Abstract
Oral Abstracts
Differential Release of Human Soluble Receptors and Related Proteins in Mineralizing Versus Non-Mineralizing Jaw Periosteal Cells
In the present study, the expression of a multitude of 119 soluble receptors and related proteins in undifferentiated and differentiating mJPCs versus nmJPCs was analysed. Mineralization capacity in mJPCs versus nmJPCs was detected by Alizarin and Von Kossa stainings. Expression pattern analysis was carried out using the human soluble receptor and related protein array of the non-hematopoietic panel (R&D Systems). Culture supernatants from undifferentiated and differentiating mJPCs and nmJPCs from day 10 and 20 of osteogenesis were used. Preliminary data revealed a differential integrin expression profile in nmJPCs vs. mJPCs. Whereas undifferentiated nmJPCs expressed higher levels of integrin ß1-ß6, the expression of all ß integrins with the exception of ITGB1 were reduced during osteogenic culturing. mJPCs expressed lower integrin ß levels, however they were inducible during osteogenesis. In contrast, higher basal expressions of integrin α3, α5, αV, α6, α9 were detected in mJPCs. Junctional adhesion molecules such as JAM-A was higher in nmJPCs, but downregulated during osteogenic culturing and expression of JAM-B and −C and desmoglein-2 was higher in mJPCs and additionally induced during osteogenesis. Among other cell-cell adhesion molecules, cadherins (cadherin-4, -11, -13, E- and N-cadherin) and clusterin expressions were clearly upregulated and induced during osteogenic differentiation in undifferentiated mJPCs in contrast to nmJPCs. Proteome arrays are suitable for the examination of the complex microenvironment which leads to osteogenesis.
Repair of Critical Bone Defect in a Rabbit Model by Autologous Adipose-Derived Stem Cells
Rabbit adipose-derived stem cells (ASCs) were isolated from interscapular adipose tissue, expanded in vitro and used for regeneration of full-thickness bone defects in the tibial crest of New Zealand rabbits. The animals have been divided in 4 treatment groups: sham, disk of hydroxyapatite (HA), ASCs, and ASCs seeded on HA-disk. Meanwhile the ASCs have been in vitro characterized.
Eight weeks after surgical interventions, gross appearance, X-ray, BMD and histological analyses were performed on all the animals of each group.
At first, each ASCs population shows a high proliferation rate, a marked clonogenic ability and osteogenic potential in the absence or in the presence of HA.
The macroscopic analyses of all the tibias show a satisfactory filling of the lesion without any significant differences in term of stiffness between groups treated with or without cells. In both scaffold-treated groups, a good osteointegration was radiographically observed. Even if HA was not completely reabsorbed, ASCs-loaded HA showed a more efficient scaffold readsorption than the unloaded disks. Hystology shows a marked osteogenic ability of the scaffold-treated defects compared to the scaffold cell-free samples, and in particular, the new formed bone was more mature and similar to native bone in presence of scaffold + ASCs.
Tuning the Mechanical Properties of Bioreducible Multilayer Films for Improved Cell Adhesion and Transfection Activity
Based on changes in film rigidity, cell adhesion characteristics and transfection activity were investigated in vitro. LbL films consisting of reducible hyperbranched poly(amide amine) (RHB) have been implemented along with DNA for investigating fibroblast adhesion on [RHB/DNA]n/2 films with varying rigidities. Molecular force probe (MFP) measurements were performed to measure the apparent Young's modulus of the films in situ. Cell adhesion and stress-fiber characteristics were investigated using total internal reflectance microscopy (TIRF-M). The average cell peripheral area, fiber density and average fiber length during 5 days of cell growth on films with either low (below 2.0 MPa) or high (above 2.0 MPa) film elastic modulus were investigated. Transfection studies were performed using gfpDNA and SEAP-DNA to investigate if changes in cell adhesion affect transfection activity. Cell proliferation and cytotoxicity studies were used to investigate cellular viability over a week. The results showed that surface modification of bioreducible LbL films of controlled thickness promotes cellular adhesion, stress-fiber growth and increased transfection activity without the need for additional adhesive protein pre-coating of the surface or chemical cross-linking of the film. In particular, it has been found that bioreducible LbL films with an apparent elastic modulus greater than 2.0 MPa have increased cell adhesion, cell spreading and transfection activity compared to films with low modulus.
Osteogenic Differentiation of Primary Stem Cells with BMP-2 in a Rotating Bed Reactor
Human mesenchymal stem cells were cultivated in a rotating bed bioreactor system (ZRP® system, Zellwerk GmbH) on the Zirconia based macroporous ceramic Sponceram® for four weeks. The ceramic discs were loaded with PLGA microspheres releasing BMP-2. In this work human mesenchymal stem cells derived from adipose tissue were used. During the cultivation glucose and lactate concentrations were measured. After the experiments histological stainings were performed, (DAPI, von Kossa and alizarine red). The mRNA of the cells was isolated and RT-PCR was performed to investigate the expression of different bone markers. The concentration of alkaline phosphatase was measured in medium samples (Sigma fastTM, Sigma). The glucose consumption increased during the cultivation indicating a continuous cell growth. The DAPI staining of the ceramics showed a homogenous spreading of the cells on the ceramic and the histological staining showed matrix calcification. The expression of typical bone markers was shown. Osteogenic differentiation of MSC on Sponceram, effected by BMP-2 released from PLGA microspheres, was demonstrated.
Injectable Hybrid Hydrogels for Bone Regeneration
In order to mimic the mineralized matrix of bone tissue, in which collagen fibrils and mineral particles provide rigidity and biological properties, the present study aimed at the development of a hybrid bone substitute for bone regenerative treatment by combining oligo(poly(ethylene glycol) fumarate) (OPF) hydrogel with calcium phosphate (CaP) crystals. The biological performance was assessed using in vitro and in vivo models. Primary rat bone marrow stromal stem cells (MSCs) were embedded in OPF hydrogels with and without CaP crystals and the in vitro biological responses were evaluated via biochemical assays and histology. In vivo bone and soft tissue responses to hybrid hydrogels were assessed as injectable formulations in the tibial medullar cavity (Figure 1) and as preset scaffolds in the subcutaneous dorsum of a guinea pig model. Cell culture studies showed poor proliferation of embedded MSCs and limited evidence of osteogenic differentiation, although mineralization was observed. Examination of histological sections showed limited cell-cell interactions and cells maintained a round shape during culture time. Assessment of in vivo bone and subcutaneous responses are ongoing and based on microCT (Figure 2), histology and histomorphometry. OPF/CaP hydrogels promote mineralization in vitro and hence appear to provide a suitable microenvironment that resembles bone tissue heterogeneity. Injectable hydrogels increase the scope of minimally invasive procedures and offer the ability of optimal defect filling for irregularly-shaped bone defects.
Injectable Scaffold with Sustained rhBMP-2 Delivery for Bone Regeneration
An in situ solidifying scaffold was prepared from thermosensitive poly(lactic-co-glycolic) acid/poly(ethylene glycol) (PLGA/PEG) particles with a glass transition temperature of 37°C. Encapsulation of model proteins into PLGA microspheres was optimised using the double emulsion (W/O/W) and single emulsion (S/O/W) Methods. The latter method was then used to encapsulate recombinant human bone morphogenetic 2 (rhBMP2) into PLGA microspheres. rhBMP2 was first micronised with HSA as a carrier protein at different ratios and then encapsulated; maintaining the amount of HSA constant and varying the rhBMP2 amount allowed for dose alteration. The activity of micronised rhBMP2 was confirmed using both an ELISA and a C2C12 cells/Alkaline Phosphatase (ALP) biological activity assay. Longitudinal studies of rhBMP2 release kinetics are currently being carried. PLGA/PEG thermosensitive particles produced an injectable formulation that solidified at body temperature. Varying the processing parameters of the two methods of protein encapsulation improved the encapsulation efficiency (EE). Moreover, an ELISA showed that micronised rhBMP2 was active at all HSA/rhBMP2 ratios, and when compared to a positive control (rhBMP2 alone), it stimulated C2C12 cell differentiation towards a bone cell-like lineage. The micronised HSA/rhBMP2 was then encapsulated into PLGA microspheres with an EE of between 60-80%. Initial results suggest that a sustained release of rhBMP2 from these particles could be achieved.
Effect of Strontium on Human Primary Bone Cells
Human osteoblasts obtained from explants cultures were grown in presence of sol-gel hydroxyapatite (HA), Sr(strontium)-substituted HA and in a strontium chloride (SrCl2) containing medium. Viability and proliferation were assayed by trypan blue exclusion and MTS tests. Cell morphology was assayed by scanning electron microscopy and proteins production was studied by Real-Time PCR and ELISA while MMP-2 activity was assessed by zymography.
Our results reveal that strontium enhances the replication capacities of bone cells without altering their viability or morphology and modify the proteins expression. We also demonstrate an increased expression of collagen I and SERPINH1 mRNA and a reduced production of matrix metalloproteinases (MMP) −1 and −2 without modifying the tissular inhibitors of MMPs (TIMPs) levels.
Our study demonstrates for the first time that hydroxyapatite particles modify the MMPs-TIMPs balance and increase bone degradation in an osteoblastic cells model. We also demonstrated for the first time that strontium has a positive effect on bone degradation by decreasing the MMP (1 and 2) production.
Reconstruction of Femoral Epiphysis with Two Different Types of Scaffolds: Preliminary Results
The technique involves the removal of the chondromalacic and the necrotic area, followed by reconstruction of the epiphysis with bioceramic cylinders (Trufit), lyophilized bone chips soaked in stromal cells and concentrated platelet gel with coverage by scaffold engineered with autologous chondrocytes. 13 patients were treated for 14 femoral epiphysis. In the first 7 cases we used as scaffolds Hyaff-11, then we replaced with Chondro-Gide for the best mechanical properties. The follow-up provides radiographic, CT and MRI of the pelvis at 1.5, 3, 6, 12, 24 month from the surgery.
The histological analysis performed on engineered scaffolds showed that chondrocytes are viable (viability between 89 and 98%) and express typical molecules of hyaline cartilage, including collagen type 2 and aggrecan. At a median follow-up of 17,2 months, the joint space was preserved similarly in both groups with pain relief in 93% of cases. The only failure was in the group treated with Hyaff-11 and is due to associated rheumatic disease. Secondly, we have a functional limitation in 4 cases with Hyaff-11 and 3 cases with Chondro-Gide, due to hip impingement secondary to problems in reconstructing the normal spherical morphology of femoral epiphysis.
Comparison of Six Bone-Graft Substitutes: Efficiency, Metabolism and Growth Behaviour of Human Mesenchymal Stem Cells in Vitro
Tutoplast®, Cerabone®, the different tricalciumphoph-ates (ß-TCP: Chronos® und Vitoss®; a-TCP BioBase®) and a silicated hydroxylapatite (Actifuse®) were used. After seeding, the number of adherent cells were determined fluoroscopically, the metabolic activity of the cells was measured in a functional assay and the differentiation of the cells was evaluated by RT-PCR. In general, a substantial difference could be determined between the scaffolds and the cell populations. MSCs alone were found to adhere best to Tutoplast, showing a high metabolic activity and expression of osteogenic proteins cbfa-1 and osteocalcin. These results were confirmed by electron microscopy, demonstrating a fibrillar structure of Tutoplast®, thus allowing a widespread adhesion. Chronos® exhibits as single scaffold a substantial adhesion and function of MSCs as well as a nice morphology, while the other tricalciumphosphates, Actifuse® und Cerabone® were less suitable. The results in the EPC group were different. Actifuse® demonstrated a high adhesion rate, followed by Biobase®. This is confirmed in the functional MTT-assay and the mRNA-Expression of endothelium-associated proteins.
Tetracyclines as Biomodulators in Bone Tissue Regeneration
This work aims to characterize the effect of 2 semi-synthetic tetracyclines in human osteogenic in vitro systems, both in presence and absence of biomaterials. Human osteoblastic cultures were established and characterized in the presence of representative therapeutic concentrations of doxycycline (1 to 25 μg/ml) or minocycline (1 to 50 μg/ml). Furthermore, the in vitro osteogenic behavior of hydroxyapatite and a glass- reinforced hydroxyapatite (GrHA) was assayed, in the presence of both tetracyclines.
Both 1 μg/ml doxycycline and minocycline increased cell proliferation, without inducing representative alterations in cell phenotype and functional activity. Higher concentrations of both agents induced dose-dependent detrimental effects in cellular behavior. Cell function was evaluated on seeded hydroxyapatite and GrHA, in the presence of 1 μg/ml doxycycline or minocycline. Both tetracyclines induced cell behavior of established cultures - proliferation was enhanced while phenotypic characteristics were maintained. The GrHA reported an improved biological behavior, comparing to HA. Results suggest that local delivery of both tetracyclines may combine an expected local antibacterial activity with a potential anabolic effect regarding osteoblastic proliferation, both in presence and absence of biomaterials.
One-Step and Two-Step Seeding Protocols of Porcine Chondrocytes on a Three-Dimensional ß-Tricalciumphosphate Matrix
Porcine chondrocytes were isolated from the stifle joint and routinely maintained in T-flasks. All steps of the seeding process were performed with cells in passage number 0. Initially chondrocytes were harvested after 7 days of cultivation and sedimented by gravity on solid ceramic discs (ChronOs®, Synthes) consisting of pure ß-TCP in a 12-well-plate. The constructs were cultivated in medium for 15 days (one-step-protocol). Following the two-step-protocol the seeded discs were cultivated under similar conditions for 10 days. Then a second seeding step was performed using chondrocytes after 17 days of cultivation. The constructs were cultivated for another 5 days. Descriptive evaluation of cell characteristics was performed using FDA-PI-staining to assess cell viability, scanning electron microscopy to visualize the cell-matrix-interface and RT-PCR to detect collagen I and II, aggrecan and versican to analyse cell quality. Visualization of the matrix surface showed an almost closed multi-layer of cells following the two-step-protocol. High cell viability occurred with only few areas indicating apoptosis. High expression of collagen II and aggrecan in contrast to very low expression of versican and low expression of collagen I indicated the presence of hyaline-like cartilage. Following the one-step-protocol a reduced cell content with minor viability became obvious. Quality assessment showed low expression of collagen II and high expression of aggrecan. A two-step-seeding protocol may be used with advantage for generating hyaline-like cartilage on a ceramic matrix.
