Abstracts-Bone Tech 2008 Meeting Abstracts

1) Plastic Compressed Collagen Reinforced with Phosphate-Based Glass for Bone Tissue Engineering

We report a technique for production of potential bone substitutes based on plastic compressed collagen (PCC) incorporating phosphate-based glass (PG) particles. PC is a technique for rapid fabrication of dense tissue-like constructs proposed for bone engineering. The chemistry of PG can be tailored. PCC-PG blending produces a bio-mimetic model of hard tissues, comprising extra-cellular matrix and mineral phases.

PC constructs were prepared1 with addition of 0.50 (P2O5) - 0.30(CaO) - 0.17 (Na₂ O) - 0.03 (Fe₂O₃) glass particles (17-20 um) in two concentrations: 1:1 and 1:4 (wt:wt) relative to total collagen (2 mg/ml). Human osteosarcoma cells (5 × 10⁵/ml) were cultured in constructs for 21 days. Cell viability was measured (0d, 7d, 14d, 21d) using Live-Dead staining. Mechanical properties were measured (compression mode) using dynamic mechanical analysis.

Compression (at day 0) did not affect cell viability, however viability decreased significantly in 1:1 and 1:4 constructs compared to zero PG controls (p < 0.05) by day 21 (70.6%, 78.3% and 88% respectively). Constructs with 1:1 showed significantly changed compressive modulus (32 ± 7 kPa) compared to collagen only (22 ± 1 kPa). At 1:4, compressive modulus was reduced to 10 ± 4 kPa.

Therefore, incorporation of PG particles into a dense collagen matrix altered compressive strength of constructs, with only modest effect on cell viability and it appears to have considerable potential for engineering bone tissue.

2) CD271: An Osteogenic Marker Distinguishing between Mineralizing and Non-Mineralizing Periosteal Cells

Our study aims at finding differentiating markers which distinguish between mineralizing and non-mineralizing periosteal-derived progenitor cells (PDPC) to improve the success of tissue engineering applications. Nerve growth factor receptor (NGFR/CD271) represents a specific marker for bone marrow stem cells. While NGFR expression is not prominent in undifferentiated PDPC, we analysed its expression during osteogenesis.

Mineralization capacity of PDPC was detected by alizarin and von Kossa stainings. We screened the expression of several stem cell surface markers in mineralizing versus non-mineralizing PDPC by flow cytometry. NGFR mRNA expression analyses followed in both cell groups by quantitative PCR. NGFR protein expression was additionally visualized by immunohistochemical stainings and semi-quantitated by western blotting.

We found two PDPC groups which act differently during osteogenesis: the mineralizing cell group was Alizarin- and von Kossa-positive (n = 6), whereas the non-mineralizing cells showed negative stainings (n = 6). By FACS-analyses, NGFR was induced during osteogenesis at higher levels in mineralizing PDPC in comparison to non-mineralizing PDPC. We obtained similar results by immunohistochemical stainings and western blot analyses. Significantly higher transcript levels were detected by quantitative real-time PCR in mineralizing PDPC in comparison to non-mineralizing PDPC.

NGFR represents a potential differentiating marker with unknown function which distinguishes between mineralizing and non-mineralizing periosteal cells during osteogenesis.

3) Upregulation of Specific ECM Components during Osteogenesis of Periosteal Cells

Jaw periosteal cells (JPC) could be an alternative to mesenchymal stem cells for the engineering of cell-based osteoinductive grafts. Harvest of the jaw periosteum is simple and causes minimal morbidity. We analysed in this study the expression of different collagen types and extracellular matrix (ECM) components during osteogenesis of JPC.

Three cell groups were analysed: 1) untreated JPC, 2) JPC treated with differentiation medium and 3) JPC treated with differentiation medium and BMP-2. JPC proliferation and gene expression analyses of the ECM components were determined at day 5, 10 and 20 of osteogenesis. Protein expression was analysed at the same time points. JPC osteogenic differentiation capacity was confirmed by alizarin and von Kossa staining and by upregulation of alkaline phosphatase expression.

