Osteogenic stimulation of osteoprogenitors by putamen ovi peptides and hyaluronic acid.

Eggshell peptides (EP) majorly contribute to rapid bone building in chicks, wherefore this paper investigated their potential for stimulating osteogenesis in vitro. In this study, the effects of EP, also called putamen ovi peptides and a combination of hyaluronic acid with EP in cell culture medium were tested towards proliferation, differentiation, gene expression and mineralization of bovine osteoprogenitors and primary human osteoblasts. The influence of EP at concentrations of 0.005 g/L, 0.5 g/L and 0.5 g/L with 0.25% hyaluronic acid was analyzed using immunocytochemical staining of bone-specific matrix proteins, namely collagen type I, osteonectin, osteopontin and osteocalcin, to prove osteoblastic differentiation. Additionally, Richardson-staining was performed. All tests revealed a superior osteoblastic differentiation with EP at 0.5 g/L after 5 days of cultivation. Hyaluronic acid alone showed controversial results and partially constrained osteoblastic differentiation in combination with EP to a level as low as for pure EP at 0.005 g/L. Of particular interest is the osteoblast-typical mineralization, as an important indicator of bone formation, which was measured indirectly via the calcium concentration after cultivation over 4 weeks. The mineralization showed an increase by a factor of 286 during the cultivation of primary human osteoblasts with hyaluronic acid and EP. Meanwhile, cell cultures treated with EP (0.5 g/L) only showed an 80-fold increase in calcium concentration.The influence of EP (0.5 g/L) on primary human osteoblasts was investigated by gene expression after 2 weeks of cultivation. Microarray and qRT-PCR analysis showed a strongly increased expression of main important genes in bone formation, bone regeneration and the physiological bone remodelling processes. Namely, BMP 2, osteopontin and the matrix metalloproteinases 1 and 9, were present during in vitro osteoprogenitor culture with EP. By explicitly underlining the potential of eggshell peptides for stimulating osteogenesis, as well as emphasizing complex and controversial interaction with hyaluronan, this manuscript is relevant for developing new functionalized biomaterials for bone regeneration.

Efficacy of Endodontic Disinfection Protocols in an E. faecalis Biofilm Model-Using DAPI Staining and SEM.

The aim of this study was to investigate the antimicrobial efficacy of different disinfection protocols in a novel Enterococcus faecalis biofilm model based on a visualization method and to evaluate the potential alteration of dentinal surface. A total of 120 extracted human premolars were allocated to 6 groups with different irrigation protocols. The assessment of the effectiveness of each protocol and the alteration of dentinal surface were visualized by using SEM and fluorescence microscopy (DAPI). A dense E. faecalis biofilm with a penetration depth of 289 µm (medial part of the root canal) and 93 µm (apical part) validated that the biofilm model had been successfully implemented. A significant difference between the 3% NaOCl groups and all the other groups in both observed parts of the root canal (p < 0.05) was detected. However, the SEM analysis revealed that the dentinal surface in the 3% NaOCl groups was severely altered. The established biofilm model and the visualization method based on DAPI are appropriate for bacterial quantification and evaluation of the depth effect of different disinfection protocols in the root canal system. The combination of 3% NaOCl with 20% EDTA or MTAD with PUI allows the decontamination of deeper dentine zones within the root canal but simultaneously alters the dentinal surface.

Is it really penetration? Part 2. Locomotion of Enterococcus faecalis cells within dentinal tubules of bovine teeth.

OBJECTIVE: The aim of the present vitro study was to examine the question whether devitalized Enterococcus faecalis (E. faecalis) cells can migrate into dentinal tubules and if that process takes place in a time-dependent manner. DESIGN: Sixty bovine root canals were incubated with devitalized and vital streptomycin-resistant E. faecalis strains after root canal enlargement (size 80, taper .02) with 3% NaOCl solution. Incubation times 7, 14, 21, 28, 35, and 42 days. Samples were processed for analysis by scanning electron microscopy (SEM) and DAPI (4',6-diamidino-2-phenylindole) staining. The penetration depth was calculated with the measurement tool of the Axio Vision program (Zeiss, Jena, Germany). Statistical analysis was performed by Kruskal-Wallis (α = 0.05) and Mann-Whitney U test (p < 0.05). RESULTS: Devitalized E. faecalis strains were able to migrate into dentinal tubules. The total number and penetration depth of devitalized E. faecalis cells was lower compared to the vital suspension of E. faecalis. It was noted, that bacterial penetration was not common to all of the dentinal tubules in the vital E. faecalis control and especially in the devitalized control. The migration took place in a time-dependent migration characteristic. CONCLUSIONS: Devitalized E. faecalis cells are still able to migrate into the dentinal tubules due to possible electrokinetic and osmotic processes. Thereby, increased exposure times lead to a time-dependent penetration characteristic. CLINICAL RELEVANCE: Since devitalized bacteria can migrate as well into dentinal tubules, the presence of bacteria within dentinal tubules cannot be interpreted as a failure of tested preparation regimens.