Primary Stability of an Osteochondral Autograft Stabilized with a Resorbable Osteoconductive Bone Cement
Biomechanic testing will be conducted on 6 pairs of cadaveric bovine femurs. For the first 3 pairs, 6 single osteochondral autografts will be inserted in a press fit fashion on one femur. On the other femur, 6 grafts will be inserted and stabilized with an osteoconductive adhesive (Kryptonite, DRG inc.). For the 3 other pairs of femurs, 4 groups of 3 adjacent grafts will be inserted in a press fit fashion on one femur whereas on the contralateral femur, those grafts will be stabilized with an osteoconductive adhesive. After a maturation period, compressive loading will be applied on the grafts. The axial load needed to sink the grafts to 1, 2 and 3mm below cartilage level will be recorded and compared using Mann-Whitney tests. Preliminary tests were done on 2 pairs of cadaveric bovine femurs. Statistically significant results were obtained at 1mm (147N vs 101N, p = 0.008), 2mm (233N vs 119N, p < 0.001) and 3mm (298N vs 185N, p = 0.003) favoring the cemented group. Our preliminary results show that single cemented grafts from bovine cadaveric specimens have a greater primary stability than similar grafts inserted in a press fit fashion.
A Novel Method for Controlling Pore Geometry, Size and Distribution in Engineered Scaffolds and Its Effects on Cell Attachment, Proliferation, and Differentiation
Poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogel discs were synthesised using dimethacrylate crosslinkers. Hydroxyapatite discs were also produced. A novel method was developed to modify the scaffolds with pores of varying sizes and distributions. Human MSCs were seeded onto the discs and cultured for up to 14 days, and the resultant cell attachment, proliferation and osteogenic differentiation were examined by measuring cell counts, Geimsa staining, RT-PCR and SEM.
A post processing method has been developed and the ability to produce polymer and ceramic samples, with engineered porosity ranging in size from sub 100 μm to an infinite size is proven. This porosity can be tailored and is reproducible, resulting in complete control of pore geometry, size and distribution with spatial separation of pores as little as 50 microns, dependent on the pore size.
Preliminary results have shown marked differences in the effect of mono modal pore sizes evenly distribution across the scaffolds. Smaller pore spacing and larger pore size in the range of 300 microns have resulted in increased proliferation of cells.
Bone Regeneration after Implantation of Nanoparticulate Hydroxyapatite and Collagen Type-1 in Osteoporotic Goats
We asked if implantation of nanoparticulate hydroxyapatite (HA) with or without collagen type-1 (col-1) will cause an increase in new bone formation. Osteoporosis was induced by bilateral ovariectomy and low-calcium diet in Chinese mountain goats. Defects were created in lumbar vertebra, femoral condyle, left and right iliac crest and filled with HA, HA/col-1 or left empty. After 42 days implanted bones were collected and used for histological, histomorphometrical, histochemical and electron microscopical analysis. Both implants were fragmented, surrounded by new formed bone and foreign body giant cells that reabsorbed the implant. Histomorphometry showed that significant more new bone was built in defects that were filled with material in correlation to empty defects. In tendency, HA implants provided more new formed bone than HA/col-1 implants. Our results showed that usage of HA and HA/col-1 implants resulted in an increased formation of new bone in correlation to empty defects. Addition of col-1 to HA did not cause any upgrade.
Biological Response of Human Osteoblasts to Ferritic Stainless Steel Surfaces
One of the most serious issues with total joint replacement operations is the failure of an implant surface to integrate with the adjacent bone cells. One solution to this problem proposed by A.E. Markaki, T.W. Clyne1 is based on the exploitation of bonded arrays of ferromagnetic fibres that when subjected to an external magnetic field are capable of imposing mechanical strains that can potentially stimulate bone cells adhesion and growth. However the use of such ferromagnetic materials needs to be evaluated in terms of their biocompatibility. Therefore the aim of the present study was to assess the response of osteoblasts to 444 ferritic stainless surfaces. A comparison is made between 316L, the non-magnetic austenitic stainless steel in common use for implants, and 444, a magnetic ferritic stainless steel. Profilometry, atomic force microscopy (AFM), scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and water contact angles techniques were used to characterize the morphology and chemistry of the surfaces. Cellular proliferation, metabolism, morphology and early osteoblastic differentiation were examined, using the CyQuant® assay, alamarBlue®, SEM imaging and an alkaline phosphatase (ALP) activity assay respectively. To quantify the effect of chemistry on cell attachment, a centrifugal field was used to detach the cells from the substrates.
The results obtained from the biological studies show that osteoblast cells respond successfully when in contact with 444 ferritic stainless steel surfaces and consequently that this material holds potential to be used in advanced bone applications.
Bone Engineering with Dental Pulp Mesenchymal Stem Cells (DPMSC): Laser Assisted Differentiation to Osteoblasts
Analyze the possible relation between 830nm GaAlAr laser radiation and the gene expression in differentiation of human dental pulp mesenchymal stem cells (DPMSC). The DPMSC were obtained according to the ethics committee of University of Sao Paulo. The cells were separated by digestion with 3mg/ml collagenase type I and 4mg/ml dispase in 4ml PBS containing 110U/ml penilillin, 100μg streptomycin for 1 hour at 37° (all from Invitrogen, CA). The solution was filtered with 70μm Falcon strainers and cultivated until sub confluent for 2 passages then cells were sorted by FACS (Becton Dickinson), San Jose, CA) with the markers CD166, CD81, CD105, CD34, CD45, CD14 and differentiated into osteoblasts using osteogenic medium composed of αMEM with 10% Fetal Bovine Serum, 10mM β-glicerophosphate, 10nM dexamethasone and 50 μM ascorbic acid-2-phosphate, cell viability tests were performed with MTT. The cells were divided into two groups: one of the groups (group GL) received 3J/cm2 low level laser radiation (830 nm/80mW) every two days from the fourth day until 22° day and the second group (group GC) was a control group that didn't received laser radiation. RT-PCR and PCR were performed to detect any variation between the two groups using primers of genes linked to bone lineage initial differentiation: runx-2 and Stro-1. The results showed variation between the two groups with expression of the gene runx-2 in the group GL before the group GC. Laser radiation can have relation with the velocity of the differentiation of DPMSC.
Effects of Electron-Beam Irradiation to the Hydroxyapatite and Tricalcium Phosphate Mixtures for the Development of New Synthetic Bone Substitutes
The aim of this study is to evaluate the effect and potential of electron beam irradiation treatment to new bone formation and healing in rat calvarial bone defects using hydroxyapatite (HA, Bongros®, Bio@ Co., KOREA) and tricalcium phosphate (β-TCP, Sigma-Aldrich Co., USA) mixed at various ratios. We used 1.0-2.0 MeV linear accelerator and 2.0 MeV superconductive linear accelerator (power 100KW, pressure 115kPa, temperature −30∼120°C, sensor sensitivity 0.1∼1.2mV/kPa, generating power sensitivity 44.75mV/kPa, supply voltage 5 ± 0.25V) with different irradiation dose such as 1, 30, 60 kGy. Structural changes in this alloplast bone material were studied in vitro, by SEM (Scanning Electron Microscopy), elementary and FE-SEM (Field Emission Scanning Electron Microscope), ATR (Attenuated Total Reflection), and CRS (Confocal Raman Spectrometer).
After sterilization with ethylene oxide, we used it as a bone graft material. Bilateral, standardized transosseous circular calvarial defects, 5.0 mm in diameter, were created in male Sprague-Dawley rats. In each experimental group, the defect was filled with HA and TCP at various ratio with type I collagen. Rats were sacrificed 2, 4 and 16 weeks post-op for radiographic (standardized plain film, Kodak Co., USA), histomorphologic (Hematoxylin and Eosin, Masson Trichrome), immunohistochemical staining (for BMP-2, -4, VEGF, and vWF), TEM (Transmission Electron Microscopy), and elementary analysis (Atomic absorption spectrophotometer, Perkin Elmer Analyst 100®). Electron beam irradiation to the artificial bone mixtures increased C, O components according to the energy amounts, and Na, Mg, Al, and P components were also present and increased slightly. Ca component was also increased, P component was kept continuously, and the ratio of P component was increased comparatively under 1 MeV electron beam irradiation. After more than 1 MeV- 60KGy electron beam irradiation, generalized bony segment was immaturely mixed under 1,000 magnifications of SEM, and definite fused bony mixtures were found under 10,000 magnifications of SEM and TEM. In conclusion, the ratio of 7 : 3 mixtures of HA and TCP after 16 weeks of rat calvarial defect model, showed very good bony regeneration ability having much endothelial cells and pericytes with lamellar bony surface texture.
Human Osteoblasts and Their Progenitors: Discovery of New Molecular Targets and Support Biomaterials for Bone Tissue Regeneration
Mesenchymal stem cells from umbilical cord (WJMSCs) and periodontal ligament (PDLs), were isolated from normal donors, cultured and differentiated towards osteoblast lineage. The characterization of their phenotype together with mature osteoblasts (hOBs) were performed by flow cytometric analysis, immunohystochemistry, and gene expression analysis. Alamar blue, MTT and Calcein/Propidium Iodide were used as viability and proliferation assays. The microencapsulation in alginate was carried out by a vibrating nozzle device. The cellular secretive profiles were obtained by Bio-plex technology. Chromatin Immunoprecipitation (ChIP) was used to detect the “in vivo” recruitment of transcriptional regulators on target gene promoters. We isolated viable, highly proliferating WJMSCs and PDLs displaying MSC-phenotype, and able to become mature osteoblasts. The cells were encapsulated in barium-alginate microbeads and maintened their ostogenic inducibility (alkaline phosphatase, RUNX2, osteocalcin mRNA expression and bone matrix deposition). In addition, a specific gene expression analysis carried out in hOBs and hMSCs, using a knockdown approach, demonstrated for the first time the role of Slug protein in the mineralization process.
Polychrome Sequence Labeling for the Investigation of Bone Remodeling Activity of Different Magnesium Alloys
Extruded pins (25 mm x 2.5 mm) of the magnesium alloys LAE442, MgCa 0.8 and fluoride coated MgCa 0.8 were implanted intramedullary into both tibiae of five rabbits each group. Three tibiae without implants served as control. Polychrome sequence labeling was performed during the observation period of six months. After histological sectioning the distances between the flourescent bar marks were measured and a calculation of the mineral apposition rate (MAR) at six different time periods was performed. In all groups with Mg-alloys, the MAR was highest in the first time period (day 4 to 31) and decreased to the end of the observation period of six months. In the first period, the bone remodelling in the fluorid-coated MgCa 0.8-alloys (MAR: 6.6 μm/d) was higher than in the MgCa 0.8 (MAR: 4.29 μm/d), LAE442 (MAR: 3.36 μm/d) and control group (MAR: 0.88 μm/d). The remodeled bone distance during six month was increased in flouride-coated MgCa 0.8 alloys (448 μm ± 33 μm) in comparison to the other alloys MgCa 0.8 (353 μm ± 143 μm) and LAE442 (284 μm ± 94 μm). With polychrome sequence labeling the increased bone remodeling activity of three different Mg-alloys in comparison to a control group could be differentiated. LAE442-alloys with lowest increase assume a better biocompatibility than the other examined Mg-alloys.
Osteoblast Mechanical Stimulation by Converse Piezoelectric Effect
The polymeric piezoelectric films used (PVDF) had an active area of 12x13 mm2, with silver ink electrodes. Film coating and sterilization processes are described. The amount of the displacement and its distribution along the piezoelectric surface was studied using Electronic Speckle Pattern Interferometry (ESPI). MCT3T3-E1 cells were cultured under standard conditions and on the surface of films, subjected to static and dynamic conditions. In dynamic conditions the substrates were deformed by applying a 5 V current, at 1Hz and 3Hz for 15 minutes. Cell viability and proliferation were addressed; nitric oxide was measured in medium after stimulation and normalized to protein content, 24 and 48 hours after seeding. Immunofluorescence studies were conducted on the cytoskeleton.
Cells under dynamic stimulation have shown significantly higher levels of nitric oxide and changes on cytoskeleton organization, although no significant differences were found between dynamic and static groups' viability. The ESPI studies showed a maximum displacement on the substrate of 0.6 μm. The present study has shown that MC3T3 osteoblast cells are able to sense and respond to minimal displacements of the substrate in a reproducible manner.
Evaluation of Degradation Behaviour and Biocompatibility of the Magnesium Alloy Zek100 by Use of In-Vivo Micro Computed Tomography
Pins of ZEK100 were implanted intramedullary into the tibiae of 20 rabbits. During the implantation periods, in-vivo microCT scans of the tibiae were performed regularly. As degradation parameters, volume and density of the implants were determined. Furthermore, pin diameters and their standard deviations characterized heterogeneity of corrosion. To quantify bone changes, volume, density and porosity of the tibia diaphysis were calculated. The volume of the implants decreased from 122.48 to 19 mm3 after 48 weeks. From week 20, the loss of volume was statistically significant. The pin diameter decreased from 2.3 to 0.83 mm although their standard deviations increased. The implants showed a significant loss of density during implantation. The bone volume around the implant increased significantly from 1.48 to 2.55 mm3/slice. However, a decrease of the bone density from 1207.6 to 1139.8 mgHA/ccm could be found during implantation. Additionally, the bone porosity rose from 4.49 to 6 %.
Magnetic Biomimetic Scaffold for a Controlled Spatial Distribution of Cells Saturated with Magnetic Nanoparticles
Human bone marrow mesenchymal stem cells (hBMSCs) were stained by carboxyfluorescein diacetate after saturation with 10 nm superparamagnetic nanoparticles covered with polyacrylic acid. Hydroxyapatite ceramic scaffold produced by foaming technique and magnetized by vacuum-assisted infiltration with magnetite nanoparticles were used. Scaffolds were loaded with cells with applying an external magnetic field (MF), incubated overnight in osteogenic medium and then visualized by fluorescent microscopy. MF was provided by permanent magnet (Br = 1.2 T). The intensity of MF inside the scaffolds was regulated by the distance from the magnet. Nonmagnetized scaffold and hBMSCs were used as control. According to experimental data, magnetic scaffolds (MagSs) provided spatial orientation and adhesion of magnetic nanoparticles (MNCs) under influence of MF. In general MNCs were attached on the MagS pore walls according to MF direction, forming a layer inside the MagS. The thickness of the layer and the concentration of MNCs in this area were dependent on MF intensity and exposition time. MNCs saturated nonmagnetic scaffold provided insignificant spatial orientation of cells under the influence of MF. In comparison with nonmagnetic scaffold, magnetic scaffold showed a more effective spatial MNCs orientation.