Gene expression analyses during osteogenesis of periosteal cells revealed a strong elevation of type VII, VIII and XI collagen gene expression at much higher extent than type I collagen. Type I, VIII and XI, but not type VII collagen seemed to be susceptible to BMP-2. Furthermore, cartilage oligomeric matrix protein (COMP) and the tissue inhibitor of metalloproteinases-4 (TIMP-4) were strongly upregulated during osteogenesis of periosteal progenitor cells.

We identified new collagen types and ECM components which seem to play an important role during osteogenesis of periosteal progenitor cells. Type I collagen provides the basic scaffolding for bone tissue. The newly identified upregulated collagen types as well as COMP and TIMP-4 probably participate to strong cross-linking of released ECM components and to inhibition of ECM degradation.

4) The Effect of Hydroxyapatite/Collagen I Composites, Bone Marrow Aspirate and Bone Graft on Fixation of Bone Implants in Sheep

Hydroxyapatite and collagen composites (HA/coll) have the potential in mimicking and replacing skeletal bones. This study attempted to determine the effect of newly developed HA/coll-composites with and without bone marrow aspirate (BMA) in order to enhance the fixation of bone implants.

Titanium alloy implants were inserted into bilateral femoral condyles of 8 skeletally mature sheep, four in each sheep. The implant has a circumferential gap of 2 mm. The gap was filled with: HA/coll; HA/coll-BMA; autograft or allograft. Allograft was served as the control groups. The observation period was 5 weeks. The sheep were euthanized and both femurs were harvested. A push-out mechanical test was performed. A two sample t-test was used. The data are given as mean ± SD.

The mechanical testing results were presented as stiffness, shear strength, strain, and failure energy. Autograft/Allograft: Stiffness (MPa) 11.8 ± 11.8/4.3 ± 3.6 (P = 0.11); strength (MPa) 1.58 ± 1.52/0.70 ± 0.6 (P = 0.15); Strain (%) 42 ± 14/45 ± 15 (P = 0.25); Failure Energy kJ/m² 232 ± 228/ 90.4 ± 29.5 (P = 0.25) HA/coll and HA/coll-BMA composite were observed with fibrous tissue between the implant and bone. Thus, mechanical testing of these samples was impossible. The autograft group showed a clear trend for greater mechanical values than allograft group but the differences were not significant. This might represent a type two error due to the low number of animals included.

We conclude that autogenous bone graft may be more efficient than allograft in the fixation of bone implants, a further study with larger sample size is suggested to reveal this issue. HA/coll composite has no effect on the early fixation of bone implants. Whether there is a long-term effect need to be investigated.

5) Phosphorous Acid Polymer Beads for Bone Repair

Osteoporosis is a major disease which affects millions of people worldwide. The risk of re-fracture in osteoporotic patients is greatly increased due to porous and brittle bone. The local administration of drug at the implant site would reduce the risk of re-fracture. The formation of electrospun bead structures could provide a solution to the problem.

A 10% (w/v) solution of polycaprolactone (PCL) was made in acetone. Electrospinning parameters: flow rate of 0.05 ml/min, voltage 20 kV and distance from needle to collector plate of 15 cm. The PCL mats produced were immersed in poly(vinyl phosphonic acid – co- acrylic acid) and rolled into a bead. Beads were heated at 50°C for 30 minutes. SEM analysis was utilised to view cells on the structures. Human osteoblast cells were seeded onto samples and cultured in media (DMEM) containing 10% FBS and 1% antibiotic for 3 days. EDX analysis confirmed the presence of phosphorous.

A novel electrospun bead has been produced which contains a phosphorous acid polymer which has the same P-C-P backbone as bisphosphonates. Osteoblasts cells cultured on the structures were seen to attach and spread on the surface after 3 days. EDX analysis confirmed the presence of the phosphorous polymer not only on the surface but also throughout the bead.

A biocompatible bead structure has been produced. The structure allows the attachment and spreading of human osteoblasts cells. To progress this study a co-culture of osteoblasts and osteoclasts will be employed to analyse the interactions between the cells.

