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Enamel matrix derivative (EMD), a decellularized porcine extracellular matrix (ECM), is used clinically in periodontal tissue regeneration. Amelogenin, EMD’s principal component, spontaneously assembles into nanospheresin vivo, forming an ECM complex that releases proteolytically cleaved peptides. However, the role of amelogenin or amelogenin peptides in mediating osteoblast response to EMD is not clear. Human MG63 osteoblast-like cells or normal human osteoblasts were treated with recombinant human amelogenin or a 5 kDa tyrosine-rich amelogenin peptide (TRAP) isolated from EMD and the effect on osteogenesis, local factor production, and apoptosis assessed. Treated MG63 cells increased alkaline phosphatase specific activity and levels of osteocalcin, osteoprotegerin, prostaglandin E2, and active/latent TGF-β1, an effect sensitive to the effector and concentration. Primary osteoblasts exhibited similar, but less robust, effects. TRAP-rich 5 kDa peptides yielded more mineralization than rhAmelogenin in osteoblastsin vitro. Both amelogenin and 5 kDa peptides protected MG63s from chelerythrine-induced apoptosis. The data suggest that the 5 kDa TRAP-rich sequence is an active amelogenin peptide that regulates osteoblast differentiation and local factor production and prevents osteoblast apoptosis.

Herein we describe the use of peptide linkers to establish a nucleic acid-based immobilization system based on biphasic calcium phosphates (BCP), with which different molecules can be immobilized at the same time in defined ratios. It consists of single-stranded oligonucleotides, anchor strands (AS) which are immobilized to the surface and conjugates of complementary strands (CS) and bioactive molecules that bind to the AS via Watson-Crick base pairing. AS immobilization can be achieved on calcium phosphate ceramics using conjugates of AS and peptides that bind specifically to the ceramic. We successfully immobilized three different peptide sequences on BCP. Among them, we identified Stath (DpSpS EEK FLR RIG RFG, phosphoserine) as the most suitable one and further immobilized Stath-AS conjugates on BCP. This immobilized AS was able to hybridize with CS. Unspecific adsorption of oligonucleotides on the BCP surface was negligible. The stability of the system was proven by short term desorption experiments. The amounts of immobilized peptides, oligonucleotides and peptide-AS conjugates were determined by an enzymatic assay using biotin-streptavidin interactions, and were found to reach surface densities that are of therapeutic relevance (0.03 pmol cm(-2)).

The influence of oxygen on neural stem cell proliferation, differentiation, and apoptosis is of great interest for regenerative therapies in neurodegenerative disorders, such as Parkinson's disease. These oxygen depending mechanisms have to been considered for the optimization of neural cell culture conditions. In this study, we used a cell culture system with an oxygen-permeable polytetrafluorethylene (PTFE) foil to investigate the effect of oxygen on metabolism and survival of neural cell lines in vitro. Human glial astrocytoma-derived cells (GOS-3) and rat pheochromacytoma cells (PC12) were cultured on the gas-permeable PTFE foil as well as a conventional non oxygen-permeable cell culture substrate at various oxygen concentrations. Analyses of metabolic activity, gene expression of apoptotic grade, and dopamine synthesis were performed. Under low oxygen partial pressure (2%, 5%) the anaerobic metabolism and apoptotic rate of cultured cells is diminished on PTFE foil when compared with the conventional culture dishes. In contrast, under higher oxygen atmosphere (21%) the number of apoptotic cells on the PTFE foil was enhanced. This culture model demonstrates a suitable model for the improvement of oxygen dependent metabolism under low oxygen conditions as well as for induction of oxidative stress by high oxygen atmosphere without supplementation of neurotoxins. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010

Polyethylene glycol hydrogels (PEG) have been used as slow release carrier for osteoinductive growth factors in order to achieve a retarded delivery. However, there have been concerns about negative effects on bone regeneration. This study aims to test whether PEG hydrogels themselves affect new bone formation (NBF), when used as a carrier during mandibular augmentation procedures. In a randomized split-mouth design, bilateral mandibular bone defects were surgically created in 12 Goettingen minipigs, and subsequently augmented, using PEG hydrogel on one side of the mandible. The contralateral sides, without PEG, served as controls. After 4 and 12 weeks, bone formation was evaluated in six animals each. A comparison of the data, using a three-way analysis of variance (ANOVA), revealed a significant effect of the healing time and the region of the graft on the distribution and enhancement of NBF (P