Characterization of Native Salmon Collagen for Application in Bone Tissue Regeneration
We demonstrate that the skin of Atlantic salmon (Salmo salar) can be used to isolate soluble native collagen in high purity. This collagen is suitable as a matrix material for application in bone tissue regeneration. Soluble collagen was prepared from skin of Atlantic salmon under varying conditions. The collagen was characterised by means of DSC, Polarimetry, HPLC, electrophoresis and amino acid analysis. In addition, soluble fish collagen was used for selfassembling experiments. From this, fibrillar collagen was obtained which was further investigated for use as a porous matrix in bone tissue engineering applications. Using the skin of Atlantic salmon, native fish collagen was extracted in high amounts. The elution behaviour of this collagen in HPLC and electrophoresis was similar to that of bovine type-I collagen. However, the ratio of the two different polypeptide chains was found to be different. Furthermore, the fragments obtained from fish collagen after hydrolysis with bromocyane displayed a lower molecular weight. The low thermal stability of salmon collagen could be increased by refibrillation. After freeze-drying fibrillar fish collagen, porous matrices were obtained that are suitable for application in bone tissue regeneration.
Repair of a Critical-Sized Cranial Defect with a Biodegradable PEG-Tetra-Norbornene Polymer Scaffold Encapsulating rhBMP-2
The goal of the study was the assessment of bone formation by implantation of light-curable, MMP-degradable scaffolds in critical-sized calvarial defects in rats. Critical-sized calvarial defects 8 mm in size were created in male albino Sprague Dawley rats age 10 weeks. Experimental groups included 1.) rats with no critical size cranial defect implant (control subjects), 2.) rats with hydrogel scaffold implants in the cranial defect, and 3.) rats with scaffold implants polymerized with recombinant human Bone Morphogenetic Protein-2 (rhBMP-2). To non-invasively assess new bone formation and to calculate new bone density, micro-computed tomography (microCT) was performed at regular intervals, allowing us to follow the regeneration of mineralized bone tissue longitudinally over the course of the study. New software was developed to align microCT data and standardize image analysis. Rats were sacrificed at after the final microCT scan and histological analysis was performed on the rat calvaria. The synthetic, MMP-degradable scaffold enhanced bone formation in the area of the defect when compared to untreated controls. The incorporation of rhBMP-2 into the hydrogel scaffolds dramatically increased both the rate of bone growth and the volume of bone regenerated over the course of the study. Excessive and aberrant bone growth was also observed in the animals that received rhBMP-2-imbedded scaffolds, whereas the scaffold alone showed no bone growth outside of the defect area. Histology correlated with the microCT findings. Light-curable, MMP-2-degradable hydrogel scaffolds are osteoconductive and lead to significantly enhanced bone formation in critical-sized calvarial defects in rats. This bone formation was enhanced by rhBMP-2, though bone formation appeared abnormal in these animals.
Human Knee Meniscal Cells: Characterization of Their Primary Culture and Response to Biomimetic Surfaces in 2D and 3D Growth Environments
To examine (1) phenotype/gene expression in human meniscal cells and their changes during monolayer expansion, and (2) the effects of biomimetic surfaces on the growth, morphology, matrix production and gene expression of passaged meniscal cells in 2D and 3D environments.
Expression of ECM, transcription factors and transforming growth factors (TGFs) in meniscal cells extracted from non-arthritic human menisci (n = 6) and monolayer primary cultures were evaluated using quantitative real-time reverse transcriptase polymerase chain reaction (RT-qPCR). The passaged cells were then seeded on coverslips or PLGA scaffolds with self-assembled biomimetic surfaces. Cellular responses were examined using RT-qPCR, DNA quantification and a dimethylmethylene blue (DMMB) assay. Meniscal cells from the inner zone of human menisci expressed a prominent level of mRNA for collagen I (COLl), and to a lesser extent collagen lI (COLll) and aggrecan. Only low levels of Sox-9, Runx-2 and TGFs were detected in the initial harvest. All the genes, except COLl and Runx-2, were downregulated during monolayer expansion and the cells lost their characteristic morphology. All the presented surfaces supported cell growth. The cells formed colonies and exhibited the highest sulfated glycosaminoglycans (sGAG)/DNA ratio and COLll gene upregulation on surfaces that composed of covalently-bound collagen l/ll and chondroitin-6-sulfate, on a background of hyaluronic acid/chitosan multilayers (C6S surface). When the cells were seeded onto scaffolds with the C6S surface, downregulation of COLl and aggrecan, and upregulation of COLll, TGFs and Runx-2 were observed over a 2-week culture period. Our results indicate that primary human meniscal cells undergo dedifferentiation during monolayer expansion. This process could be partially reversed by culturing cells on the biomimetic C6S surface.
The Effect of Surface Biomimetic Apatite Nanostructure of Chitosan/Nano-hydroxyapatite Scaffold on Protein Adsorption and Osteogenic Differentiation of Rat Bone Marrow Stem Cells
The new genipin-cross-linked chitosan/nano-hydroxyapatite (Hap) composite scaffold was constructed through a nano-crystallon induced biomimetic mineralization method in simulated body fluid (SBF). Fetal bovine serum (FBS) was used as a model protein to assess the difference in protein adsorption. To evaluate the surface effect on osteogenic differentiation of rat bone marrow stem cells (rat BMSCs) at both the protein and gene levels, alkaline phosphatase (ALP) activity was measured, and quantitative real time PCR (RT-PCR) analysis was performed for mRNA expression of osteogenic differentiation makers, including osterix, osteopontin (OPN), and osteocalcin (OCN). Unlike the other potential bone tissue scaffolds, this new scaffold has the intrinsic fluorescent property on the basis of good biocompatibility. Compare to genipin-cross-linked chitosan framework, the higher amount of protein absorbed was found on the chitosan/nano-HAp composite scaffold due to higher surface energy of nanostructure. Moreover, the result showed the ALP activity of rat BMSCs on chitosan/nano-HAp composite scaffold was higher than chitosan framework. Similar, chitosan/nano-HAp composite scaffold with apatite nanostructure induced higher mRNA expression of osterix, OPN, and osteocalcin (OCN).
Interactions between Bone and Titanium New Insights into the Unique Marriage between Metal and Biology
From coating procedures with HA it is known that between the surface of Ti-Implants and the HA coating a thin 4 μ layer consisting of CaTiO3 (perovskit) develops. In 2004 Yashima et al. (1) showed that calcium in the CaTiO3 crystal is being bond ionically instead of covalently as was previously thought. This would enable the calcium ion at the bone to implant interface to take part in the process of constant bone remodeling. The aim of this study was to prove or disprove the existence of a CaTiO3 layer being formed under biological conditions. Fully osseointegrated implants were removed, air dried and its surface investigated by x-ray diffractometry at the DESY synchrotron in Hamburg/Germany.
The results clearly show the existence of CaTiO3 at the implant surface. There was no indication of the existence of TiO2. For the first time powder x-ray diffractometry has proven CaTiO3 to be formed under biological conditions at the surface of osseointegrated titanium implants. Simultaneously the TiO2 layer obviously is being modified to form perovskit. The extraordinary strength of the status of osseointegration can be explained by the fact that bone calcium ions of bone are integrated into the titanium oxide crystals thus forming an earth crust abundant molecule called perovskit, while at the same time take part in the dynamic process of bone remodelling.
Assessment of Stimulated Collagen Synthesis and Maturation in Osteoblast Cultures in Cell Sheets and on Scaffolds
The study compared the adequacy of various tests and used them to show the efficacy of the proposed method for the stimulation of collagen production in human bone derived cells (HBDCs) culture. Col1 expression (real-time PCR), protein itself (ELISA, collagen1), collagen maturation (hydroxyproline content, HYP) and fibril arrangement (TEM) were evaluated in HBDC culture. Additionally, picosirius-based quantitative assays (SR) were performed simultaneously after 7, 14, 21 days of culture. Ascorbate or lactate influence on collagen production indicated by each test was examined. Morphological observations were done in optical and electron microscopes (SEM, TEM). The set of tests was performed in HBDC cultures on elecrospun microfibrous polylactate (PLA).
The enhanced Col1 expression was accompanied by increased values in both HYP and ELISA tests. SR results indicated that proteins other than collagen 1 with SR-affinity were synthesized in high quantities. Sensitivity of the tests was verified in response to ascorbate addition. Controlled stimulation of collagen synthesis and maturation by using lactate was achieved, which may be valuable in bone TE. Gradually growing tissue-like structures and collagen fibril arrangement (TEM) were found in both cell sheets and PLA scaffolds. At the protein level, HYP was more reliable than ELISA and SR. Advantages and drawbacks of the tests are discussed.
Developing an Off-The-Shelf Tissue Engineering Strategy with Using Highly Angiogenic and Osteogenic Human Fetal Mesenchymal Stem Cells and a Biaxial Rotating Bioreactor
We have previously reported human fetal mesenchymal stem cells (hfMSC) as a superior stem cell source for bone tissue engineering (BTE) application, with low immunogenicity, great proliferative and osteogenic capacities. Additionally, we developed a biaxial rotating (BXR) bioreactor combining a perpendicular biaxial rotation movement with media perfusion, with proved efficacy to generate tissue engineered bone grafts (TEBG). We systematically compared this BXR bioreactor with three most commonly used systems: Spinner Flask (SF), Perfusion and Rotating Wall Vessel (RWV) bioreactors, for their efficacies for BTE. Furthermore, we generated TEBG using hfMSC, BXR bioreactor and a bioactive polymeric scaffold and evaluated its efficacy in a rat femoral defect model. The BXR bioreactor achieved higher levels of cellularity and confluence (1.4-2.5x) in large 785mm3 macroporous scaffolds not achieved in the other bioreactors operating in optimal settings. BXR bioreactor treated-scaffolds experienced earlier and more robust osteogenic differentiation on von Kossa staining, ALP induction (1.2-1.6x) and calcium deposition (1.3-2.3x). BXR bioreactor enabled superior cellular proliferation, spatial-distribution and osteogenic induction of hfMSC over other commonly used bioreactors, demonstrating its superior capacity for BTE. The implantation of TEBG into rat femoral defect successfully treated the critical-sized defects with increased vascularization and new bone formation, demonstrated the highly osteogenic and angiogenic nature of hfMSC and the culture efficacy of BXR bioreactor.
In Vitro Biomimetic Mineralization of Native Three-Dimensional Collagen Framework for Bone Regeneration
PADM framework was extracted from porcine skin, on which NanoHAp was fabricated to form PADM-HAp composite scaffold through a two-step biomimetic mineralization process. Mesenchymal stem cells (MSCs) were seeded on the scaffold and implanted in rat muscle. The tissue with implants were harvested 2, 4 and 8 weeks later and evaluated histologically.
X-ray diffraction patterns and SEM results indicated HAp nano-structure formed on the scaffold. In vitro cell culture shows that MSCs can proliferate on the scaffolds with and without HAp fabrication. After implantation in rat muscle, both PADM framework and PADM-HAp composite scaffold exhibit good in vivo biocompatibility. New bone appears 4 weeks later after implantation. It shows that the new bone tissue can be induced more easily in PADM-HAp scaffold than that in pure PADM. After implantation for 8 weeks, the amount of new bone tissue becomes larger and larger. The new inorganic phase formed in vivo are quiet different from the flake-like HAp fabricated in vitro.
It is suggested that both PADM and HAp-PADM has good in vivo biocompatibility. The fabrication of HAp as a calcium source benefits the new bone formation.
Poster Presentations
How Does Implantation of CFRP Cage Influence Cervical Kinematics? – A Retrospective Analysis
A retrospective analysis of operative cervical spine treatment by ACDF (anterior cervical discectomy and fusion) with a CFRP (carbonfiber reinforced polymer) -cage interposition. There was a follow-up of 43 patients aged 38 to 70 (53 ± 10 years) over mean time of 7,17 ± 4,80 month, treated with ACDF with CFRP-cage interposition (33 single-, 8 bi-, 2 multi-level) by use of autologous bone graft, 14 by additional use of hydroxyapatite bone paste (12 ActiFuse™, 2 Nanostim™). Angle measurement included the C2-C7-COBB-angle and the angles of the treated and adjacent segments.
The mean C2-C7 angle remained lordotic from pre- to postoperative and increased by 0,58° (preop: 13,40° ± 12,54°; postop: 13,99° ± 11,09°). For the mono-treated segments, there was an increase of the mean angle (4,20° ± 4,79°). For the adjacent segments, the mean angles decreased (superior: 0,60° ± 5,28°; inferior: 0,14° ± 3,13°). Using synthetic bone paste, there was an increase of the mean C2-C7 angle (3,76° ± 5,10°), without, it decreased (0,95° ± 5,65°). For the bi-levels, the mean angle of the superior treated segments increased (4,90° ± 5,42°), the inferior decreased (3,10° ± 7,94°).
For the treated segments as for C2-C7, the mean angle was lordotic and increased. That means a stabilisation of the treated segments and an improvement of the statics of cervical spine. The increase of these angles can be traced especially to the use of synthetic bone paste, because for the cases without, the mean C2-C7 angle decreased.
Composite Chitosan/Hydroxyapatite Scaffolds for Bone Tissue Engineering
The present study aims at optimizing a composite scaffold through the manipulation of the characteristics of a polymeric (chitosan) and a ceramic (hydroxyapatite) phase. Chitosan (CH) and Hydroxyapatite (Hap) are both biocompatible but Hap addition is though to overcome CH drawbacks respecting mechanical strength and bioactivity. Hap granules obtained by spray drying nanosized Hap particle suspensions were incorporated in CH solution and subsequently freeze dried. Synthesized HA fibers were also used as alternative reinforcement of CH scaffolds. Techniques including SEM, FTIR, UV, ICP and compressive mechanical tests were used to access the characteristics of the scaffolds. Bioactivity studies were performed in SBF solution. Results showed that scaffolds porosity is highly interconnected with inorganic phase uniformly distributed in the polymeric matrix. The scaffolds exhibit bioactivity and enhanced mechanical strength upon HA particles incorporation.