6) Evaluation of Cells Isolation and BMPs Presence on Different Cryopreserved Bone Tissue

The main difference between autologous bone and other types of allografts is both the lack, in the latter, of viable cells that can contribute to the healing and their potential to induce immunological reactions. Moreover, in these grafts the presence and distribution of BMPs, factors known to regulate the differentiation of mesenchymal cell precursors, is not yet elucidated.

The aim of this study is to evaluate the cells viability and BMPs distribution in frozen bone allografts to select a proper cryopreservation method.

In vitro growth of cells extracted from samples of human femoral head allografts -frozen in different conditions (−80 and −180°C)- was studied. Total RNA was extracted from bone samples preserved at two different temperatures. The analysis of bone markers expression was performed by reverse transcription polymerase chain reaction (RT-PCR). The BMPs were localized by immunohistochemistry assays using anti-BMPs antibodies.

An efficient method for extraction of cells from hard tissue has been set up. The bone cells derived from the two samples showed a similar gene expression profile with respect to the osteoblast-related genes. Immunohistochemistry analysis confirmed the presence of BMPs in all the bone sections.

This study confirmed that cells from frozen bone allografts have the potential to grow in vitro and to maintain osteoblast phenotype. Moreover, the bone matrix contains BMPs that are fundamental for the osteointegration and for the migration of mesenchymal cells. The two procedures of cryopreservation studied didn't show significant differences with respect to the parameters analyzed.

7) Production and Characterisation of Nanoparticulate Calcium Hydroxyapatite

According to the sol-gel technique, thiethyl phosphite (P(OC₂H₅)₃) was hydrolyzed with distilled water (DW). Calcium nitrate (Ca(NO₃)₂ was dissolved in DW and this solution was added to the hydrolyzed phosphate sol. The mixed solution was kept static at 45 C for 2 h (aging time). The aged sol was then subjected to thermal treatment (drying time). The dried gel was calcined by applying a constant heat rate of 10 C/min up to 500 C, and then further calcined for 10 min. The final product was ball-milled to get a fine powder texture. A quantity of the produced HA was mixed with a deflocculating agent at a 3:1 ratio to determine if even smaller particles can be formed. The nano-HA was characterised using X-ray diffraction (XRD), scanning electron microscopy (SEM) and transmission electron microscopy (TEM).

The XRD results showed that there was a good match between the experimental and theoretical HA spectra. The manufactured HA was also compared with commercially available HA. Factors such as the aging and drying time, and the temperature play an important role in the crystallinity and morphology of the HA. SEM results indicated that HA formed agglomerates of smaller particles, which were further investigated with TEM. These particles were found to have dimensions between 50 nm and 70 nm. In the samples that deflocculant had been used, individual HA particles appeared to be as small as 10 nm. The deflocculant appears to affect the morphology and distribution of the HA particles.

8) Osteogenic Differentiation of MG-63 Cells Induced by Mechanical Strain in a Three Dimensional Collagen Scaffold

The aim of this work was to study the effect of mechanical strain on the osteogenic differentiation of MG-63 cells in a three dimensional collagen scaffold. The cells were stimulated for 15 minutes, 60 minutes, two hours, four hours and eight hours. The stimulations were performed once and thrice. Furthermore, a continuous stimulation was applied where the previous stimulations were performed in series. Parallel to the stimulations as a control cells were cultivated on the collagen scaffold without stimulation. After the experiments the viability of the cells was determined by an MTT assay. The viability was not affected by the mechanical stimulation. The mRNA of the cells was isolated and a PCR was performed to investigate the expression of bone markers. The expression of typical bone markers was found. It was noticeable that each stimulation scheme had different influence on the cells. The expression of bone markers varied in the different stimulations. The activity of alkaline phosphatase was examined by an AP-activity test and was verified for almost all stimulations. There was no difference between the AP activities of the stimulated und unstrained cells. The synthesis of alkaline phosphatase and RUNX2 were tested with immunological staining. RUNX2 was not observed in any experiment. Alkaline phosphatase was substantiated in the most experiments. The mineralisation of the extracellular matrix was studied by an alizarinred/calcein-double staining. Especially by the threefold stimulations and the unstrained cells an explicit mineralisation was recognized. Recapitulatory, thrice two hours of strain induced strongest effects and enhanced osteogenic differentiation.