Aim To test the hypothesis that a synthetic hydroxyapatite/β-tricalcium phosphate (HA/TCP) construct combined with polyethylene glycol (PEG) hydrogel including recombinant human bone morphogenetic proteins-2 (rhBMP-2) enhances new bone formation compared with bone morphogenetic proteins-2 (BMP-2) delivered using the HA/TCP construct alone. Material and Methods Bilateral mandibular partial thickness 20 × 8 × 8 mm (L × W × H) alveolar defects were surgically created in the edentulated posterior mandible in 18 female minipigs. Randomized into two groups of nine animals each, the alveolar defects either received HA/TCP or HA/TCP/PEG with or without BMP-2 (105 μg/defect) in contra-lateral sites using a split-mouth design. Primary outcome, bone density (%) within four regions of interest, was evaluated following a 4-week healing interval when the animals were killed for histometric analysis. Results Bone morphogenetic proteins-2 loaded onto HA/TCP constructs significantly enhanced new bone formation compared with HA/TCP controls. Adding PEG apparently obstructed BMP-2 induced bone formation. Conclusion Polyethylene glycol compromises the osteogenic effect of BMP-2.

This in vitro study examined (a) the anti-bacterial efficacy of a pulsed erbium-doped yttrium aluminum garnet (Er:YAG) laser applied to Streptococcus sanguinis or Porphyromonas gingivalis adhered to either polished or microstructured titanium implant surfaces, (b) the response of osteoblast-like cells and (c) adhesion of oral bacteria to titanium surfaces after laser irradiation. Thereto, (a) bacteria adhered to titanium disks were irradiated with a pulsed Er:YAG laser (λ = 2,940 nm) at two different power settings: a lower mode (12.74 J/cm(2) calculated energy density) and a higher mode (63.69 J/cm(2)). (b) After laser irradiation with both settings of sterile titanium, disks were seeded with 10(4) MG-63 cells/cm(2). Adhesion and proliferation were determined after 1, 4, and 24 h by fluorescence microscopy and scanning electron microscopy. (c) Bacterial adhesion was also studied on irradiated (test) and non-irradiated (control) surfaces. Adhered P. gingivalis were effectively killed, even at the lower laser setting, independent of the material's surface. S. sanguinis cells adhered were effectively killed only at the higher setting of 63.69 J/cm(2). Laser irradiation of titanium surfaces had no significant effects on (b) adhesion or proliferation of osteoblast-like MG-63 cells or (c) adhesion of both oral bacterial species in comparison to untreated surfaces. An effective decontamination of polished and rough titanium implant surfaces with a Er:YAG laser could only be achieved with a fluence of 63.69 J/cm(2). Even though this setting may lead to certain surface alterations, no significant adverse effect on subsequent colonization and proliferation of MG-63 cells or increased bacterial adhesion was found in comparison to untreated control surfaces.

For clinicians, soft connective tissue integration (STI), one of the critical issues for dental implant success, is usually tested using the fibroblasts monolayer regime. Therefore, we aimed at an extension of this regime by employing interactive gingival fibroblast-keratinocyte cocultures (CCs) as an in vivo-like test platform. In the extended regime, 13 STI-relevant genes were analyzed in response to five different titanium implant biomaterial surfaces. The genes quantitated by real-time polymerase chain reaction were categorized as pro supportive or contra supportive, that is, nonsupportive for cell growth on an engineered surface. Monocultures had higher levels of contra supportive gene expression, but the fibroblast-keratinocyte CC had two out of five of the titanium surfaces with more pro supportive gene expression than contra supportive gene expression. We defined this change from contra supportive gene expression to pro supportive gene expression by developing the "relative supportive difference" index. Hence, interactive CCs exhibit valuable supportive effects on the expression of STI-relevant genes, possibly via physiological cell-to-cell-interactions. Our results render interactive gingival CCs suitable as a test platform for dental implant-related STI under more in vivo-like conditions.

Dental implants are prone to bacterial colonization which may result in bone destruction and implant loss. Treatments of peri-implant disease aim to reduce bacterial adherence while leaving the implant surface intact for attachment of bone-regenerating host cells. The aims of this study were to investigate the antimicrobial efficacy of gaseous ozone on bacteria adhered to various titanium and zirconia surfaces and to evaluate adhesion of osteoblast-like MG-63 cells to ozone-treated surfaces. Saliva-coated titanium (SLA and polished) and zirconia (acid etched and polished) disks served as substrates for the adherence of Streptococcus sanguinis DSM20068 and Porphyromonas gingivalis ATCC33277. The test specimens were treated with gaseous ozone (140 ppm; 33 mL/s) for 6 and 24 s. Bacteria were resuspended using ultrasonication, serially diluted and cultured. MG-63 cell adhesion was analyzed with reference to cell attachment, morphology, spreading, and proliferation. Surface topography as well as cell morphology of the test specimens were inspected by SEM. The highest bacterial adherence was found on titanium SLA whereas the other surfaces revealed 50–75% less adherent bacteria. P. gingivalis was eliminated by ozone from all surfaces within 24 s to below the detection limit (≥99.94% reduction). S. sanguinis was more resistant and showed the highest reduction on zirconia substrates (>90% reduction). Ozone treatment did not affect the surface structures of the test specimens and did not influence osteoblastic cell adhesion and proliferation negatively. Titanium (polished) and zirconia (acid etched and polished) had a lower colonization potential and may be suitable material for implant abutments. Gaseous ozone showed selective efficacy to reduce adherent bacteria on titanium and zirconia without affecting adhesion and proliferation of osteoblastic cells. This in vitro study may provide a solid basis for clinical studies on gaseous ozone treatment of peri-implantitis and revealed an essential base for sufficient tissue regeneration.