Biofunctionalization of Polylactic Acid Scaffolds with Different Peptides and Coating Variants
PLLA scaffolds were coated directly with linear and cyclic RGD-peptides and indirectly via a poly-L-lysin spacer (PLL). Reagents such as EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide) and DCC (N,N'-dicyclohexylcarbodiimid) were tested. Additionally coupling of the peptide to PLL was carried out using SPDP (sulfosuccinimidyl 6-(3'-[2-pyridyldithio]-propionamido)hexanoate). 2D cell adhesion was analyzed by fluorescence microscopy. Proliferation of cell seeded scaffolds was measured by a colorimetric assay. Gene expression analysis was carried out using osteogenic and ECM pathway gene arrays. DCC showed better coating efficiency than the water soluble EDC. For indirect coating, mechanical entrapment of PLL in contrast to covalent linking of PLL was examined and the latter one showed to be the better coating variant. In 2D cell adhesion assays, best results were obtained for cyclic and PLL RGD-peptides. 3D proliferation assays (8, 16 days) showed enhanced cell proliferation for cell seeded scaffolds which were directly coated with linear peptide and for those which were coated indirectly with covalent linked PLL in combination with linear peptide. 3D gene expression analysis of osteogenic differentiated cells showed different patterns regarding ECM components, related integrins and osteogenic markers expression.
Development of a 3D Cell Culture System with Sensors and Actuators for Stimulation and Manipulation
We developed a 3D cell culture system to investigate the calcification and formation of bone as the anabolic part of bone turnover. Osteoblast-like cells were used as model system to obtain medically relevant in vitro data. A fluidic channel system was imprinted in poly-dimethylsiloxan (PDMS) on a polycarbonate carrier or a glass sensor layer. Because of the non biological character of PDMS, other materials have also been tested for the fluidic part such as alginate and agar-gelatin mixtures. It could be shown that the cells grow on alginate and on agar-gelatin. Because of the softer structure of alginate the agar-gelatin-mixture was preferred for fluidic imprinting. The glass sensor layer carried interdigitated electrode structures (IDES) as well as oxygen and pH-sensors. The sensors allow us to observe adhesion, viability and proliferation, even in substrates that are not optically transparent. The sensor-layer chips were produced by thin film technology on glass wafers using platinum as sensor material.
Cell Adhesion Forces in Dependence on Different Surface Materials With the Same Roughness Factor
We consider the dependence of cell adhesion forces on varying surface materials by Single-Cell Force Spectroscopy (SCFS), which is a special application of Atomic Force Microscopy (AFM). For SCFS, single cells are captured by pressing the AFM cantilever on the cells. The cantilever was functionalized by fibronectin, to ensure cantilever-cell adhesion in the investigation of the adhesion behavior of connective tissue fibroblasts L-929. The surface detachment forces of the cantilever-adhered fibroblasts were probed at a constant pulling speed over a time range of 60 s. Different materials were chosen which are used in 3D-multisensor structures for pH-, IDES- (interdigitated electrode structure), and O2-sensors in a bone cell culture system [platinum and borosilicate] or in scaffolds for cell guidance [PDMS, polycarbonate]. All materials had the same roughness factors.
Generation of Tissue Engineered Bone Flap with a Vascular Pedicle in Beagle Dogs
Firstly, BMSCs of beagle dogs were isolated by a Ficoll-Paque PLUS solution and expanded in an osteogenic culture medium for 2 weeks. The osteogenic characteristics of induced BMSCs were evaluated by immunocytochemistry staining. Secondly, the induced BMSCs were seeded on the coral hydroxyapatite (CHA) scaffold with a prefabricated inclined groove and were 3D cultured for 1 week. Lastly, the engineered construction was implanted into subcutaneous site of hind leg of beagle dogs with the arteria saphena and saphenous vein dissected and embedded in the groove of the scaffold (experimental group, n = 12). Engineered construction without vessels embedded was used as control (n = 8). Vascularization and bone formation were assayed by computed tomography (CT), angiography and histological observation at 1, 3, 5, and 6 months after implatation. Angiography examination showed a microvascular network forming at 1 month implantation and gradually becoming dense in engineered constructions of experimental group. CT scanning and histology study demonstrated the level of engineered bone calcification and CHA scaffold degradation increasing in both groups after implantation. However, more significant calcification of engineered bone were observed in experimental group comparing with that in control group after 3 months.
Treatment of Avascular Necrosis of Femoral Head in an Ovine Model Using Autologous Mesenchymal Stem Cells
Animals were divided in 3 groups: the first group was subjected to core decompression (gold standard) whilst the other two groups were treated with the construct, with and without MSC, respectively. Bone marrow cells were harvested from sternum and mononuclear cells (MNC) were isolated on a Ficoll density gradient and MSC expanded for twenty days. Then particulated cancellous bone scaffold was colonized by the “ex vivo” expanded cells. The construct was formed using fibrin glue and immediately introduced into the animals. The remaining cells were used for quality controls, differentiation assays and phenotypical characterization that confirmed their MSC identity. After 6 weeks, sheep were euthanatized and femoral heads were analyzed by Hematoxilin and Eosin staining of histological sections. These analyses evidenced the presence of newly generated bone in the test group treated with cells, which grew forming new bone around the scaffold and survived until the end of the treatment. The test group treated without cells showed limited formation of new bone, and the gold standard group showed a fibrotic tissue that developed without bone formation.
Bone Tissue Engineered Construct for the Treatement of Critical Size Segmental Tibial Defect in Sheep
Animals were divided in 3 groups: the first group was subjected to autologous bone replacement (gold standard) whilst the other two groups were treated with the construct, with and without MSCs, respectively. Bone marrow cells were harvested from the sternum and mononuclear cells (MNC) were isolated on a Ficoll density gradient and, subsequently, adherent MSCs were expanded for twenty days. Then particulated cancellous bone scaffold were colonized by the “ex vivo” expanded cells. The construct was consolidated by using fibrin glue and immediately introduced into the animals where a critical size segmental tibial defect (2.2-2.5 mm) was surgically produced and stabilized. The remaining cells were used for, quality controls, differentiation assays and phenotypical characterization. Sheep were euthanatized at 3 months post-surgery and tibiae were analyzed by Hematoxilin and Eosin staining of histological sections. Group treated with whole construct showed both ossification and, importantly, mechanical properties comparable with the gold standard group. In contrast, poorer ossification and mechanical properties were observed on the group treated without cells.
Comparison of Different Secured Allograft, an In Vitro and In Vivo Study
We tested in vitro MSC cell seeding efficiency and viability, osteogenic gene and protein expression and in vivo ectopic bone formation. Cell seeding efficiency and viability were equivalent on the three different scaffolds. Tutoplast®, fresh- and irradiated-frozen bone are able to significantly induce osteoblastic gene expression of BSP (2 to 38 fold), osteopontin (300 to 19.000 fold), osteocalcin (2 to 20 fold) and BMP2 (10 to 274 fold) of seeded MSC. However, a higher osteoblastic gene expression and BMP2 protein synthesis are observed when fresh-frozen bone is used. Semi-quantitative analysis of in vivo ectopic bone formation indicated a higher number of area of bone formation when fresh frozen-bone is used (p < 0.05). However, the frequency of scaffold able to induce ectopic bone formation is not significantly different. In conclusion, secured processed allograft Tutoplast® is able to support a high frequency of bone formation almost as good as fresh-frozen bone.
Native Bone Collagen for Tissue Regeneration Applications
Collagen type I was extracted from tibias and femurs of New Zeland's rabbit bones and pulverized in a mill refrigerated with liquid nitrogen to obtain a fine powder, which was decalcified using a solution of 0.5M EDTA/4M guanidine hydrochloride (10% w/v) at pH 7.5 during 2 weeks at 4°C, and solubilised with 0.5M acetic acid/pepsin (0.5% w/w)[1]. The supernatant (soluble collagen) was centrifuged and freeze-dried, while the insoluble part was preserved at 4°C. Finally, the soluble collagen was immersed into a buffered solution (pH 9.2, 50mM glycine, 200mM KCl) inducing the growth and organization of collagen fibrils [2]. Collagen was crosslinked using enzyme microbic transglutaminase and biomineralized by the biomimetic method[3]. The morphology and composition (SEM-EDS, scanning electron microscopy), the chemical interactions (FTIR, infrared spectroscopy) and the thermal properties (DSC, differential scanning calorimetry) were evaluated.
Microfabrication of Polymeric Scaffolds for Tissue Engineering Applying Two Photon Polymerization
A screening of various polymer precursors for their ability to be polymerized and structured by the 2PP-method was performed selecting methacrylated oligocaprolactone and polyglycerine macromers as building blocks for the bone phase and polyethylene glycol (PEG) based macromers for the cartilage phase. Scaffolds with regular 3D-structure and varying pore sizes were fabricated by 2PP and analyzed by scanning electron microscopy (SEM). The scaffolds were submitted to cell culture applying chondrocytes in order to investigate the biocompatibility of the materials. Qualitative analysis by confocal laser scanning microscopy (CLSM) revealed a strong cell adhesion for caprolactone- and PEG-urethane methacrylate-based materials and furthermore a preference of the cells to pore sizes between 55 and 80 μm.
Bone Formation and Neovascularization Mediated by Mesenchymal Stem Cells and Endothelial Cells in Critical-Sized Calvarial Defects
4-mm-diameter collagen sponge discs were seeded with spheroidal endothelial aggregates and/or osteogenically differentiated human MSCs. Constructs were implanted in a 4-mm-diameter calvarial critical-sized defect of SCID mice. Mice were randomly divided into 4 groups: 1: defect filled with collagen sponge alone; 2: defect filled with collagen sponge seeded with EC-spheroids; 3: defects filled with collagen sponge seeded with MSCs; 4: defect filled with collagen sponge seeded with EC-spheroids and MSCs. After 6 weeks, implants were retrieved and analyzed for the formation of a human EC-derived vasculature and for bone regeneration. Implantation of human spheroidal endothelial aggregates resulted in the formation of a complex three-dimensional network of human blood vessels. The human vasculature matured by recruiting mouse mural cells and was connected to the mouse circulation. Implantation of human osteogenically differentiated MSCs resulted in a statistically significant increase in new bone formation (group 3 versus group 1: p < 0.05; group 4 versus group 1: p < 0.005). However, new bone formation in MSC-seeded implants was not improved by co-seeding of EC-spheroids.
Micro-topographies for Composite Bone Implants
Several micro- and nano-topographies were fabricated initially on planar acrylate resin samples. The fabrication process includes the production of master structures in quartz or silicon wafers by photolithography and colloidal lithography, an intermediate structure replication in PDMS and a final replication by casting in resin. These planar samples were then tested in vitro as a model for the more complex curved surface of a real bone implant. Proliferation studies made on planar samples using an osteosarcoma cell line (SaOs-2) indicate that the cell grows well and colonizes the structured surfaces. Moreover, proliferation on the structured resin disks appears much better than on control surfaces made of titanium alloys currently used in conventional bone implants. One specific micro-structure was selected to be used in in vivo tests on mini-pigs. Textured 3D samples for implantation were fabricated using a conic mould with a layer of structured PDMS for resin casting. The truncated cones obtained have micrometric structures on the lateral surface and have been implanted into mini-pig femurs. Twelve weeks after the operation a series of analyses will be made on the femurs. The results of push-out strength, μCT imaging, histomorphometry and SEM/EDXA of hard-tissue sections will indicate whether or not the micro-structured implant gives better osseointegration than titanium and unstructured resin samples.
Biomimetic Growth of Hydroxyapatite Nanocrystals Inside Self-Assembled Collagen Fibers
Collagen type I was obtained from rat tail tendons using a H3PO4, the direct nucleation of inorganic phase (hydroxyapatite-HA) inside collagen fibers consist of a neutralization synthesis with an aqueous suspension of Ca(OH)2 until a hydrogel was formed. After 1 week of drying at 50°C a white dust was obtained, Genipin was added as a crosslinked agent, following by drying at 50°C during 3 days. Samples were analyzed using scanning and transmission electron microscopy (SEM-TEM), energy dispersive spectrophotometry (EDS) and differential scanning calorimetry (DSC). The SEM-EDS results suggest that effective nucleation of HA inside self-assembled collagen fibers occurred. TEM micrograph showed a preferentially highly aligned collagen nanostructure like bone. DSC results showed the typical denaturation temperature of collagen triple helix (65°C) and a fusion temperature (155°C) corresponding to the self-assembled collagen fibers presented in bone.
Healing of an Atrophic Non-Union of a Canine Radius Using an Bioartificial Bone Graft and Dynamized Plate-Osteosynthesis
To fuse an atrophic non-union of a canine radius-ulna fracture using a ß-tricalciumphosphate (ß-TCP)/bone marrow-composite-graft supported by dynamization of plate osteosynthesis. A 7 year old female Siberian Husky dog was referred for treatment of comminuted serial fractures of the left metatarsus and a comminuted radius-ulna-fracture of the left forelimb after being involved in a car accident. 7 months after initial treatment using plate-osteosynthesis a non-viable atrophic non-union of the left radius occurred. Revision surgery was performed using ß-TCP-granules and a cylindrical ß-TCP graft soaked with autologous blood and bone marrow harvested from the ipsilateral iliac crest to fill the defect zone. Surgery was completed by debridement and refreshment of the fracture ends. In addition dynamization of the fracture stabilization by removal of central screws (No. 3, 4 and 5) was performed to biomechanically initiate osteoneogenesis by switching load from the plate to the fracture site to enhance micromovement. 20 months after revision surgery complete biodegradation of the ß-TCP-matrix and osseous fusion of the non-union as well as ongoing remodeling became obvious radiographically and histologically. No site effects occurred. Healing of a canine non-viable atrophic non-union can be provided by the use of ß-TCP-granules and a cylindrical ß-TCP-graft loaded with autologous bone marrow.
Development of 3D Scaffolds for Bone Tissue Engineering Based on Mineralized Collagen of Marine Origin
We present collagen isolated from fish skin as an alternative source for preparation of porous mineralized collagen 3D scaffolds, suitable for bone tissue engineering applications (project funded by German Federal Ministry for Education and Research). Collagen type I was extracted from skin of Atlantic salmon, lyophilized for transportation and solubilized in acetic acid. The fibril reassembly was monitored by turbidity measurements; parameters like pH, collagen concentration, NaCl concentration and temperature were varied to optimize conditions for simultaneous fibril reassembly and mineralization. Porous scaffolds were prepared by freeze drying and chemical cross linking of mineralized salmon collagen and characterized by FTIR, SEM and mechanical investigations First cell culture experiments with human MSC were conducted. Porous 3D scaffolds of biomimetically mineralized salmon collagen showed similar properties compared to those made from bovine collagen. It was confirmed by FTIR that the mineral phase formed during the preparation process is hydroxyapatite, the mineral phase of bone. The scaffolds revealed an interconnected porosity with pore diameters up to 200 μm and are stable under cyclic compression. The biocompatibility of the novel material in vitro was validated by cultivation of hMSC.