9) Yield of Adipose-Derived Stromal Vascular Fraction From Liposuction or Lumbar Resection for Regenerative Medicine

Stromal vascular fraction (SVF) represents a rich source of adipose tissue-derived multipotent stem cells (ADSCs), ideal candidates for use in regenerative medicine. Aim of this work is to determine the yield of SVF from adipose tissue obtained by Coleman's liposuction or dorsal resection as a function of age, gender and Body Mass Index (BMI). Besides, SVF phenotipical characteristics were evaluated in order to verify their consistency with those typical of progenitor stem cells.

Material and methods: a total number of 37 informed subjects (14 males and 23 females) were enrolled for adipose tissue recovery. The mean age of patients undergone subumbilical liposuction (n = 9) was 31.6 ± 19.19 y, BMI 23.5 ± 5.56; the age of patients submitted to standard microdiscectomy (n = 28) was 58.3 ± 18.8 y; BMI 27.7 ± 5.34. SVF was obtained following Zuk isolation procedure. Harvested cells were counted under the microscope, and their vitality was assessed by the trypan blue exclusion technique. SVF yield was calculated, and the cell number and volume specific live cell number was estimated.

Results indicate that Coleman's liposuction technique leads to higher recovery rates of stromal vascular fraction with respect to resection, taking into account gender, body mass index and age of subject. The high recovery rates should be confirmed by the SVF selection by plating, typization, as well as differentiation potentiality for stem cell regenerative therapy.

10) Genetic Chondrogenic Potenciality of HUVEC and Wharton's Jelly Cells Isolated from Human Umbilical Cord

To evaluate the chondrogenic potential of human umbilical cord stem cells (umbilical vein endothelial cells -HUVEC- and Wharton jelly cells -HWJC-) we compared the genetic pathways expressed by those cells in comparison with human chondrocytes using high-density oligonucleotide microarrays.

HUVEC and HWJC stem cells were isolated from human umbilical cords, whereas hyaline cartilage chondrocytes were enzymatically isolated from small biopsies of the articular surface of the knee joint. Once isolated, a gene expression analysis was performed in the three types of cells by using Affymetrix Human Genome U133 plus 2.0 arrays. Genes with an average expression above 1000 F.U. were selected.

2092 genes were overexpressed by HWJC, with 2047 genes overexpressed in HUVEC and 1962 in chondrocytes cultures. All cell types showed a significant enrichment in genes with a role in cell metabolism, cytoskeleton, biosynthesis, cell growth, synthesis of cytoplasm components, nucleotide synthesis, collagen synthesis and secretion, etc. Only HWJC and chondrocytes expressed several genes with a role in cell adhesion to the substrate, with HWJC expressing cell-cell adhesion genes as well. Expression of genes with a role in extracellular matrix formation was similar for HUVEC and chondrocytes, with higher number of gene pathways in HWJC. Collagen was preferentially secreted by chondrocytes followed by HWJC.

Our gene expression analysis suggests that the chondrogenic potential of HWJC is higher than that of HUVEC cells and supports the use of these cells for the development of hyaline cartilage by guided transdifferentiation.

11) AMSC and Osteoblast Response to Three Different Synthetic Resorbible Polymers (PCL, PLGA AND PLLA-PCL) Normally Used For Bone Tissue Engineering Strategies

In order to increase the knowledge about key aspects in functional substitutes development for bone tissue engineering strategies (TE), this work studies some interaction processes that occur in cells when are supported by different type of scaffolds. In this study, we investigate the response of human adipose mesenchymal stem cells (AMSC) and osteoblast, the mayor cell types used in bone TE, to three synthetic resorbable polymers that are being used to develop bone substitutes: policaprolactone (PCL), polilactic and poliglycolic acid copolymer (PLGA) and polilactic acid and policaprolactone copolymer (PLLA-PCL).

