Effect of gabapentin-lactam and gamma-aminobutyric acid/lactam analogs on proliferation and phenotype of ovine mesenchymal stem cells.

PURPOSE: Classic tissue engineering consists of three components: scaffold, cells, and growth or differentiation factors. Currently, expensive bone morphogenetic proteins are the most common substance used for hard tissue regeneration. An alternative could be gamma-aminobutyric acid/lactam (GABA-lactam) analogs. MATERIALS AND METHODS: The effects of gabapentin-lactam, cis- and trans-8-tertbutyl-GABA-pentinlactam (trans-TB-GBP-L), and phenyl-GABA-lactam were tested in this study on ovine mesenchymal stem cell (MSC) proliferation. MSCs were selected from bone marrow aspirate concentrate by plastic adherence and amplified. Aliquots of the cells were incubated in medium, with four different concentrations of the GABA-lactam analogs dissolved in dimethyl sulfoxide. Cells in medium with and without dimethyl sulfoxide served as controls. Cell proliferation was tested with a nonradioactive assay. Before and after GABA-lactam analog influence, the MSC character was evaluated by the ability of the cells to differentiate into osteoblasts, chondrocytes, and adipocytes. RESULTS: Proliferation was significantly increased under the influence of the analogs, depending on their concentration. MSCs cultured in 1 nmol/L trans-TB-GBP-L showed the highest proliferation rate. The MSC character was not altered. CONCLUSIONS: GABA-lactam analogs could be suited to stimulate MSC proliferation for tissue engineering applications. Further in vivo studies are necessary to evaluate the possible clinical potential of GABA-lactam analogs for hard tissue regeneration.

Cocultivation of human oral keratinocytes and human osteoblast-like cells.

Head and neck reconstruction transplants often require a bony structure but also tissue for the intraoral lining. This is why oral keratinocytes and osteoblast-like cells are essential cell types for combined tissue engineered transplants for defects in the field of craniomaxillofacial surgery. Therefore, we isolated oral keratinocytes and osteoblast-like cells from human tissue samples and cocultivated both cell types on the same carrier. Cell proliferation and morphological analysis showed that the contemporaneous cultivation of human oral keratinocytes and human osteoblast-like cells is possible.The successful in vitro cocultivation of hard and soft tissue derived cells on the same carrier will be an important advancement for developing hard and soft tissue reconstruction therapies especially in the oral cavity.

Human saliva exposure modulates bone cell performance in vitro.

Various situations encountered by a clinician during the daily routine including surgical periodontitis therapy, dental implant insertion, or tooth extraction involve the contact of saliva with the jaw bone. However, there are only sparse data concerning the influence of saliva on bone cells. Saliva specimens were incorporated within culture medium and administered to murine MC3T3 osteoblasts, of which the morphology (REM), proliferation (EZ4U), and differentiation (qRT-PCR, alkaline phosphatase activity, extracellular matrix calcification) were assessed. Simultaneously, the composition of saliva media was analyzed with respect to the content of lactoferrin, activities of classical salivary enzymes, and the ability to provoke inflammatory cytokine production (enzyme-linked immunosorbent assay) in MC3T3 osteoblasts. The morphology, proliferation, and expression of differentiation-associated genes were seriously handicapped by saliva contact. Saliva-touched cells exhibited less alkaline phosphatase but normal levels of extracellular matrix mineralization. Saliva-containing culture media featured physiological activities of salivary enzymes and considerable amounts of lactoferrin but almost completely lacked salivary alkaline phosphatase and unspecific proteases. Upon saliva incubation, MC3T3 osteoblasts did not release noteworthy levels of interleukin-1 beta or tumor necrosis factor alpha. Although saliva is generally considered to vitalize oral tissues, this study reveals that it harms osteoblast-like cells more due to the presence of salivary enzymes than by triggering of inflammation. This issue is clinically relevant because it broadens the understanding of the bone cell fate within the rather complex cosmos of the oral cavity thereby providing a basis for clinical decision making and treatment guidelines. It seems to be reasonable to restrict the contact period between saliva and bone.