Assessment of High Molecular Weight Hyaluronic Acid on Peripheral Blood Monocytes
Enzymatic degradation was investigated using 1mg/ml Durolane© subjected to 10 μl of 10mM bovine testicular hyaluronidase under appropriate conditions, and the resulting solution was analysed using PAGE (polyacrylamide gel electrophoresis). Secondly, ELISpot analysis was used to assess memory T-cell release of INF-? in 1 ng/ml and 0.1 ng/ml of CD3 on exposure to a range of concentrations of Durolane©. The initial experiment confirmed that the changes in HA (hyaluronic acid) structure to manufacture Durolane© did not prevent enzymatic break-down, with distinct changes in molecular weight being observed. The second set of experiments demonstrated a dose-related decrease in memory T-cell response to stimulation by CD3 when mixed with Durolane© at concentrations of 50 μg/ml, 100 μg/ml, 500 μg/ml and 1 mg/ml.
Mechanical Properties of Calcium Silicate/Biopolyester Ternary Composites During Degradation
The aim of this study was to determine changes of the mechanical properties of nanostructured calcium silicate (CS)/ poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) composites during degradation in simulated body fluid (SBF) at 37°C to choose materials suitable for the fabrication of bone tissue scaffolds. CS/PHBV films, which contained 5 wt% of CS and 0, 5, 10, 20 or 30 wt % of poly(lactide-co-glycolide) (PLGA) were fabricated by solvent casting. After each week of degradation Young's modulus, ultimate strength and elongation at break (tensile tests), surface morphology (SEM), changes of weight of the samples and pH of the SBF were determined. Additionally, attachment of MG63 cells to different materials was studied. It was observed that the degradation rate of the composites was influenced by the PLGA content. After five weeks in SBF the composite containing 5 wt% of PLGA exhibited the lowest loss of ultimate strength (20% of initial) as well as an increase of 18% of Young's modulus and very good cell attachment.
Comparison of the Stem Cells for Bone Regeneration: Long Bone Versus Mandibular Bone
We isolated mesenchymal stem cells (MBMSC (mandibular bone marrow stem cell), LBMSC (long bone marrow stem cell)) from mandible and iliac bone with a different developmental process. Characteristics of these stem cells and efficiency of bone regeneration was confirmed.
Immunocytochemistry proved the existence of stem cells in these cell populations using STRO-1 as a stem cell marker. The cell colony forming efficiency, self-renewal capabilities, cell growth rate, and ability to differentiate into other tissue were compared. Mineralization in the osteogenic cultures was measured. Also we investigated bone regeneration by transplating these stem cells into the dorsal subcutaneous tissue and the skull defect of immunocompromised mice. Both MBMSC and LBMSC showed characteristics of stem cell such as self-renewal, colony formation, and expression of marker molecule. The MBMSC was definitely superior to the LMBSC in all respects. In particular, MBMSC showed high mineralization efficiency and perfectly regenerated bone defect.
Addition of BMP-2 or BMP-6 to Dexamethasone, Ascorbic Acid, And Beta-Glycerophosphate May Not Enhance Osteogenic Differentiation of Human Periodontal Ligament Cells
Human periodontal ligament cells (PDLCs) were obtained from third molar teeth and were cultured with D-MEM/F-12 containing 10% fetal bovine serum. PDLCs were exposed to (1) BMP-2 + AA (ascorbic acid); (2) BMP-6 + AA; (3) osteogenic supplements; (4) osteogenic supplements + BMP-2; or (5) osteogenic supplements + BMP-6 to evaluate their effect on cell proliferation and osteogenic differentiation. Osteogenic differentiation was evaluated by mineralization assays including alizarin red staining and quantitative calcium assay, quantitative alkaline phosphatase (ALP) assay and real-time polymerase chain reaction (PCR) analysis for the expression of collagen type I, Runx2, osteopontin, and osteocalcin in PDLCs. Cell culture with BMP-2 or BMP-6 + AA increased ALP activity of PDLCs, suggesting the osteo-inductive effect of BMP-2 and BMP-6. However, longer duration of culture showed neither of the BMPs induced in vitro mineralization of PDLCs. In contrast, osteogenic supplements were able to induce significant increase in ALP activity with or without the combination of BMP-2 or BMP-6 of PDLCs and significant upregulation in osteopontin expression was observed.
The Effect of Chitosan-Gelatin Scaffolds Pore Size on Amniotic Epithelial Cell Attachment for Bone Tissue Engineering
We evaluated the effect of chitosan, chitosan-gelatin and gelatin scaffold with different pore size on AEC attachment and differentiation for bone tissue engineering. Porous scaffolds were obtained by using different pre-freezing temperature (−20°C, −80°C and −196°C) of freeze-drying technique. Samples were analyzed by scanning electron microscope (SEM) and porosity was measured. After isolation of AEC from amniotic membrane, attachment of these cells was tested using MTT-Formazan assay. Results show that the chitosan-gelatin scaffolds with ∼120μm pore size can be utilized as a good matrix for AEC culture.
The Effect of Bioabsorbable Polymer-Types for Delivery of Adipose Derived Stromal Vascular Fraction (SVF) on Bone Regeneration
Rabbits were divided into three groups (control: without PLGA-types and SVF, group-1: PLGA particle-type with SVF, group-2: PLGA block-type with SVF). The long bone defects were created on 16 mature white new zealand rabbits ulna with size 15 mm x 4 mm. After implantation of control, group-1, and group-2, rabbits were sacrificed and the implants were retrieved at 8 and 12 weeks. The effect of bioabsorbable polymer-types was investigated by X-ray, microCT, and H&E staining. From the results of microCT at 8 weeks, the bone volume fraction (BVF) of control, group-1, and group-2 was 24.09%, 28, 92% and 56.6%, respectively. Further, at 12 weeks, BVF of control, group-1, and group-2 was 44.22%, 54.51%, and 61.87%, respectively. It is noted that PLGA block-type scaffold was more effective than PLGA particle-type scaffold and control. Also, from the result of H&E staining, the bone area from PLGA block-type scaffold observed a larger black area than PLGA particle and control.
Human Umbilical Cord Tissue-derived Mesenchymal Stem Cell-like Cells: Cells with Potential for Tissue Engineering Applications
We isolated a population of plastic-adherent mesenchymal stem cell-like cells from human umbilical cord (UC) tissue under xeno-free culture conditions. The cells exhibit a high proliferative potential, express several mesenchymal stem cell markers, including CD44, CD73, CD90 and CD105 (negative for CD31, CD34 and CD45), can be differentiated at least towards the adipogenic and chondrogenic lineage and demonstrate immunoprivileged and immunomodulatory properties. Mesenchymal stem cells can be cultivated on biocompatible materials and differentiated to target tissues. The selection of the scaffold is a critical parameter. The ideal scaffold has to mimic the desired physiological and biochemical parameters. In addition, the scaffold needs to favour cellular attachment. According to the bone structure, a highly porous, open pored and fully interconnected geometry is desired. Therefore different materials have been tested for their application in bone Tissue Engineering.
Heparinized Scaffolds with Pores of Inverted Colloidal Crystals for Chondrogenesis
Monodispersed polystyrene microspheres were self-assembled by floating in the medium containing ethylene glycol, dried, annealed and infiltrated with heparin/chitin/chitosan gels. The results indicated that the colloidal template was in a structure of hexagonal arrays. In addition, the regularity of the organized pores in the scaffolds reduced when the concentration of ethylene glycol decreased. An increase in the weight percentage of heparin enhanced the viability of bovine knee chondeocytes (BKCs) in inverted colloidal crystal (ICC) matrices. Over 4 weeks of cultivation, the amount of cartilaginous components including BKCs, glycosaminoglycans (GAGs) and collagen enhanced with time. Moreover, an increase in the weight percentage of heparin promoted the production of BKCs, GAGs and collagen in ICC constructs. Histological and immunochemical staining of the cultured ICC constructs revealed minor differences in BKCs, GAGs and type II collagen between the peripheral and core regions.
Silk Scaffold Fabricated by Pressing Method
Silkworm cocoon was cut to small pieces and treated with soap and CaCO3 solution twice. The degummed cocoon was washed thoroughly in warm distilled water and then dried at room temperature and stored in desiccators prior to use. Cocoon was dissolved in mixture solvent (CaCl2, EtOH, and water), dialysis using cellulose tube, and then freeze-dried. Silk scaffold was prepared from lyophilyzed silk fibroin with hydrolytic press. Surface morphology, structural properties, degradability were examined and HT-1376 cell was cultured on the scaffold. SEM and photomicrographs showed that silk scaffold has interconnected pore. X-ray diffraction results showed that silk scaffold has stable crystalline structure. The degradation of silk scaffold depended on the fabricated composition. It could be degraded in the human body by enzyme, protease slowly. Biocompatibility of silk scaffold was examined and well attachment and proliferation on the scaffold surface. Therefore, silk scaffold prepared by pressing method might be used as a novel tissue engineering scaffold.
In Vivo Evaluation of an Injectable Premixed Radioopaque Calcium Phosphate Cement
We evaluated biocompatibility of a novel premixed calcium phosphate cement containing ZrO2 in a rat model of vertebral defect. The powder part of the cement consisted of β-tricalciumphosphate (β-TCP), monocalcium phosphate monohydrate (MCPM) in equimolar amounts, then 20% (w/w) ZrO2 was added. The powder was then mixed with glycerol with a powder/liquid ratio of 4.2 g/ml. 1 ml syringes were prefilled with the sterilized mixture under aseptic conditions. Wistar rats were anesthesized and a drill hole was prepared in a tail vertebra and filled with a spacer for 12 weeks. After removal of the spacer the cavity was filled with poly-methyl-metacrilate (PMMA) or premixed TCP-based bone cement (TCP). Osteoblast activity was investigated in vivo using nanoSPECT-CT using the adjacent intact vertebra as a control. Osteointegration was evaluated with microCT at 12 weeks after implantation. The trend towards increasing osteoblast activity was higher in the TCP group than that of the PMMA group. CT scans of the vertebrae showed that PMMA completely filled the hole and a sclerotic demarcation line and thin gap was visible between the bone and the polymer. In contrast, the TCP-based bone cement inegrated into the regenerating bone and resorption-remodelling was evident while the material still filled the hole after 12 weeks. The premixed calcium phosphate cement containing 20 % ZrO2 was found to have excellent handling, good radio-opacity and good biological activity as a filler of bone cavities.
Effects of Different Chain Extenders on the Properties of Polyoxamer-Based Hydrogels as Cell Carriers for Tissue Engineering
We assessed the rheology and biocompatibility of biodegradable temperature responsive hydrogels containing different chain extenders. Hydrogels were made by dissolving polyurethanes based on polyoxamers using different chain extenders: diisocyanatobutane (BDI) and hexamethylendiisocyanate (HDI). Rheological measurements were performed with a rotational rheometer in oscillating mode with constant frequency and amplitude running temperature ramps. Injectability was analyzed with a TextureAnalyzer. To assess the cell survival hydrogels were populated with immortalized hMSCs and the percentage of living cells was determined via live/dead-assays after 24 h. With increasing polymer concentration the gels stabilize faster, but cell survival decreases. In a 1:8 dilution gels are liquid at room temperature and solid at 37°C. Injection forces are about 2N at room temperature. Cell survival after 24 h in syringe was 81 ± 7% in BDI- and 58 ± 20% in HDI-based gels. A dilution of 1:8 was identified as the optimum of handling properties, hardening and cell survival. Both gels are very promising candidates for hydrogel based cell injection, due to their biocompatibility and rheological behaviour.
Coculture with Endothelial Cells Promotes Expansion of Osteoblasts – To Be Used in Bone Tissue Engineering
Human bone derived cells (HBDCs) and human umbilical vein endothelial cells (HUVEC) were cocultured in direct contact at the ratio of 1:1, 4:1 or 1:4 or were separated with an insert (ratio 1:1). Both cell types were detected by labelling with different quantum dots. Cell number was determined by DNA measurement (Picogreen) and by FACS, alkaline phosphatase (ALP) activity was determined in a p-nitrophenol-based assay. Our observations confirmed the increasing ALP activity in direct cocultures of osteoblasts-ESs comparing to osteoblast monocultures and a lack of paracrine effect of endothelial cells on ALP activity of osteoblasts. Both phenomena were reported also by other authors. Increasing ALP activity was found for all cell proportions although not to the same extent. Both HBDCs and HUVECs could be found in the coculture after 7 days in culture, independently on the initial cell proportion, which was not the case as reported by Unger et al. when human dermal microvascular endothelial cells were investigated in coculture with osteoblasts. What is new, a significant enhancement of HBDC expansion was observed in coculture, i.e. 12× growth of HBDCs number after a week comparing to 4× in monoculture. Potential usefulness of the results for tissue engineering is discussed.
Coculture of Human Endothelial Progenitor Stem Cells and Bone Marrow-Derived Human Fetal Mesenchymal Stem Cells Potentiates Osteogenesis through Paracrine Activity In Vitro and Induces Neovasculogenesis within Tissue Engineered Bone Grafts In Vivo
We hypothesized that the interaction between Endothelial Progenitor Cells (EPC) and human fetal mesenchymal stem cells (hfMSC) will lead to enhanced osteogenesis. Co-culture of umbilical cord blood EPC and hfMSC at low passages, and osteogenic differentiation were investigated in both mono-layer culture and after loading onto osteo-conductive macroporous PCL-TCP scaffolds. EPC:hfMSC-loaded scaffolds were studied after both static and dynamic culture, and after subcutaneous implantation to study its effects on osteogenesis and vasculogenesis. EPC was able to enhance osteogenic differentiation in hfMSC in 1:1 co-culture, with earlier induction of ALP (Day 7 vs Day 10) and higher calcium deposition (1.9×; p < 0.001) compared to hfMSC alone. This effect was observed only in osteogenic differentiation medium, but not in basal medium. The osteogenic enhancement phenomenon was induced by soluble factors as EPC conditioned media (1:1 and 1:10) was able to recapitulate this effect on hfMSC in bone induction medium, with 1.8-2.2X higher ALP levels and 1.4-1.5X increase in extracellular calcium deposition (p < 0.01). Ectopic implantation resulted in higher numbers of CD31 positive vessels in EPC:hfMSC over hfMSC alone. Dynamic culture of EPC:hfMSC scaffolds resulted in more prominent trabecular-like networks of ECM and increased calcium deposition (1.3X; p < 0.001) compared to static culture. Histological sections of ectopic implantations in mice demonstrated capillary-like extensions with 1.8X higher capillary-like structures in dynamically cultured scaffolds. EPC enhanced hfMSC osteogenic differentiation significantly through paracrine activity, and enhanced vasculogenesis in vivo.