One step 3D printing of surface functionalized composite scaffolds for tissue engineering applications.

their surface properties. A main challenge in this area is the development of processing routes enabling for a simple but efficient surface design of complex shaped geometries. Against this background, this work aimed at the implementation of self-assembly principles for surface functionalization of 3D-printed poly(lactic-co-glycolic acid) (PLGA)-based constructs with macro- and microporous geometries via precision extruding deposition. METHODS: Three-component melts from PLGA, CaCO3 and amphiphilic polymers (poly(2-oxazoline) block copolymer) were printed and their bulk and surface properties were studied. RESULTS: Melts with up to 30 mass % of CaCO3 could be successfully printed with homogeneously distributed mineral particles. PLGA degradation during the printing process was temperature and time dependent: the molecular weight reached 10 to 15% of the initial values after ca. 120 min of heat exposure. Filament surfaces from melts containing CaCO3 show an increasing microroughness along with increasing CaCO3 content. Surface roughness and amphiphilic polymer content improve scaffold wettability with both factors showing synergistic effects. The CaCO3 content of the melts affected the inner filament structure during in vitro degradation in PBS, resulting in a homogeneous mineral particle-associated microporosity for mineral contents of 20 mass % and above. CONCLUSIONS: These results provide novel insights into the behavior of three-component melts from PLGA, CaCO3 and amphiphilic polymers during precision extruding deposition and show for the first time that self-assembly processes can be used to tailor scaffolds surface properties under such processing conditions.

Odontoblast-like differentiation and mineral formation of pulpsphere derived cells on human root canal dentin in vitro.

BACKGROUND: The revitalization or regeneration of the dental pulp is a preferable goal in current endodontic research. In this study, human dental pulp cell (DPC) spheres were applied to human root canal samples to evaluate their potential adoption for physiological tissue-like regeneration of the dental root canal by odontoblastic differentiation as well as cell-induced mineral formation. METHODS: DPC were cultivated into three-dimensional cell spheres and seeded on human root canal specimens. The evaluation of sphere formation, tissue-like behavior and differentiation as well as mineral formation of the cells was carried out with the aid of optical light microscopy, immunohistochemical staining and scanning electron microscopy (SEM). RESULTS: Spheres and cells migrated out of the spheres showed an intense cell-cell- and cell-dentin-contact with the formation of extra cellular matrix. In addition, the ingrowth of cell processes into dentinal tubules and the interaction of cell processes with the tubule walls were detected by SEM-imaging. Immunohistochemical staining of the odontoblast specific matrix proteins, dentin matrix protein-1, and dentin sialoprotein revealed an odontoblast-like cell differentiation in contact with the dentin surface. This differentiation was confirmed by SEM-imaging of cells with an odontoblast specific phenotype and cell induced mineral formation. CONCLUSIONS: The results of the present study reveal the high potential of pulp cells organized in spheres for dental tissue engineering. The odontoblast-like differentiation and the cell induced mineral formation display the possibility of a complete or partial "dentinal filling" of the root canal and the opportunity to combine this method with other current strategies.

Is it really penetration? Locomotion of devitalized Enterococcus faecalis cells within dentinal tubules of bovine teeth.

OBJECTIVE: The aim of the present study was to evaluate the penetration characteristics of devitalized and vital E. faecalis cells into root dentinal tubules. DESIGN: Thirteen root canals were incubated with devitalized (4days, 7days, 14days, 28days) and vital (28days) E. faecalis strains (streptomycin-resistant strains) after root canal enlargement (size 80, taper 0.02) with 3 % NaOCl solution. The smear layer was intentionally removed with 20 % EDTA before inoculation. Samples were processed for analysis by scanning electron microscopy (SEM) and DAPI (4',6-diamidino-2-phenylindole) staining. DAPI was conducted for fluorescence microscopic visualization of the bacterial penetration into dentinal tubules. The penetration depth was calculated with the measurement tool of the Axio Vision program (Zeiss, Jena, Germany). RESULTS: Devitalized E. faecalis strains were able to penetrate into dentinal tubules of the root canal. Apikal penetration depths of the devitalized cells were 100.67µm±26.54µm after 7days, 230.67µm±111.5µm after 14days and 266.5µm±92.63µm after 28days of incubation. The total number and penetration depth of E. faecalis cells was lower compared to a vital suspension of E. faecalis (1002.45µm) after 28days. It was noted that bacterial penetration was not common to all of the dentinal tubules in the vital E. faecalis control and especially in the devitalized control. CONCLUSIONS: Increased exposure times of devitalized bacteria into root canals lead to an increased number of penetrated dentinal tubules as well as to a deeper penetration.