Bone engineering-vitalisation of alloplastic and allogenic bone grafts by human osteoblast-like cells.

Human osteoblasts on non-sintered hydroxyapatite and demineralised bone matrix (DBX) were analysed in vitro to find out whether they would be suitable for reconstruction of bones in oral surgery. Human osteoblasts were isolated from the jaw during routine dental operations and seeded onto the two biomaterials. Cells were characterised by assay of alkaline phosphatase, detection of type 1 collagen, and production of osteocalcin. After 21 days of cultivation, the cell/biomaterial constructs were examined by scanning electron microscopy, thin sections, and propidium iodide/fluorescein diacetate staining. The osteoblasts formed a vital multiple cell layer on DBX within 3 weeks of cultivation. On hydroxyapatite, the cells showed no tendency to proliferate or migrate onto the synthetic biomaterial, or to form well-spread and viable cell constructs. These findings suggest that surface morphology or the presence of osteoinductive factors may have an important role in the adhesion and proliferation of osteoblasts. Human DBX can be colonised by human osteoblast-like cells in vitro, indicating the potential of allogeneic carriers for future procedures in bone engineering.

Change in diet and oral hygiene over an 8-week period: effects on oral health and oral biofilm.

The aim of the study was to monitor changes in oral health and oral biofilm composition in vivo during an experiment simulating prehistoric lifestyle and diet and poor oral hygiene. Thirteen subjects lived for a period of 8 weeks under Neolithic conditions. The following clinical parameters were recorded before and after the project: gingival and plaque index (Löe and Silness, Acta Odontol Scand 21:533, 1963; Silness and Löe, Acta Odontol Scand 22:121-135, 1964), probing pocket depth, and bleeding upon probing. In addition, supragingival plaque samples were collected both before and after the project and were analysed quantitatively using multiplex fluorescence in situ hybridization and confocal laser scanning microscopy. The following plaque bacteria were evaluated: Streptococcus spp., Veillonella spp., Fusobacterium nucleatum, and Actinomyces naeslundii. The plaque index increased significantly from 1.12 up to 1.55 over the 8-week period (gingival index before, 0.46; after, 0.93; p < 0.05). A strong correlation of both indices was recorded before (r = 0.77) and after (r = 0.83) participation in the study. Each of the children in the study showed a progression of carious lesions and/or new areas of demineralisation. The probing pocket depth and bleeding upon probing were not affected. All subjects yielded an intra-individual shift in biofilm composition. The proportion of F. nucleatum decreased across all subjects. The proportion of Veillonella spp. increased among the children. Poor oral hygiene and change of diet lead to an increase in oral plaque and gingival inflammation. The inter-individual comparison indicated a shift in bacterial composition.

Bone regeneration in sinus lifts: comparing tissue-engineered bone and iliac bone.

Lifting of the sinus floor is a standard procedure for bony augmentation that enables dental implantation. Although cultivated skin and mucosal grafts are often used in plastic and maxillofacial surgery, tissue-engineered bone has not achieved the same success. We present the clinical results of dental implants placed after the insertion of periosteum-derived, tissue-engineered bone grafts in sinus lifts. Periosteal cells were isolated from biopsy specimens of periosteum, resuspended and cultured. The cell suspension was soaked in polymer fleeces. The cell-polymer constructs were transplanted by sinus lift 8 weeks after harvesting. The patients (n=35) had either one or both sides operated on. Seventeen had a one-stage sinus lift with simultaneous implantation (54 implants). In 18 patients the implants were inserted 3 months after augmentation (64 implants). Selected cases were biopsied. A control group (41 patients: one stage=48 implants, two stage=135 implants) had augmentation with autologous bone only. They were followed up clinically and radiologically for at least 24 months. Both implants and augmentation were significantly more successful in the control group. Failure of augmentation of the tissue-engineered bone was more common after large areas had been augmented. Eleven implants were lost in the study group and only one in the control group. Lifting the sinus floor with autologous bone is more reliable than with tissue-engineered transplants. Although lamellar bone can be found in periosteum-derived, tissue-engineered transplants, the range of indications must be limited.