Magnesium Alloys as Degradable Biomedical Implants: A Theoretical Risk Assessment
Toxicological data were obtained from US/UK public health agencies, and the NOAEL approach was utilized for risk assessment. ASTM upper abundance limits of alloy components were adopted. Implants were assumed to degrade evenly and completely in one year in 60kg human. Calibration was made to assume full absorption. Threshold implant masses based on component elements in an alloy were individually calculated; and the lowest value taken as the threshold implant mass of that alloy.
Threshold implant masses for aluminium-containing magnesium alloys were near or below one gram, much less than other alloys; thus their use as multiple bone fixation devices necessitated added caution. No authoritative NOAEL or equivalents were identified for rare earth metal group or zirconium; although qualitative documentations on their toxicological effects were found.
Silk/Regenerated Silk Composite Scaffolds for Bone Tissue Engineering
Natural silk (NS) and regenerated silk (RS) were combined to achieve better mechanical properties in the 3D porous form. RS has been made using mulberry-silk cocoons. RS/NS composite scaffolds have been fabricated by the freeze-drying technique. Silk protein extract was evaluated by FTIR and XRD methods, which showed sharp amide peaks in 1655 cm-1 and 1530 cm-1 wavelength in FTIR spectrum pattern confirming existence of fibroin. The fabricated 3D scaffolds were morphologically analyzed by scanning electron microscopy and showed an inter-connective spongy structure. Mechanical characterizations were also carried out by a universal testing machine. Results showed that the RS/NS composites have improved mechanical properties compared with scaffolds fabricated with either RS or NS alone. Compressive strength of 1.8Mpa and elastic modulus of 3.4Mpa are measured for this porous structure, which is considerably higher in comparison with the other reported the biodegradable porous scaffolds with the compressive strength of 0.05 to 0.7Mpa. In addition, In vitro tests including cell viability, cell proliferation and adhesion have been carried out and confirmed biocompatibility of this scaffold.
Collagen Scaffolds from Shark Cartilages (CHILOSCYLLIUM PUNCTATUM) for Tissue Engineering
The 2.3% cartilage collagen solution (isoelectric point, pI of 7.03) was prepared in 0.05 M acetic acid solution, pH 3.6, and lyophilized to obtain the scaffolds. Carbodiimide (2.2 %w/w of collagen mass) was used for cross-linking collagen. Microstructure and pore size of the scaffolds were investigated by scanning electron microscopy (SEM). Surface charge was analyzed by zeta potential measurement. The swelling ratio of the scaffolds was observed in phosphate buffer saline (PBS) at 37°C. Cross-linking of the collagen scaffolds was confirmed by using Fourier transform infrared spectroscopy (FT-IR). Cytotoxicity of the scaffolds was observed on MC3T3-E1 cells using MTT assay. The scaffolds were porous with average pore size of 240.25 ± 93.73 mm. Interconnecting pores were observed under SEM. The zeta potential of the scaffolds was found as 25.46 ± 0.53 mV. This may be the result of the positive charged collagen in acetic acid solution at the pH under the pI. Swelling ratio of the scaffolds was found as 4.58 ± 0.38 g/g scaffold. Shifting of the bands of FT-IR spectra indicates amide bonding of the scaffolds after cross-linking. No cytotoxicity of the scaffolds on MC3T3-E1 cells was observed using MTT assay. A novel source of collagen from cartilages of the marine shark presented in this study can be useful for fabrication of matrices and scaffolds.
Improvement of Osteogenic Power of PCL-Composite Scaffolds for Mesenchymal Stem Cells
We used a method of phase inversion together with an appropriate solid porogen to realize matrices of polycaprolactone (PCL) with controlled porosity and mechanical characteristics. To improve cell adhesion, proliferation, and osteogenic differentiation of rabbit mesenchymal stem cells (rMSC), synthetic hydroxyapatite and rat bone extracellular matrix were added to PCL scaffolds. PCL scaffolds were obtained by the phase inversion technique. PCL was dissolved in tetrahydrofuran (THF) at room temperature followed by addition of water to the homogenous solution in a weight ratio 1:0.5. The obtained gel was mixed with Ca2+ alginate threads as a porogen (PCL-A) or charged also with hydroxyapatite (PCL-HA) or bone powder (PCL-BP) as cell adhesion promoters. The mixture was transferred into a mold and incubated at −30°C for 24 h until solidification. The solid replica was removed and washed with ethanol at −30°C to extract the THF solvent and with sodium phosphate buffered solution at 4°C to remove the porogen and the ethanol. PCL scaffold without porogen (PCL-WP) was used as a negative control. Rabbit MSC viability on PCL matrices was evaluated by MTS assay, whereas cell differentiation was assessed by determination of alkaline phosphatase activity (ALP) and quantitative analysis of alizarin red staining. Morphological analysis was carried out by SEM before and after cell seeding. Porosity and mechanical properties were evaluated also by tomography (μCT). Scaffolds with good interconnected structure and physical integrity were prepared successfully. In comparison with PCL-WP negative control (25% ± 7.81), PCL matrices prepared with HA (PCL-HA), or BP (PCL-BP) or only porogen (PCL-A) showed by μCT analysis a total porosity of ∼65% ± 8.06. The observation with electron microscopy revealed differences among matrices as micro-(5-10 μm), as macro-(50-200 μm) porosity and as mean pore dimension (70 μm). Moreover, the tomography analysis revealed that PCL-HA scaffolds are more resistant to mechanical stress in comparison with PCL-BP (p < 0.05) or PCL-A (p < 0.05). The biological tests performed using rMSCs demonstrated that the osteogenic potentialities of PCL-HA and PCL-BP are increased in comparison with PCL-WP and PCL-A, as confirmed by the higher ALP expression and the increased of mineralized extracellular matrix observed after one week differentiation induction.
In Vivo and In Vitro Study of a PLA Fiber Reinforced β-TCP Composite Cage in an Ovine Anterior Cervical Intercorporal Fusion Model
This study combines a pilot in vivo study of spinal fusion in sheep and in vitro biocompatibility and osteoconductive tests with adipose stem cells (ASCs) of composite material used in the fusion. The in vivo study was conducted first. The composite implant consisted of a homogenous mixture of 70 wt% of poly-(70L/30DL)-lactide and 30 wt% of β-tricalcium phosphate with a hollow interior for autologous bone graft. Implants were surrounded by continuous fibers made of poly-(96L/4D)-lactide (PLA96) for the mechanical support and reinforcement of the device. In vitro samples were similar excluding the fiber reinforcement. One animal was lost preoperatively due to an anesthetic complication. CT images showed interbody fusion inside the control devices. The composite implants showed multiple cracks and sclerotic endplates but no loose or migrated fragments. Histologically, a moderate foreign body reaction developed in the composite implant group. The in vitro studies showed the similarity between human and sheep ASC viability on the composite samples. Osteogenic differentiation was detected only with human ASCs.
A Design of the Highest DICAM-Coated Silk Fibroin Scaffold for Development of Artificial Bone
Recombinant DICAM (Dual Ig domain containing Cell Adhesion Molecule) protein had been expressed and purified using the BEVS (Baculovirus Expression Vector System) system and had applied to the 3D Silk Fibroin scaffold. DICAM mediates cell adhesion via αvβ3 integrin and is found to be expressed ubiquitously in various organs and cell lines. In addition, αvβ3 integrin is one of key regulators for angiogenesis and osteoclast differentiation. The rBV (recombinant Baculovirus) plasmid was generated by inserting expression cassettes encoding DICAM and the EGFP reporter gene into the standard Baculovirus transfer vector pBacGus-4X in an orientation such that the direction of transcription was convergent. DICAM gene expression was driven by the polyhedron promoter, and, therefore, should be expressed at high levels in insect cells late in infection. Using each 3D Silk Fibroin scaffold coated with purified recombinant DICAM protein, cell adhesion assay was performed with C2C12 cell. The highest DICAM-coated Silk Fibroin scaffold showed the highest adhesion activity to the C2C12 cell. Consistently, low levels of DICAM-coated scaffold showed low retention of C2C12 cell. This result strongly indicates that our newly developed scaffold increase cell adhesion rates and results in DICAM mediated cell adhesion. In the previous study, Silk Fibroin helps to osteoblast cell differentiation.
A Novel Fabrication and High Performance of 3-Dimensional Highly Porous Silk Nanofiber Scaffold Prepared by Electrospinning
We have developed a modified electrospinning method to make a 3-D nanofibrous silk fibroin scaffold (NSFS) with high porosity, which is also important for cell adhesion and proliferation. In this new method, as-spun nanofibers are collected in a coagulation bath and formed in a dispersed state. The dispersion is solidified by freeze-drying, resulting in a freely-shaped sponge-like 3-D nanofibrous scaffold. After evaluating the morphological structures and cell culture analyses of this 3-D scaffold, we have concluded that seeded cells would easily adhere and proliferate on 3-D NSFS because of its high porosity compared with 2-D type. To test the newly developed 3-D NSFS, we seeded an osteoblast cell line, MC3T3-E1, on to both 2-D and 3-D NSFS and compared cell adhesion and proliferation between groups for 7 days using scanning electron microscopy, MTT colorimetric assay and immunoblotting.
Mathematical Model of Cartilage Transplant in Perfused Bioreactor
We describe a continuous modelling and simulation framework aimed at monitoring and steering a recently developed bioreactor for cartilage production based on mesenchymal stem cells (MSCs). This bioreactor provides online-access to the cell density by acoustic measuring sections. An appropriate oxygen distribution throughout the bioreactor is essential for a successful expansion and chondrogenic differentiation of the cells. We use experimentally validated knowledge on the oxygen dependence of MSC organisation in order to simulate this distribution. The aim of the present study is to verify in a spatially three-dimensional setting that our modelling framework is suitable for monitoring the relevant processes in the bioreactor and consequently can support optimization of protocol for cartilage formation in the bioreactor.
Synergistic Effect of Stromal Cell-Derived Factor-1 and Bone Morphogenetic Protein-2 Released from Gelatin Hydrogels for In Vivo Bone Regeneration
Gelatin hydrogels with or without incorporation of stromal cell-derived factor-1 (SDF-1), bone morphogenetic protein-2 (BMP-2), or mixed SDF-1 and BMP-2 were implanted in a critical 6-mm ulna defect of Wistar rats. The bone regeneration was analyzed by soft x-ray, micro-computed tomography (μCT), and histological examinations. An ectopic bone formation model with the hydrogels was prepared to evaluate the expression of CXCR4 (a cell-surface receptor of SDF-1), Runx2, and osteocalcin genes. In vivo recruitment of mesenchymal stem cells by the hydrogels was also examined for GFP-chimeric mice. In addition, following the hydrogels incorporating 125I-labeled SDF-1 and BMP-2 were implanted into back subcutis of ddY mice, the release profile of both factors was assessed. Gelatin hydrogels incorporating mixed SDF-1 and BMP-2 showed stronger bone regeneration in a critical defect than the empty hydrogels and those incorporating either SDF-1 or BMP-2. The phenomenon was experimentally supported by a significant up-regulation of CXCR4, Runx2, and osteocalcin genes 3 days and 4 weeks after implantation. Histological studies showed the ectopic bone formation induced by the hydrogels incorporating mixed SDF-1 and BMP-2. SDF-1 released enhanced the recruitment of GFP-positive cells around the site of hydrogels implanted. The release study demonstrated a controlled simultaneous release of SDF-1 and BMP-2 from the gelatin hydrogels. Dual release of SDF-1 and BMP-2 from the gelatin hydrogels showed the synergistic effect on bone regeneration through both the enhanced of cell recruitment and up-regulated osteogenic genes.
Assessment of the Effects of the Biosilicate Scaffold on Bone Regeneration in Tibias of Rats
The aim of this study was to investigate the effects of Biosilicate® scaffolds on bone regeneration of tibial callus of rats. Twenty male Wistar rats were randomly distributed into two groups of 10 animals each: control group (bone defects without any fillers) and Biosilicate® group (bone defects filled with the Biosilicate scaffold). A 2.5-mm-diameter non-critical size bone defects was surgically created at the upper third of the left tibia. Immediately afterwards, bone cavities were completely filled with the scaffold l in the treated animal groups. On day 20 after the surgical procedure, animals were euthanized and tibias were harvested for analysis. A descriptive qualitative histopathological evaluation of the total area of the bone defect was performed by a pathologist. Also, the biomechanical properties of the tibia were determined by a three-point bending test in an Instron® Universal Testing Machine. The maximum load at failure (N), structural stiffness (N/mm) and energy absorption (J) were obtained. Statistical analysis was made using the one-way analysis of variance (ANOVA), complemented by Kruskal-Wallis test. Values of p < 0.05 were considered statistically significant. The results pointed out intense new bone formation surrounded by highly vascularized connective tissue presenting a slight osteogenic activity, with primary bone deposition was observed in the treated group compared to control group.
The Effects of Biosilicate® and Low Level Laser Therapy on Tibial Bone Consolidation in Osteopenic Rats: a Histological Study
It was used 40 female osteopenic rats, divided into 4 groups: osteopenic fractured control (OC); osteopenic fractured treated with Biosilicate® (FBio), osteopenic fractured treated with laser (OL); osteopenic fractured treated with Biosilicate® and laser (OFLBio). Biosilicate® is an osteogenic biomaterial developed by our research group which is a novel fully-crystallized bioactive glass-ceramic of the quaternary P2O5-Na2O-CaO-SiO2 system (Biosilicate®, patent application WO 2004/074199). Two months after the surgery of ovariectomy (to induce osteopenia), a 2.5-mm-diameter bone defect was created in the tibia of the rats. Immediately afterwards, bone cavities were completely filled with the corresponding biomaterial in the treated animal groups. The laser used was a GaAlAs laser, 830 nm, 100 mW, 120 J/cm2. The laser irradiation was performed during 12 sessions. On day 14 after the surgery, the animals were euthanatized and the left tibias were defleshed and prepared for a histological analysis. The specimens were embedded in paraffin and sectioned with 5 micron thickness. Histological sections were stained with hematoxylin and eosin (H&E) and they were performed under a light microscope. Changes occurred in the surgical area, presence of fibrous granulation tissue, signs of bone neoformation and bone union were investigated per animal. Results: Animals of the control group presented a high concentration of fibrous granulation tissue and a lower presence of neoformed bone. In the groups laser and laser and Biosilicate® it could be observed a lower presence of granulation tissue and a high presence of neoformed bone compared to control. Some of the animals of the OL and OFLBio groups showed signs of fracture union. The animals treated with Biosilicate presented histological findings similar to the controls.