Nowadays, intervertebral disc (IVD) degeneration is one of the principal causes of low back pain involving high expense within the health care system. The long-term goal is the development of a medical treatment modality focused on a more biological regeneration of the inner nucleus pulposus (NP). Hence, interest in the endoscopic implantation of an injectable material took center stage in the recent past. We report on the development of a novel polyurethane (PU) scaffold as a mechanically stable carrier system for the reimplantation of expanded autologous IVD-derived cells (disc cells) to stimulate regenerative processes and restore the chondrocyte-like tissue within the NP. Primary human disc cells were seeded into newly developed PU spheroids which were subsequently encapsulated in fibrin hydrogel. The study aims to analyze adhesion properties, proliferation capacity and phenotypic characterization of these cells. Polymerase chain reaction was carried out to detect the expression of genes specifically expressed by native IVD cells. Biochemical analyses showed an increased DNA content, and a progressive enhancement of total collagen and glycosaminoglycans (GAG) was observed during cell culture. The results suggest the synthesis of an appropriate extracellular matrix as well as a stable mRNA expression of chondrogenic and/or NP specific markers. In conclusion, the data presented indicate an alternative medical approach to current treatment options of degenerated IVD tissue.

Glass-ceramics featuring special properties can be used as a basis to develop biomaterials. It is generally differentiated between highly durable biomaterials for restorative dental applications and bioactive glass-ceramics for medical use, for example, bone replacements. In detail, this paper presents one biomaterial from each of these two groups of materials. In respect to the restorative dental biomaterials, the authors give an overview of the most important glass-ceramics for clinical applications. Leucite, leucite-apatite, lithium disilicate and apatite containing glass-ceramics represent biomaterials for these applications. In detail, the authors report on nucleation and crystallization mechanisms and properties of leucite-apatite glass-ceramics. The mechanism of apatite nucleation is characterized by a heterogeneous process. Primary crystal phases of alpha - and beta -NaCaPO4 were determined. Rhenanite glass-ceramics represent biomaterials with high surface reactivity in simulated body fluid, SBF, and exhibit reactive behaviour in tests with bone cells. Cell adhesion phenomena and cell growth were observed. Suitable colonization and proliferation and differentiation of cells as a preliminary stage in the development of a material for bone regeneration applications was established. The authors conclude that the processes of heterogeneous nucleation and crystallization are important for controlling the required reactions in both biomaterial groups.

In recent years, enamel matrix derivative (EMD) has garnered much interest in the dental field for its apparent bioactivity that stimulates regeneration of periodontal tissues including periodontal ligament, cementum and alveolar bone. Despite its widespread use, the underlying cellular mechanisms remain unclear and an understanding of its biological interactions could identify new strategies for tissue engineering. Previous in vitro research has demonstrated that EMD promotes premature osteoblast clustering at early time points. The aim of the present study was to evaluate the influence of cell clustering on vital osteoblast cell-cell communication and adhesion molecules, connexin 43 (cx43) and N-cadherin (N-cad) as assessed by immunofluorescence imaging, real-time PCR and Western blot analysis. In addition, differentiation markers of osteoblasts were quantified using alkaline phosphatase, osteocalcin and von Kossa staining. EMD significantly increased the expression of connexin 43 and N-cadherin at early time points ranging from 2 to 5 days. Protein expression was localized to cell membranes when compared to control groups. Alkaline phosphatase activity was also significantly increased on EMD-coated samples at 3, 5 and 7 days post seeding. Interestingly, higher activity was localized to cell cluster regions. There was a 3 fold increase in osteocalcin and bone sialoprotein mRNA levels for osteoblasts cultured on EMD-coated culture dishes. Moreover, EMD significantly increased extracellular mineral deposition in cell clusters as assessed through von Kossa staining at 5, 7, 10 and 14 days post seeding. We conclude that EMD up-regulates the expression of vital osteoblast cell-cell communication and adhesion molecules, which enhances the differentiation and mineralization activity of osteoblasts. These findings provide further support for the clinical evidence that EMD increases the speed and quality of new bone formation in vivo.