Development and evaluation of an internet-based blended-learning module in biomedicine for university applicants--Education as a challenge for the future.

BACKGROUND: Biomedical science, especially biomaterials, is an expanding field in medicine. Universities are being challenged to gain the best students for a later academic career. Pre-university assessment of pupils has become crucial to reach this aim. Blended learning is an emerging paradigm for science education even though it has not yet been rigorously assessed, especially in the pupil/undergraduate situation. The aim of the study was to develop and preliminarily test a blended-learning system in biomedicine for university applicants. METHODS: An internet-based blended-learning module in material science was developed in close collaboration between a university (Biomaterials Department, Dresden TU), a German Gymnasium and an internationally oriented medical college (IMC®, Münster). Forty pre-university students were taught by this learning module composed of school education and internet-based knowledge transfer and involved in the evaluation of the utility of this learning tool. Finally, the students took first-year university examinations in order to evaluate the success of this kind of education. RESULTS: The internet-based blended-learning module as a combination of e-learning tutorials and live online lectures which was applied in phase 3 of this study was developed on the basis of the findings of both pre-university studies. The results of the learning behavior regarding the number of invokes and the dwell time of the individual pages of the pre-university learning material, the results of the online evaluation and the results of the pre-phase examination were successively used to optimize the next phase. At the end of the pre-university learning, seven of eight participants were able to pass the first-year university examination followed by nationally accepted credit award. CONCLUSION: Internet-based blended-learning module proved to be suitable to prepare students for biomedical university education while also giving them the possibility to assess their qualifications for studying biomedicine and subsequent scientific careers. Moreover, the module can help universities to find the best students.

Eggshells as natural calcium carbonate source in combination with hyaluronan as beneficial additives for bone graft materials, an in vitro study.

INTRODUCTION: In bone metabolism and the formation especially in bone substitution, calcium as basic module is of high importance. Different studies have shown that the use of eggshells as a bone substitute material is a promising and inexpensive alternative. In this in vitro study, the effects of eggshell granulate and calcium carbonate towards primary bovine osteoblasts were investigated. Hyaluronan (HA) was used as artificial extracellular matrix (ECM) for the used cells to facilitate proliferation and differentiation and to mimic the physiological requirements given by the egg in vivo. METHODS: Hyaluronan, eggshells, a combination of hyaluronan and eggshells and CaCO3 were applied to the cells as additive to the used standard medium (modified High Growth Enhancement Medium) in a concentration of 0,1 g/l. The effect of the additives in the culture medium was examined by proliferation tests, immunohistochemical staining (anti-collagen type I, anti-osteopontin, anti-osteonectin and anti-osteocalcin) and kinetic oxygen measurements. RESULTS: Our investigations revealed that all investigated additives show beneficial effect on osteoblast activity. Cell proliferation, differentiation and the metabolic activity of the differentiated cells could be influenced positively. Especially in the case cell cultures treated with eggshells the strongest effects were detected, while for the hyaluronan compared with eggshells, a weaker increase in cell activity was observed. CONCLUSION: In summary, it can be stated that the investigated components come into consideration as beneficial supplements for bone graft materials especially for maxillo facial surgery application.

Dentin-like tissue formation and biomineralization by multicellular human pulp cell spheres in vitro.

INTRODUCTION: Maintaining or regenerating a vital pulp is a preferable goal in current endodontic research. In this study, human dental pulp cell aggregates (spheres) were applied onto bovine and human root canal models to evaluate their potential use as pre-differentiated tissue units for dental pulp tissue regeneration. METHODS: Human dental pulp cells (DPC) were derived from wisdom teeth, cultivated into three-dimensional cell spheres and seeded onto bovine and into human root canals. Sphere formation, tissue-like and mineralization properties as well as growth behavior of cells on dentin structure were evaluated by light microscopy (LM), confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX). RESULTS: Spheres and outgrown cells showed tissue-like properties, the ability to merge with other cell spheres and extra cellular matrix formation; CLSM investigation revealed a dense network of actin and focal adhesion contacts (FAC) inside the spheres and a pronounced actin structure of cells outgrown from the spheres. A dentin-structure-orientated migration of the cells was shown by SEM investigation. Besides the direct extension of the cells into dentinal tubules, the coverage of the tubular walls with cell matrix was detected. Moreover, an emulation of dentin-like structures with tubuli-like and biomineral formation was detected by SEM- and EDX-investigation. CONCLUSIONS: The results of the present study show tissue-like behavior, the replication of tubular structures and the mineralization of human dental pulp spheres when colonized on root dentin. The application of cells in form of pulp spheres on root dentin reveals their beneficial potential for dental tissue regeneration.