Proliferation assay was measured by MTT method using polymer extracts. Cell adhesion and morphology in the polymers were observed by SEM and gene expression of bone development target genes (osteocalcin, osteopontin and osteonectin) were analysed by RT-PCR.

We have observed that PLGA increases AMSC proliferation compared to control, while no effects were observed in populations cultured with PCL and PLLA-PCL extracts. In osteoblasts, while PCL and PLGA decreased proliferation rate, PLLA-PCL extracts induce cell proliferation. On the other hand, SEM images showed a good cell adhesion and appropriate morphological aspect adoption when cultured over all polymers assayed. Regarding cell differentiation, all three polymers induce an inactivation of osteocalcin gene expression of AMSC. In contrast, in osteoblasts, osteocalcin and osteopontin gene expression were induced by PCL and PLGA while no changes were observed with PLLA-PCL.

In AMSC and osteoblasts, both proliferation activity and bone differentiation related key genes expression were altered depending on polymer type. The scaffold composition can determine the differentiation progress leading and bioactivity in new tissue development in regeneration process. This fact must be considered in TE strategies for bone regeneration.

12) Antibiotics Loading on Cyclodextrins Polymer (Poly-Cds) Functionalized Hydroxyapatite for Prolonged Delivery

Perioperative infection is one of the most common complications for bone-graft surgery. Systemic antibiotic (ATB) treatments only produce low drug diffusion into surgical sites due to the blood-bone barrier. An alternative solution is combining ATB with bone-graft materials for augmenting local drug concentration at targeted sites. This study aimed at achieving a controllable and sustained local drug delivery for ATB-loaded implant materials.

Widely applied bone-substitute material – Hydroxyapatite (HA) has been functionalized with 1,2,3,4-butanetetracarboxylic acid (BTCA) crosslinked hydroxy-propyl-β-cyclodextrin polymer (poly-BTCA-HPβCD), which could include different bioactive molecules to give a sustained release in vivo. We attempted incorporating poly-HPβCD into micro-porous HA under thermal fixation processing with their monomer mixture or soluble polymer. The HPβCD-functionalized HA (HPβCD-HA) has been characterized by infrared-spectroscopy (FTIR) and thermogravimetric analysis (TGA). Two common-used ATBs: ciprofloxacin and vancomycin have been loaded on HPβCD-functionalized and non-functionalized HA by vacuum impregnation. The kinetic release profile of ATB has been investigated in vitro in phosphate buffered saline (PBS) and assessed by UV-visible spectrophotometry.

TGA showed a grafting yield of around 1.6 ± 0.1 wt% into HA under our optimized HPβCD grafting processing. FTIR further confirmed the presence of poly-HPβCD in functionalized HA. Kinetic releasing study of ATB-loaded HA in PBS showed significantly increased initial-burst amount and prolonged releasing profiles (up to 96 hours) for both ciprofloxacin and vancomycin on HPβCD-HA comparing with those of non-functionalized HA.

Poly-cyclodextrins-functionalization on HA might be an effective drug-delivery model of loading different drug molecules for infection-prevention or cancer-chemotherapy.

13) Intervertebral Disc Regeneration by Adipose Derived Stem Cell

The degenerative pathologies of the intervertebral disc have a remarkable social impact in the industrialized countries and can provide serious disabilities in the population. The current treatment consists in conservative treatments (such as symptomatic pharmacological therapies and physiokinetic therapy) and surgical treatments (intervertebral fusion, total disc replacement, nucleus pulposus (NP) replacement or surgical exeresis). Recent advances in cell therapy foresee the possibility of regenerating the damaged disc: the autologous disc tissue can be withdrawn, in vitro regenerated and re-implanted. The aim of this work was to verify if autologous adipose-derived adult stem cells (ADASc) can improve the quality of an in vitro reconstructed nucleus pulposus tissue.

A three dimensional (3D) co-culture of NP cells and adipose tissue non-adipocyte fraction cells (nAF) was assessed in a previously developed alginate 3D culture system following the GMP guidelines, to assure patient safety for clinical studies. Morphology investigation of cultured and co-cultured cells was performed by transmission electron microscopy and by immunofluorescence for Collagen Type I, Aggrecan, CD90, CD34 and vimentin.

