with threshold levels of 7 and 3. The study revealed a significant increase in dentine fluorescence after laser irradiation. Conclusion: The results of the present in vitro study indicate that a fluorescence threshold level of 7 or 8 units can guide an Er:YAG laser to a complete removal of carious dentine.

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 3.3u2009×u2009105) 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.

Full‐mouth disinfection for the treatment of adult chronic periodontitis

BACKGROUNDnIn an attempt to enhance treatment outcomes, alternative protocols for anti-infective periodontal therapy have been introduced.nnnOBJECTIVESnTo evaluate the effectiveness of full-mouth disinfection or full-mouth scaling compared to conventional quadrant scaling for periodontitis.nnnSEARCH STRATEGYnData sources included electronic databases, handsearched journals and contact with experts. The Cochrane Oral Health Group Trials Register, the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE and EMBASE were searched. Reference lists from relevant articles were scanned and the authors of eligible studies were contacted to identify trials and obtain additional information. Date of most recent searches: December 2006: (CENTRAL) (The Cochrane Library 2006, Issue 4).nnnSELECTION CRITERIAnRandomised controlled trials were selected with at least 3 months follow up comparing full-mouth scaling and root planing within 24 hours with (FMD) or without (FMS) the adjunctive use of an antiseptic (chlorhexidine) with conventional quadrant scaling and root planing (control). The methodological quality of the studies was assessed within the data extraction form, mainly focusing on: method of randomisation, allocation concealment, blindness of examiners and completeness of follow up.nnnDATA COLLECTION AND ANALYSISnData extraction and quality assessment were conducted independently by multiple review authors. The primary outcome measure was tooth loss, secondary outcomes were reduction of probing depth, bleeding on probing and gain in probing attachment. The Cochrane Collaboration statistical guidelines were followed.nnnMAIN RESULTSnThe search identified 216 abstracts. Review of these abstracts resulted in 12 publications for detailed review. Finally, seven randomised controlled trials (RCTs) which met the criteria for eligibility were independently selected by two review authors. None of the studies included reported on tooth loss. All treatment modalities led to significant improvements in clinical parameters after a follow up of at least 3 months. For the secondary outcome, reduction in probing depth, the mean difference between FMD and control was 0.53 mm (95% confidence interval (CI) 0.28 to 0.77) in moderately deep pockets of single rooted teeth and for gain in probing attachment 0.33 mm (95% CI 0.04 to 0.62) in moderately deep single and multirooted teeth. Comparing FMD and FMS the mean difference in one study for gain in probing attachment amounted to 0.74 mm in favour of FMS (95% CI 0.17 to 1.31) for deep pockets in multirooted teeth, while another study reported a mean difference for reduction in bleeding on probing of 18% in favour of FMD (95% CI -33.74 to -2.26) for deep pockets of single rooted teeth. No significant differences were observed for any of the outcome measures, when comparing FMS and control.nnnAUTHORS CONCLUSIONSnIn patients with chronic periodontitis in moderately deep pockets slightly more favourable outcomes for pocket reduction and gain in probing attachment were found following FMD compared to control. However, these additional improvements were only modest and there was only a very limited number of studies available for comparison, thus limiting general conclusions about the clinical benefit of full-mouth disinfection.

The stage of native biofilm formation determines the gene expression of human β‐defensin‐2, psoriasin, ribonuclease 7 and inflammatory mediators: a novel approach for stimulation of keratinocytes with in situ formed biofilms

BACKGROUND/AIMSnAntimicrobial peptides such as human beta-defensin-2 (hBD-2), psoriasin (PSO), and ribonuclease 7 (RNase 7) play an important role in innate immunity. The aim of the present study was to test the hypothesis that epithelial cells show a differential gene expression pattern of antimicrobial peptides (hBD-2, PSO, RNase 7) and inflammatory mediators such as interleukin-8 (IL-8) and 5-lipoxygenase (5-LO) in response to different stages of naturally formed biofilms.nnnMETHODSnEpithelial cells were cultured from biopsies obtained from five healthy individuals. Native bacterial biofilms were taken from the same subjects that donated the gingival biopsies. To obtain different stages of biofilm formation, polymer disks were attached to prostheses and carried intraorally for 1, 3, 5, and 9 days. The expression of genes for hBD-2, PSO, RNase 7, 5-LO, and IL-8 was examined using semi-quantitative reverse transcription-polymerase chain reaction. The bacterial composition of the individual biofilms was defined using a microarray system (Parocheck), which showed the presence of 20 different bacterial species that are associated with plaque formation.nnnRESULTSnThe expression of the messenger RNAs of hBD-2, RNase 7, and 5-LO was upregulated as a result of the exposure to early biofilm stages, whereas the gene expression of IL-8 was increased in response to matured biofilms. Inter-individual differences in the innate immune response were observed.nnnCONCLUSIONnThe results of the present study showed a time-dependent messenger RNA expression of antimicrobial peptides (hBD-2, RNase 7), 5-LO, and IL-8 in oral epithelial cells responding to different stages of biofilm formation.

Fluorescence‐controlled Er:YAG laser for caries removal in permanent teeth: a randomized clinical trial

The aim of this randomized clinical study was to compare the efficacy of a fluorescence-controlled erbium-loaded yttrium aluminum garnet (Er:YAG) laser with conventional bur treatment for caries therapy in adults. Twenty-six patients with 102 carious lesions were treated using either the Er:YAG laser, at threshold levels of 7, 8, 9, and 10 [U], or rotary burs. Both techniques were applied to each lesion at separate locations. After treatment, dentine samples were obtained using a carbide bur. The viable counts of Streptococcus mutans (SM) and lactobacilli (LB) [expressed as colony-forming units (log10 CFUs)], treatment time, pain, vibration, and sound intensity were determined. The median numbers of CFUs for SM and LB were not statistically different between laser and bur treatment at threshold levels 7 and 8 [U]. At threshold levels 9 and 10 [U], the median number of CFUs for LB [1.11 (range: 0.00-2.04)] were significantly higher following laser treatment than following bur treatment [0.30 (range: 0.00-0.60)]. The results indicate that treatment with a fluorescence-controlled Er:YAG laser at threshold levels of 7 and 8 removed caries to a level similar to that achieved using conventional bur treatment, with clinically irrelevant amounts of remaining bacteria. Although more time consuming, laser treatment provided higher patient comfort than bur treatment.

Cavity size difference after caries removal by a fluorescence-controlled Er:YAG laser and by conventional bur treatment.

To determine the extensions of cavities prepared conventionally by bur or by a fluorescence-controlled Er:YAG laser. Sixty-five human teeth with dentine caries were bisected through the caries lesion and were treated by a fluorescence-controlled Er:YAG laser in a non-contact or a contact mode or by a rotary bur. The specimens were subjected to histological staining and a quantitative evaluation of cavity area (mm2) by computer-assisted alignment. Data were tested for statistical significant differences by the Wilcoxon test (pu2009<u20090.05). Twenty-three out of 29 cavities were smaller after caries removal with the non-contact laser compared to the bur. For a threshold level of seven, a cavity size difference of 1.63 (1.86) mm2 was calculated compared to a cavity size difference of 5.35 (5.05) mm2 after bur excavation. The differences were statistically significant (pu2009=u20090.029). No significant differences were observed between the cavity size differences after excavation with the non-contact or the contact laser handpiece. Residual bacteria within the cavity floor were found only in low numbers after all treatments. The present in vitro study indicates that caries removal by a fluorescence-controlled Er:YAG laser using a threshold level of seven resulted in less dentine loss than preparations by a bur.