A novel method for in vivo visualization of the microcirculation of the mandibular periosteum in rats.

OBJECTIVE: The periosteum plays an important role in bone physiology, but observation of its microcirculation is greatly limited by methodological constraints at certain anatomical locations. This study was conducted to develop a microsurgical procedure which provides access to the mandibular periosteum in rats. METHODS: Comparisons of the microcirculatory characteristics with those of the tibial periosteum were performed to confirm the functional integrity of the microvasculature. The mandibular periosteum was reached between the facial muscles and the anterior surface of the superficial masseter muscle at the external surface of the mandibular corpus; the tibial periosteum was prepared by dissecting the covering muscles at the anteromedial surface. Intravital fluorescence microscopy was used to assess the leukocyte-endothelial interactions and the RBCV in the tibial and mandibular periosteum. Both structures were also visualized through OPS and fluorescence CLSM. RESULTS: The microcirculatory variables in the mandibular periosteum proved similar to those in the tibia, indicating that no microcirculatory failure resulted from the exposure technique. CONCLUSION: This novel surgical approach provides simple access to the mandibular periosteum of the rat, offering an excellent opportunity for investigations of microcirculatory manifestations of dentoalveolar and maxillofacial diseases.

Investigation of biomaterials by human epithelial gingiva cells: an in vitro study.

INTRODUCTION: In modern medicine and dentistry the use of biomaterials is a fast developing field of increasing interest. Especially in dentistry the interaction between biomaterials like implant materials and the soft tissue in the oral cavity is in the focus of daily research. In this context the high importance of testing materials and their surfaces concerning their biocompatibility towards corresponding cells is very likely. For this purpose this study investigates cells derived from human gingival biopsies on different materials and surfaces. METHODS: Cells in this study were cultivated out of human biopsies by a grow out explant technique and were sub cultivated on titanium, zirconium dioxide and collagen membrane specimens. To characterise the cells on the material surfaces used in this study immunohistochemical and histological staining techniques as well as different methods of microscopy (light microscopy and SEM) were applied. RESULTS: With the aid of the explant technique and the chosen cell cultivation method it was possible to investigate the human gingiva derived cells on different materials. The data of the present study show that the human gingival cells attach and proliferate on all three tested materials by exhibiting characteristic gingival keratinocyte protein expression even after long periods of culture e.g. up to 70 days. CONCLUSIONS: It could be shown that the three tested materials titanium, zirconium dioxide and collagen membrane (and their special surfaces) are good candidates for the application as materials in the dental gingival environment or, in the case of the collagen membrane as scaffold/cell-carrier for human gingival cells in tissue engineering.

Computer-aided approach for customized cell-based defect reconstruction.

Computer-aided technologies like computer-aided design (CAD), computer-aided manufacturing (CAM), and a lot of other features like finite element method (FEM) have been recently employed for use in medical ways like in extracorporeal bone tissue engineering strategies. Aim of this pilot experimental study was to test whether autologous osteoblast-like cells cultured in vitro on individualized scaffolds can be used to support bone regeneration in a clinical environment. Mandibular bone defects were surgically introduced into the mandibles of Göttinger minipigs and the scaffold of the defect site was modelled by CAD/CAM techniques. From the minipigs harvested autologous bone cells from the porcine calvaria were cultivated in bioreactors. The cultured osteoblast-like cells were seeded on polylactic acid/polyglycolic acid (PLA/PGA) copolymer scaffolds being generated by rapid prototyping. The bone defects were then reconstructed by implanting these tissue-constructs into bone defects. The postoperative computerized topographic scans as well as the intraoperative sites demonstrated the accurate fit in the defect sites. The individual created, implanted scaffold constructs enriched with the porcine osteoblast-like cells were well tolerated and appeared to support bone formation, as revealed by immunohistochemical and histological analyses. The results of this investigations indicated that the in vitro expanded osteoblast-like cells spread on a resorbable individualized, computer-aided fabricated scaffold is capable of promoting the repair of bone tissue defects in vivo. The shown results warrant further attempts to combine computer modelling and tissue engineering for use in different ways in bone reconstructive surgery.