Results indicate that co-culture of NP and nAF improves the quality of the in vitro reconstructed tissue in term of extracellular matrix production and 3D cell organization. Technological resources are available for NP cell encapsulation intended for regenerating the intervertebral disc.

14) Three Dimensional Co-Colture of Chondrocytes and Adipose Stromal Vascular Fraction from Knee Hoffa's Body for Cartilage Regenerative Therapy

The use of chondrocytes in cartilage tissue engineering has been restricted by the difficulties during the first cell adhesion to the scaffold. Culture of adipose stromal vascular fraction (SVF) from human Hoffa's Body knee fat (HBF) could be an intriguing strategy to obtain a physiological autologous feed-layer in tissue engineering for cartilage regenerative therapy. The aim of this study is to investigate if the Hoffa's body-derived SVF could promote chondrocytes adhesion to the surface of a fibroin scaffold.

The HBF samples were obtained during surgery for knee articular diseases and knee articular cartilage harvested from cadaver donors. Biopsies were suspended in PBS and forwarded to a GMP cell factory at a temperature of 4°C. Tissues were digested in collagenase, and cell suspensions were filtered and centrifuged. Silk fibroin scaffold was seeded with SVF, incubated for 1 week, and then seeded with chondrocytes; co-culture were performed for 21 days. Culture of chondrocytes alone were performed in the same condition as a control.

Silk fibroin provides a stable, uniform, smooth surface, which promotes SVF cell adhesion and proliferation within 7 days culture. After chondrocyte seeding, an homogeneous coating of the scaffold can be observed within 3 days of co-colture. Co-culture of SVF and chondrocytes improves the quality of the in vitro reconstructed tissue in term of cell density and extracellular matrix production, respect to the control.

15) Neoangiogenic Effect of Unfocused Shock Waves on a Human Endothelial Cell Line

Unfocused Shock Waves (uSW) can induce soft tissue regeneration in vivo through various mechanisms, whose precise nature is still not fully understood. The aim of this study was to verify the hypothesis that endothelial cell proliferation and neoangiogenesis are the main processes involved in uSW regenerative therapy.

A three dimensional (3D) culture model on Matrigel was developed using a Human Microvascular Endothelial Cell line: 3D cell cultures were stimulated with an uSW lithotripter and after 12 hours of incubation vessel-like structures were quantified by counting the capillary connections. Gene expression analysis was performed: the cells were cultured until confluence, uSW treated and subsequently incubated at 37 °C for 3 hours. Negative controls were carried out using non-treated cells incubated in the same conditions.

Significantly higher capillary connections were obtained after uSW treatment with respect to the control and a strong 3-hours down-regulation of both BAX (pro-apoptotic) and CDKN₂C (cyclin-dependent kinase inhibitor) genes was observed. Our results seem to indicate that endothelial cells can be responsive to shock wave stimulation, which causes proliferation and the formation of vessel-like structures, probably due to a reduction in pro-apoptotic gene and cell cycle inhibitor expression. This study - whilst producing intriguing hypotheses about the mechanism by which uSW are effective in tissue healing and regenerative therapy (the mechano–transduction phenomenon) - seems itself to be a premise for employing uSW in advanced cell therapies and tissue engineering.

16) High Density Co-culture of Primary Osteoblasts with Mesenchymal Stem Cells: An Environment for Bone Formation

Engineering bone grafts with osteoprogenitor cells is a promising and innovative approach for developing future strategies for the treatment of large bone defects. Adult mesenchymal stem cells (MSCs) possess osteoprogenitor potential and provide an attractive potential donor source having. However, they require exogenous growth factors to undergo osteogenic differentiation. The aim of this study was to evaluate the osteogenic induction potential of primary isolated osteoblasts from the same donor on MSCs in high density co-cultures.

Osteoblasts and MSCs were co-cultured at a ratio of 10%/90%, 30%/70% and 50%/50%. Co-cultures were then treated with or without a specific osteogenic induction medium in monolayer and high density cultures for up to 21 days.