Comparative in vitro study of the proliferation and growth of ovine osteoblast-like cells on various alloplastic biomaterials manufactured for augmentation and reconstruction of tissue or bone defects.

In this in vitro study ovine osteoblast-like cells were cultured on seven different alloplastic biomaterials used for augmentation and for reconstruction of bone defects in dental and craniomaxillofacial surgery. The aim of this study was to examine the growth behaviour (viability, cell density and morphology) of ovine osteoblast-like cells on the investigated biomaterials to get knowledge which biomaterial is qualified to act as a cell carrier system in further in vivo experiments. The biomaterials were either synthetically manufactured or of natural origin. As synthetically manufactured biomaterials Ethisorb, MakroSorb, PalacosR, and PDS film were used. As biomaterials of natural origin BeriplastP, Bio-Oss and Titanmesh were investigated. The cell proliferation and cell colonization were analyzed by a proliferation assay and scanning electron microscopy. Osteoblast-like cells proliferated and attached on all biomaterials, except on Beriplast. On Ethisorb the highest cell proliferation rate was measured followed by PalacosR. Both biomaterials offer suitable growth and proliferation conditions for ovine osteoblast-like cells. The proliferation rates of Bio-Oss, MakroSorb, PDS-film and Titanmesh were low and SEM examinations of these materials showed less spread osteoblast-like cells. The results showed that ovine osteoblast-like cells appear to be sensitive to substrate composition and topography. This in vitro study provides the basis for further in vivo studies using the sheep model to examine the biocompatibility and the long-term interaction between the test material and tissue (bone regeneration).

Clinical application of tissue-engineered transplants. Part I: mucosa.

OBJECTIVES: The study series aims at testing the feasibility of the clinical application of tissue-engineered oral mucosa. The preliminary results were gathered over a period varying from 6 months to 12 years depending on the surgical method. METHODS: Tissue-engineered oral mucosa was used to cover defects in various surgical procedures like vestibuloplasty (n=42), freeing of the tongue (n=10), prelaminating the radial flap (n=5) and reconstruction of the urethra (n=16). In all interventions small samples of oral mucosa were harvested, cut into small pieces, resuspended in culture medium and seeded into a culture flask. Cultured keratinocytes were transferred onto membranes which then were used to cover mucosal defects in the oral cavity. RESULTS: To gain a graft of 15 cm(2) size a mucosa biopsy of 4-8 mm(2) and 40 ml autologous patients serum is needed. Tissue-engineered oral mucosa was applied successfully in all four surgical methods. Six months after transplantation a regular epithelial layering with a histological delimitation of the stratum, epithelial crest and a strong basal membrane appeared. According to the reception site the tissue engineered oral mucosa differentiated in several ways. CONCLUSION: Tissue-engineered oral mucosa fulfils the requirements for clinical routine. With view to healing time and outcome it does not appear to be superior to regular harvested oral mucosa transplants. Because of a smaller harvesting defect and primary wound closure at the actual operation site the patients' convenience is increased. Thus this method reduces morbidity and advances the quality of life.

Influence of iron restriction on Chlamydia pneumoniae and C. trachomatis.

Iron is an essential metabolite for pathogenic bacteria, and the specificity exhibited by bacteria for host-iron chelates may be correlated with host and tissue tropism. The effect of iron restriction on Chlamydia pneumoniae and C. trachomatis was studied by use of the iron-chelating compound deferoxamine. Growth of C. pneumoniae was inhibited much more than that of C. trachomatis and the effect of iron restriction largely depended on the cell line used for propagation. This might reflect differences in tissue tropism of the two chlamydial species. As iron levels are usually higher in men than in women, this might also be connected with the higher prevalence rate of C. pneumoniae antibodies in males, observed in all populations studied so far.