Biocompatibility of Collagen Scaffolds from Skin of a Novel Marine Source
The collagen scaffolds were fabricated by freeze-drying of 2.3% collagen slurry in 0.05 M acetic acid solution, pH 3.6 cross-linked at 4°C with carbodiimide (2.2% w/w of collagen). Microstructure and pore size of the scaffolds were investigated by scanning electron microscopy (SEM). Surface charge of the scaffolds was analyzed by zeta potential measurement. The swelling ratio of the scaffolds was determined in phosphate buffer saline (PBS) at 37°C. Fourier transform infrared spectroscopy (FT-IR) of the scaffolds was determined. Cytotoxicity of the scaffolds on MC3T3-E1 cells was observed using MTT assay. The collagen scaffolds were obtained with average pore size of 204.13 ± 37.69 mm. SEM photos indicate interconnecting porous structure of the scaffolds. The zeta potential of the scaffolds was found as 25.26 ± 0.65 mV. The positive charged matrices and scaffolds have been reported to be appropriate for cell-polymer interaction. Swelling ratio of the scaffolds in PBS was found as 5.79 ± 0.15 g/g scaffold. The FT-IR spectra indicate shifting of FT-IR bands of amide bonding of the scaffolds after cross-linking. The cytotoxicity assay revealed the well defined biocompatible scaffolds to the MC3T3-E1 cells.
Biocompatibility Testing of a Wear-Resistant Cobalt Chromium Alloy
Immunological compatibility was evaluated by measuring T cell specific cytokines following culture of peripheral blood mononuclear cells on modified cobalt chromium (mCoCr). Additionally, proliferation and viability of Saos-2 cells was determined after culture on mCoCr. Furthermore, thrombogenic activity and haemolysis were analysed by determination of the activation dependent thrombocyte surface marker CD62P and free haemoglobin in the supernatant, respectively. Results were compared to base CoCr (CoCr) alloy and polystyrene (PS). Determination of spontaneous released cytokines revealed reduced concentrations of IFN-gamma and IL-10 while TNF-alpha was increased in mCoCr samples compared to CoCr and PS. Compared to CoCr and PS Saos-2 cells showed a decrease in proliferation and viability when cultured on mCoCr. Furthermore, activation of thrombocytes and lysis of erythrocytes in mCoCr samples was comparable to CoCr and PS. The PIII increases the hardness of the CoCr surface but alters the secretion of T cell specific cytokines as well as proliferation and viability of Saos-2 cells. The modification through PIII caused an increased Co2+ release from the material. It is likely that the elevated Co2+ concentration plays a critical role in the alteration of cell properties.
The Biocompatibility of a Novel Bioactive Glass-Like Material
The here evaluated glass-like material is defined through silsesquioxane units which form polymeric strands that are cross-linked eventually. The molecular morphology of the resulting solids features enhance fracture toughness which allow for the generation of open porous scaffolds. Saos-2 as a relevant osteogenic cell line and peripheral blood cells were used to determine the biocompatibility of the material. Saos-2 cells were cultured on bioactive glass constructs for 6 days. They well attached to the material. Showed their typical morphology and proliferated. After 6 days, cells covered approximately 90% of the material surface. Compared to the tissue culture polystyrene control, however, decreased mitochondrial activity was found on bioactive glass. Peripheral blood cells were used to survey immunological compatibility, thrombogenic and haemolytic activity of the material at different time points. Cytokine secretion of lymphocytes in contact with bioactive glass and tissue culture polystyrene in response to immunogenic stimulation was determined. We found reduced concentrations of TNF-alpha, IL-2 and IL-10 when lymphocytes were cultivated on bioactive glass. The material induced neither thrombocyte activation nor haemolysis. Taken together, the investigated bioactive glass showed good cellular biocompatibility.
Assessment of New Bone Formation after Alveolar Distraction Osteogenesis Using Fractal Dimension Analysis: Quantitative Study
Alveolar distraction osteogenesis (ADO) technique can be used for enhancing the shape, volume, and mechanical strength of the alveolar bone for the dental implant placement. The fractal dimension analysis (FDO) is a kind of tools to quantify structural information of natural objects. Because bony trabecular pattern of distracted callus resembles typical fractal structures, we tried to evaluate the radiographic changes after ADO quantitatively using FDO. Total 14 distraction sites of 11 patients who underwent alveolar distraction osteogenesis (without any complications) were checked with panoramic radiographs during 3 years. The fractal dimension was calculated by means of a tile-counting method in the region of interest (ROI) centered on the distracted callus bone. The fractal dimension in the distracted callus bone changed markedly, the initial fractal dimension value decreased after full distraction stage but increased gradually according to the time lapse (p < 0.05). After 1 year, the value of ROI became very similar as the pre-distracted alveolar bone fractal dimension value in most of patients. Fractal dimension can be used as an indicator to evaluate the callus healing process after alveolar distraction osteogenesis. Alveolar distraction osteogenesis is also useful technique for augmentation of atrophied alveolar ridge.
Effect of Electron Beam Irradiation to the Cellulose Membrane for the Guided Bone Regeneration
We have developed cellulose membrane (CM) from Styela clava skin, a native sea squirt, to make a bone regeneration membrane. We used 0.5-1.5 MeV linear accelerator and 2.0 MeV superconductive linear accelerator with different irradiation dose such as 1, 10, 20, 40 kGy. Structural changes were studied in vitro, by SEM (Scanning Electron Microscopy), elementary analysis using FE-SEM (Field Emission Scanning Electron Microscopy), amino acid analysis, ATR-IR and XPS-ESCA analysis.
Very small amounts of peptide fragment derived from this CM, such as CCP (Cellulose Crosslinking Protein) could be lost its own structure. Scissioning of carbohydrate polymers was observed in SEM findings, and uniform turgor pressure to keep own directions of CM was changed to be elongated after E-beam irradiation. Arrangement of microtubules can also be changed indirectly, so cellulose and enzyme complexes were able to be migrated in the plasma membrane. Tensile strength of CM can be changed because any polysaccharides cross-link was able to lose their own resistance to compression. CCP was lost after electron beam (E-beam) irradiation, and cross-linking binding of each fibrils was detached each other. So clinical application of E-beam irradiated CM as the biodegradable or resorbable membrane in the bone regeneration field could be suggested.
In Situ Bone Tissue Engineering - Migration Potential and Osteogenic Gene Expression Profile of Human Periosteal Cells
We have developed clinically applied 3D cell culture techniques comprising the use of bioresorbable biomaterials combined with periosteal cells (PCs) mixed with fibrinogen that facilitate the formation of bone. First, in vitro migration of PCs toward different concentrations of chemokines was investigated in a chemotaxis assay. Then, isolated and cultivated PCs were mixed with human fibrinogen and encased in PGLA fleeces. After 28 days of osteogenic induction, matrix mineralization and cell viability were measured. RNA from culture-expanded PCs and 3D bone grafts were used for gene expression profiling using Affymetrix GeneChips and were subsequently analyzed by pattern discovery for differentially expressed genes. For the first time, we showed a chemotactic effect of chemokines (CXCL8, CXCL12, CXCL13, CCL2 and CCL9) on PCs. After 28 days of differentiation matrix formation and cell viability was confirmed. Moreover, the genome-wide microarray data displayed the entire expression profile of undifferentiated and in 3D culture differentiated PCs. About 900 genes were differentially regulated including known and new bone marker genes. We could demonstrate that PCs migrate upon stimulation with chemokines and in our 3D culture system form functional bone tissue. Therefore, PCs fulfill all prerequisites for in situ bone Tissue Engineering.
Hydroxyapatite Coated Microporous Nano-Cellulose for Bone Healing Applications
The porous and crystalline morphologies of the nano-cellulose scaffolds were analyzed using SEM. The deposited mineral was analyzed with SEM/EDX, X-ray crystallography, ESCA and FTIR. The mechanical properties were determined using compression measurements. The mineralized microporous nano-cellulose scaffolds were seeded with MC3T3-E1 osteoprogenitor cells, human adult mesenchymal stem cells and fetal human mesenchymal stem cells. Cultures were continuously analyzed with MTS assay and ALP assay to determine relative cell number and degree of differentiation respectively. Results from SEM and SEM/EDX confirmed hydroxyapatite (HAP) crystals deposited onto the nano-cellulose, resulting in an environment resembling native bone tissue's mineralized extra cellular matrix. Preliminary results from the cell study indicate increased cell proliferation and differentiation on porous mineralized scaffolds compared to non mineralized scaffolds. The HAP modified microporous nano-cellulose scaffolds are a promising biomaterial for bone repair applications.
Visualization of Collagen Type I Production by Osteoprogenitor Cells in Microporous Nano-Cellulose Scaffolds
Nano-cellulose biosynthesized by bacteria is an attractive 3D biomaterial scaffold for bone tissue engineering due to its biocompatibility and good mechanical properties. Wax porogens have been introduced during the cultivation process of the bacteria and were thereafter leached out leaving an interconnected microporous structure. Such structures are thought to facilitate osteoprogenitor cell ingrowth and formation of extracellular matrix (ECM). Coherent Anti-Stokes Raman Scattering (CARS) microscopy was used for the visualization of cell integration, proliferation and differentiation and Second Harmonic Generation (SHG) microscopy was used for imaging of cellulose and collagen fibers. The presence of collagen type I fibers were verified and quantified by ELISA, and alkaline phosphatase activity was measured to verify osteoblastic differentiation. We have shown that osteoprogenitor cells can proliferate and differentiate into microporous nano-cellulose scaffolds. CARS and SHG microscopy, in addition to visualize the cells in the pores, also provide evidence of extracellular matrix production. The cell assays are ongoing and preliminary SHG results indicate collagen production.
In Vitro-Generated Extracellular Matrix Enhances Osteogenic Properties of Hydroxyapatite In Vivo
MTT assay, protein assay, Western Blot analysis, immunohistochemistry and scanning electron microscopy were used to assess cellular viability, proliferation and extracellular matrix (ECM) synthesis onto synthetic hydroxyapatite (HA) microparticles after 21 days of culture. The osteogenic properties of the decellularized HA-ECM scaffolds were assessed in vivo using a rat calvarial critical size defect model. Newly formed bone, biomaterial-tissue interface as well as local inflammatory response were evaluated at 12 weeks postoperatively. HA microparticles supported cellular adhesion, growth and ECM production in vitro, and resulted in enhanced bone repair in defects treated with HA-ECM scaffold, compared to the HA alone, despite the higher local inflammatory response. The tissue-biomaterial interface composition differed between bone-integrated and nonintegrated HA microparticles. Our results highlight the potential of in vitro-generated ECM as a viable scaffold for bone tissue engineering applications. ECM deposited onto HA enhances its osteogenic capacity by modulating the local inflammatory response in a repair-favorable way.
Calcium Phosphate Coating on Dense and Porous Titanium Substrates
Commercially titanium (Ti) plates and porous Ti, produced by powder metallurgy, were used as substrates. The samples were pre-treated for surface bioactivation by means of a 1M NaOH solution followed by heat-treating at 200°C. Then the samples were immersed in a simplified solution (SS), which contains salts of calcium and phosphorus, for 7, 14 or 21 days. Surface topography of the samples without calcium phosphate (CaP) coating was performed by confocal scanning optical microscopy. Phase characterization of the CaP coatings was achieved by low angle x-ray diffractometry and the CaP microstrucure was identified by scanning electron microscopy with energy dispersive x-ray. Optical metallographic quantitative analysis was used to determine the total porosity.
The porous samples presented close micropores, interconnected macropores and porosity in the range of 60 to 70%, achieving the requisites for implant surfaces and scaffolds applications. CaP precipitations with distinct morphologies were obtained in dense and porous samples, immersed for 7, 14 and 21 days in SS solution. Different CaP microstructure types were observed.
The Effect of Adult MSC on Bone Regeneration and Osseointegration of Dental Implants Following a Sinus Augmentation Procedure
For this pilot study 6 patients providing a severely and symmetrically resorbed alveolar ridge in the posterior maxilla and requiring implant treatment were included. A bone marrow aspirate (approx. 10ml) from the proximal tibia was harvested during bilateral sinus augmentation procedure. At the randomly selected test side the BMA was added to the bone substitute (porous bovine bone mineral, BioOss). At the control side the augmentation procedure was performed with the bone substitute only. Approximately 2ml from the BMA was checked for MSC quality and content.
Biopsies were taken from both sides 3 and 6 months after bone augmentation and analysed with histomorphometry. CT scans, intraoral radiographs and OPTGs are used for radiographic evaluation of the periimplant bone volume and height at the augmented sites.
Array of Tests for Determination of Cytotoxicity and Cell Proliferation in Cultures for Tissue Engineering
Viability, cell proliferation and cell death were evaluated in rapidly and slowly proliferating human fibroblasts, osteosarcoma cell line (MG-63) and endothelial cells (HUVEC) exposed to cadmium chloride (CdCl2) which is a well characterized cytotoxic agent. Neutral red (NR) and 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) tests were performed to measure cell viability. Proliferation was assessed by DNA synthesis (BrdU assay) or Ki-67 expression measurement. The percentage of apoptotic/necrotic cells was measured by dead-live staining and activity of lactate dehydrogenase. (Reference values of DNA (PicoGreen, crystal violet) and protein content (Pierce) were measured.) We found discrepancy between CdCl2 concentration and time of culture and the cytotoxicity indicated by the employed assays. This may be due to the diverse sites of action in a cell targeted by the applied tests (eg. up-regulation of lisosomal activity by CdCl2 resulted in false positive NR results). Also, we observed that the results differed between the tested cell types.
Alginic Acid Gel for Bone Marrow Cell Seeding to Form Bone in Porous Hydroxyapatite Scaffolds
Generally, bone marrow cells (BMCs) at 1×107 cells/ml or higher concentration are needed for osteogenesis in porous hydroxyapatite (HA) scaffold in vivo. BMCs were obtained from bone shaft of femurs of 6-week-old male Fischer 344 rats. Cylindrical porous HA scaffolds were received alginic acid gel with BMCs at 1×106 cells/ml concentration. The other scaffolds were immersed in culture medium with BMCs at the same concentration. The scaffolds were respectively implanted in subcutis of 7-week-old Fischer 344 rats for 8 weeks. Serially cut paraffin sections were made from the removed specimens. Percentage of pores including bone to total pores was calculated in each of randomly selected 10 sections from every scaffold. The results were statistically analyzed by Welch's test (P < 0.01). The ratio of pores with bone was over 70 % of total pores in the scaffold with BMCs in alginic acid gel. The ratio was less than 5 % in the scaffold immersed in culture medium with BMCs. The difference was clearly shown. Bone was formed in most of pores in HA scaffold by BMCs in alginic acid gel. It is concluded that alginic acid gel may contribute to in vivo osteogenesis conspicuously by BMCs in the porous HA scaffold.