Osteogenesis was not observed in untreated monolayer co-cultures. In contrast high density co-cultures demonstrated enhanced osteogenesis as determined by ultrastructural evaluation with electron microscopy. There was no difference between treated, untreated or pure osteoblast cultures. Immunoblotting demonstrated the presence of collagen type I, β1-Integrin, induction of the MAPKinase pathway (Shc, Erk1/2) and Cbfa-1 production in the co-cultures during osteogenesis. The quality of osteogenesis was proportional to the quantity of osteoblasts present in the co-cultures. Co-cultures consisting of 50% osteoblasts and 50% MSC showed markedly increased osteogenesis, compared to pure osteoblast cultures. The presence of as little as 10% osteoblasts in the co-cultures strongly increased osteogenesis compared to MSCs control cultures; treatment with the induction medium further enhanced osteogenesis in these co-cultures.

High density co-cultures of MSCs and primary osteoblasts combined with a three dimensional environment strongly promote osteogenesis in vitro. This approach may have practical benefits for future bone tissue engineering strategies.

17) Aminosugars in Regenerative Medicine: Chitin Derivative as Coating Material for Titanium Implants

Early loosening of joint implants is caused, in part, by inadequate fixation. Achieving a biomechanically stable bone-implant interface is, therefore, crucial in order to decrease and possibly avoid implant loosening. Coating implants with a material that will enhance the implant fixation and accelerate the integration, could reduce the risk of implant loosening. The objective of this study is to coat titanium with chitosan films with the aim of accelerating bone-implant integration.

Chitosan films were prepared using high molecular weight chitosans with deacetylation degrees (DD) of 70%, 87% and 94%, dissolved in acetic-, lactic- and citric acid. Commercially pure titanium was treated with hot alkali and heat treated to 600°C in order to obtain better coating adhesion. In vitro test were conducted with human mesenchymal stem cells (hMSCs) on titanium dip coated in acetic acid chitosan solutions.

Lactic acid chitosan films were thicker than acetic acid chitosan films, except for DD 70%. The swelling index (=thickness after swelling in MQ/ thickness after neutralisation) for all films was between 1,6-2,8. The swelling index was higher for lactic acid than acetic acid, except for DD 87%. Coating of titanium was successful with all types of chitosan solutions. Initial studies with hMSCs suggested that these cells were able to thrive on all the chitosan coatings.

It is hypothesized that by coating a metal implant with a chitosan film, implant fixation and integration will be accelerated, resulting in faster healing and a reduced risk of implant loosening.

18) Experimental Scaffolds for Bone Tissue Engineering

The development of two scaffolds for bone tissue engineering, (i) an injectable, cell-encapsulated scaffold, (ii) a 3-D cell-seeded porous scaffold incorporating human osteoblasts (HOB) and human endothelial cells (HUVEC).

Carbonated hydroxyapatite (CHA) was produced through precipitation using simulated body fluid (SBF). HA/CHA-Alginate cell encapsulated scaffolds were obtained using aqueous sodium alginate and cells, then complexed with CaCl₂ solution to yield the injectable composite. Efficacy of HA/ CHA–alginate composite as a potential cell carrier was investigated.

A metacalcium phosphate-based porous scaffold (VM2) was fabricated by addition of poly(vinyl alcohol) and eliminating at sintering temperature. Subsequently, 5 × 10⁴ HOBs and 5 × 10⁴ HUVECs per ml of medium were seeded simultaneously on the scaffolds and cell viability, proliferation and differentiation capacity evaluated.

FTIR spectrum of the CHA-Alg scaffolds showed expected peaks of phosphate, carbonate and hydroxyl groups. Live/Dead staining confirmed cell viability and proliferation for over 21 days.

The VM2 scaffolds average pore size was 80 mm. Cell attachment and proliferation (HOB's & HUVEC's) matched controls, with cells observed within macropores, anchoring to the bone mineral-like crystals. HOB/HUVEC co-cultures were stained with long-term CellTracker™ dyes, cell viability of both cell types confirmed for over 3 weeks.