Dental Scaffold for Tooth Extracation Socket Regeneration
This study aims to evaluate the safety and efficacy of a bioresorbable 3-dimensional (3D) polycaprolaconte (PCL) scaffold in maintaining bony architecture of the alveolar ridge following tooth extraction. Normal post-extraction healing response of an alveolar ridge is resorptive in nature and this decrease in alveolar ridge volume may preclude optimum dental implant placement. Bone grafting prodecures may then be necessary, resulting in additional surgery, morbidity and cost.
In Vitro Biomimetic Construction of Porcine ADM-HAp Composite Scaffold for Bone Regeneration
Porcine acellular dermal matrix (PADM) framework was extracted from porcine skin, on which Nano hydroxyapatite (Hap) was fabricated to form porcine acellular dermal matrix-hydroxyapatite (PADM-Hap) composite scaffold through a two-step biomimetic mineralization process. Mesenchymal stem cells (MSCs) were seeded on the scaffold and implanted in rat muscle. The tissue with implants were harvested 2, 4 and 8 weeks later and evaluated histologically. X-ray diffraction patterns and SEM results indicated HAp nano-structure formed on the scaffold. In vitro cell culture shows that MSCs can proliferate on the scaffolds with and without HAp fabrication. After implantation in rat muscle, both PADM framework and PADM-HAp composite scaffold exhibit good in vivo biocompatibility. New bone appears 4 weeks later after implantation. It shows that the new bone tissue can be induced more easily in PADM-HAp scaffold than that in pure PADM. After implantation for 8 weeks, the amount of new bone tissue becomes larger and larger. The new inorganic phase formed in vivo are quiet different from the flake-like HAp fabricated in vitro.
Abstract Author Index
by abstract number
Åberg, J., P-26
Acham, S., P-51
Agata, H., P-20
Aguirre, M., P-7, P-8
Ahlbrand, L., P-1
Alexander, D., O-1, P-3
Almeida, M., P-2
Alt, V., O-14
Angrisani, N., O-21
Ardjomandi, N., O-1, P-3
Aro, H., P-34
Arrigoni, E., O-2
Azevedo, L.H., O-16
Bae, J.-H., P-22
Baumann, W., P-4
Beck, U., P-5
Benjakul, S., P-32, P-41
Berg, A., P-11
Bernhardt, A., O-23, P-16
Blacklock, J., O-3
Boehm, S., P-23
Böhm, S., O-4
Bongio, M., O-5
Bonk, S., P-5
Bonk, S.M., P-4
Bormann, D., O-19, O-21
Bossinia, P.S., P-40
Bostelmann, R., P-1
Boussahel, A., O-6
Bouthors, S., O-7
Brackmann, C., P-48
Braux, J., O-7
Brini, A.T., O-2
Brooks, R.A., O-15
Bruinink, A., P-18
Bullerdiek, J., O-11
Burchielli, S., P-10
Busacca, M., O-8
Cai, L., P-6
Caminal, M., P-7, P-8
Campbell, E.C., P-17
Cao, Y., P-6
Capela-Silva, F., O-20
Chan, J., O-29, P-29
Chen, L., O-26, O-30, P-55
Cheung, E.N.M., O-14
Cheung, L.W.H., O-14
Chevallier, N., P-9
Chiono, V., P-10
Chong, M. SK, P-29
Choolani, M.A., P-29
Chow, S.K.H., O-14
Ciach, T., O-28
Ciardelli, G., P-10
Clyne, T.W., O-15
Conconi, M.T., P-33
Cooper-White, J.J., O-25
Coquelin, L., P-9
Costa, M.E.V., P-2
da Rocha, M. Navarro, P-50
Dallari, D., O-8
Dalzoppo, D., P-33
de Andrade, M.C., P-50
de Girolamo, L., O-2
de Oliveira, M. Varella, P-50
Dediu, V., O-22
Del Piccolo, N., O-8
Di Liddo, R., P-33
Dinnes, D.L.M., O-25
Domeneghini, C., O-2
Eckervogt, V., O-11
Elter, P., P-5
Enejder, A., P-48
Engqvist, H., P-26
Eo, M.Y., O-17, P-44, P-45
Esteves, M., P-7, P-8
Fantasia, R., O-8
Feijoo, J.L., P-14
Feltrin, G., P-33
Fernandes, M.H., O-10
Fernández, A., P-8
Ferreira, A.M., P-10
Fialaire-Legendre, A., P-9
Fiedler, S., P-11
Finkenzeller, G., P-12
Frank, J., O-9
Frantzén, J., P-34
Frias, C., O-20
Frieß, W., P-27
Furuya, H., P-38
Galle, J., P-37
Garcia, J., P-7, P-8
Gatenholm, P., P-47, P-48
Geis, R., O-23
Gelinsky, M., O-23, P-16
Gentile, P., P-10
Gerisch, A., P-37
Giazzon, M., P-13
Gimsa, J., P-4, P-5
Giraudo, C., P-33
Gomes, P.S., O-10
Gonzalez, G., P-14
Gonzalez-Paz, R.J., P-14
Goo, T.-W., P-35
Goranov, V., O-22
Gosheger, G., O-11
Grandi, C., P-33
Guillaume, C., O-7
Gwellaouen, P-9
Haimi, S., P-34
Han, S.M., P-25
Haranava, Y., O-22
Hatlapatka, T., P-23
Hauschild, G., O-11, P-15
Hayashi, H., P-53
Heimann, L., O-14
Heino, H., P-34
Henrich, D., O-9
Henrichs, M., O-11
Hildebrand1, G., P-11
Hoffmann, J., O-1
Hoyer, B., O-23, P-16
Hu, J., P-6
Hu, S., O-30, P-55
Huehnerschulte, T.-A., O-21
Huguet, P., P-7, P-8
Hulsart, G., P-26
Hutchison, P.D., P-17
Hutmacher, D.W., P-54
Idaszek, J., P-18
Ip, W.Y., P-30
Jallot, E., O-7
Jankowska-Steifer, E., O-28
Jansen, J.A., O-5
Jo, Y.-Y., P-19, P-35
JO, Y.Y., P-25
Kakigi, H., P-53
Kalaszczyńska, I., P-52
Kalbacher, H., P-3
Kamprad, M., P-42, P-43
Kang, D.C., P-25
Kang, J.Y., O-17, P-44, P-45
Kang, S.-W., P-19, P-35
Kang, S.W., P-25
Kasper, C., O-4, P-23
Keogh, C., O-13
Khanna-Jain, R., P-20
Khatami, F., P-21
Ki, C.S., P-36
Kim, H.-J., P-22
Kim, H.J., P-36
Kim, H.K., P-22
Kim, J.W., P-36
Kim, S.E., P-22
Kim, S.M., O-17, P-44, P-45
Kim, S.R., P-35
Kim, S.Y., P-36
Kirmeier, R., P-51
Kiss, M.-O., O-12
Klein, C., P-3
Kleinke, P., P-43
Koob, S., P-12
Koon, Y.S., P-54
Kop, A., O-13
Koziak, K., P-28, P-52
Kress, S., P-23
Kröger, T., P-4
Kumar, A., P-4
Kuo, Y.-C., P-24
Kweon, H.Y., P-19, P-25
Kweon, H.-Y., P-35
Kwon, K.Y., P-44
Lacza, Z., P-26
Lambertini, E., O-18
Landi, E., O-22
Lankenau, A., O-3
Laquerriere, P., O-7
Laurent-Maquin, D., O-7
Lavigne, P., O-12
Lee, J.H., O-17, P-44, P-45
Lee, K.-G., P-19, P-35
Lee, K.G., P-25
Leeuwenburgh, S.C.G., O-5
Leicht, U., P-27
Leszczynska, J., P-28
Leung, K.-S., O-14
Levasseur, A., O-12
Lewandowska-SzumieŁ, M., O-28, P-28, P-52
Liefeith, K., P-11
Liley, M., P-13
Lips, K.S., O-14
Lira-Olivares, J., P-14
Liu, C., P-6
Liu, D., O-26, O-30, P-55
Liu, H., O-26, O-30, P-55
Liu, X., P-6
Liu, Y., P-29
Loch, A., P-46
Lora, S., P-33
Lui, Y.F., P-30
Lutz, J., P-42
Ma, H., P-6
Machluf, M., O-4
Malheiro, V.N., O-15
Mändl, S., P-42
Marcacci, M., O-22
Marchiori-Silva, V., O-16
Mariner, P., O-24
Markaki, A.E., O-15
Marques, A.T., O-20
Martínez, M.J., P-8
Marzi, I., O-9
Matthey, N., P-13
Matthias, P., P-27
Maurer, A., P-3
Meyer, M., O-23, P-16
Meyer-Lindenberg, A., O-19, O-21
Miettinen, S., P-20, P-34
Mikos, A.G., O-5
Mobini, S., P-31
Möhwald, H., O-3
Moretti, P., P-23
Morrison, D., O-13
Munz, A., O-1
Myers, P.T., O-25
Myoung, H., O-17, P-44, P-45
Nastruzzi, C., O-18
Nedelec, J.-M., O-7
Niknejad, H., P-21
Nilasaroya, A., O-13
Nogueira Zambone Pinto, K., P-39
Noris-Suarez, K., P-10
Noris-Suárez, K., P-14
Ohgushi, H., P-53
Ortolani, A., O-22
Oungbho, K., P-32, P-41
Oupický, D., O-3
Pacha, D., P-8
Paganin, P., P-33
Pälli, A., P-34
Pankotai, E., P-26
Panseri, S., O-22
Parizotto, N.A., P-39, P-40
Park, J.M., O-17, P-44, P-45
Park, S.W., P-25
Park, S.-W., P-35
Park, Y.H., P-36
Parnigotto, P.P., P-33
Pathak, P., P-37
Paulmann, C., O-27
Payer, M., P-51
Peirovi, H., P-21, P-31
Peitl-Filhoc, O., P-40
Penolazzi, L., O-18
Pereira, L.C., P-50
Petit, Y., O-12
Piva, R., O-18
Pla, A., P-7, P-8
Potes, J., O-20
Powis, S.J., P-17
Przybillab, J., P-37
Rah, D.K., P-36
Rahman, C., O-6
Rani, N., O-8
Ratanavaraporn, J., P-38
Reifenrath, J., O-19
Reinert, S., O-1, P-3
Reis, J., O-20
Ren, N., O-26, O-30, P-55
Rennó, A.C., P-40
Rennó, A.C.M., P-39
Ribeiro, A.A., P-50
Ribeiro, M.S., O-16
Ringe, J., P-46
Rittershaus, D., O-21
Rosal, M., P-7, P-8
Rose, F.R.A.J., O-6
Roseti, L., O-8
Rossi, C., O-18
Rothe, H., P-11
Russo, A., O-22
Samadikuchaksaraei, A., P-31
Sándor, G.K.B., P-20
Sangsen, Y., P-32, P-41
Santos, C., P-2
Schade, R., P-11
Schäfer, A., O-11
Scheper, T., O-4
Schieker, M., P-27
Schlögl, W., P-27
Schmaeschke, O., P-2
Schnabelrauch, M., P-11
Schneider, H., P-42, P-43
Schnettler, R., O-14
Schröck, K., P-42, P-43
Schultheiss, J., O-9
Schulz-Siegmund, M., P-42, P-43
Seebach, C., O-9
Sen, H.K., P-54
Seo, M.H., O-17, P-44, P-45
Shakesheff, K.M., O-6
Shelyakova, T., O-22
Silva, C.F., P-2
Simões, J.A.O., O-20
Sittinger, M., P-46
Sobral, J.M., O-15
Solati-Hashjin, M., P-31
Song, H.R., P-22
Song, X., O-30, P-55
Spear, R.L., O-15
Stachel, I., O-23, P-16
Stagni, C., O-8
Stanco, D., O-2
Stark, G.B., P-12
Steiger, H.-J., P-1
Stich, S., P-46
Stigler, B., O-14
Streubel, S.-O., O-24
Stubbe, M., P-4
Sundberg, J., P-47, P-48
Suuronen, R., P-20
Święszkowski, W., P-18
Tabata, Y., P-38
Tan, D., P-54
Tan, G.-K., O-25
Tcacencu, I., P-49
Teoh, S.H., O-29, P-54
Teoh, S.-H., P-29
Thomann, M., O-19
Tim, C., P-39
Tin, G.B., P-54
Torio-Padron, N., P-12
Torreggiani, E., O-18
Tour, G., P-49
Tsai, Y.-T., P-24
Tsuji, N., P-53
Upu, Y., O-4
Valentini, F., O-22
van den Beucken, J.J.J.P., O-5
van Griensven, M., O-4
Vecchiatini, R., O-18
Velard, F., O-7
Vélez, R., P-7
Vetter, A., O-3
Vives, J., P-7, P-8
Volkmer, E., P-27
Vuorinen, A., P-20
Wang, G., O-26, O-30, P-55
Wang, J., O-26, P-6
Wang, Q., P-6
Wang, T., O-30, P-55
Weder, G., P-13
Weihe, T., P-5
Wendel, M., P-49
Weszl, M., P-26
Wiese, H., P-27
Wildburger, A., P-51
Wilhelm, K., O-9
Windhagen, H., O-19
Windhager, N., P-51
Windhager, R., P-51
Wirthmann, A., O-27
Wójtowicz, J., O-28, P-52
Woo, S.O., P-25
Wyrwa, R., P-11
Xiao, R., P-6
Xie, F., P-6
Yan, L., P-6
Yeo, J.-H., P-19
Yeo, J.H., P-25
Yin, H., P-6
Yook, T.L., P-54
Yoshikawa, M., P-53
Yuen, C.K., P-30
Yun, Y.P., P-22
Zaborowska, M., P-47, P-48
Zanottoc, E.D., P-40
Zhang, Z., P-29
Zhang, Z.-Y., O-29, P-54
Zhao, H., O-26, O-30, P-55
Zheng, L., O-26
Zinn, M., P-18