Conclusion: Successful production of cell-seeded CHA/Alginate with maintenance of viability. VM2 porous scaffolds confirmed HOB/HUVEC stability, with osteogenic and angiogenic potential, thus necessitating further investigation for use in regenerative medicine.

19) Rapid Prototyping: Porous Titanium Alloy Scaffolds Produced by Selective Laser Melting (SLM) for Bone Tissue Engineering

Selective Laser Melting (SLM), a method used in the nuclear, space and racing industries, allows the creation of customized titanium alloy scaffolds with highly defined external shape and internal structure (pore size) using rapid prototyping as supporting external structures within which bone tissue can grow. Human osteoblasts were cultured on SLM-produced 3D titanium alloy (Ti6Al4V) mesh scaffolds to demonstrate biocompatibility using Scanning Electron Microscopy (SEM) and fluorescence microscopy after cell vitality staining. The occlusion of pores of different widths (0.45–1.2 mm) by osteoblasts was evaluated. SEM investigations showed osteoblasts with well-spread morphology and multiple contact points. Cell vitality staining confirmed osteoblast vitality. 1 week after seeding, only 0.45 mm pores showed any occlusion by osteoblasts (21%). At 3 weeks the occlusion of 0.45 mm, 0.5 mm, 0.55 mm, 0.6 mm and 0.7 mm pores was 79%, 67%, 67%, 44% and 10%, respectively. At 6 weeks the occlusion of 0.45 mm and 0.5 mm pores had risen to 100%, but no increase was observed for 0.55 mm, 0.6 mm and 0.7 mm pores. No pore occlusion was observed on pores of width 0.9 – 1.2 mm. In summary, the scaffolds are biocompatible and pore size influences pore overgrowth.

20) Ceramic Scaffolds Produced by Rapid Prototyping: Characterisation and Biocompatibility Investigations

Ideally, scaffolds used as bone replacement materials should fit the defect site exactly and have tailored porosity and pore size. Commonly used ceramic materials in general are not easily formed to individual patient needs. Using rapid prototyping methods, three-dimensional ceramic scaffolds of hydroxyapatite (HAP) and tricalcium phosphate (TCP) have been produced with defined external shape and internal structure by sintering ceramic powder. The goal of this study was to evaluate the in vitro biocompatibility of these new HAP and TCP scaffolds compared to the material BioOss®, which is widely used in oral and maxillofacial surgery. To this end, human osteoblasts were cultured in eluate from the aforementioned scaffolds and also seeded onto scaffolds directly.

Using commonly used biochemical tests (LDH, MTT, WST) and cell vitality assessment by fluorescence microscopy after staining with fluorescin diacetate (FDA) and propidium iodide (PI), it was found that the new ceramic HAP scaffolds produced by rapid prototyping have superior biocompatibility for osteoblasts than the material BioOss®. However, new ceramic TCP scaffolds produced by rapid prototyping appear to be less biocompatibile than BioOss®. Scanning electron microscopy (SEM) revealed colonisation and spreading of osteoblasts on HAP and TCP scaffolds. It can be concluded that HAP scaffolds in particular are biocompatible for use as bone substitute materials.

21) Polymer-Ceramic Composite Scaffolds for Osteochondral Tissue Engineering

One popular approach in osteochondral tissue engineering for the treatment of osteochondral defects is the use of polymer-ceramic composite scaffolds. The purpose of this study was to evaluate the in vitro biocompatibility for osteoblasts of a new composite scaffold type, Osteochondral Plugs (Biocomposites Ltd., U.K.). These consisted of Poly-L-lactid Acid (PLLA) fibres, Polycaprolactone (PCL) and Tricalcium Phosphate (TCP) sprayed with Hydroxyapatite (HAP). Average pore size was approximately 200-250 μm. Two versions of Plugs were compared. Version 1 contained CaSO₄, whereas Version 2 was CaSO₄-free. Plugs were seeded with human osteoblasts. Cell morphology was assessed using scanning election microscopy (SEM). Cell vitality was evaluated using fluorescence microscopy after staining with fluorescin diacetate (FDA) and propidium iodide (PI). Standard tests for biocompatibility (WST, LDH, MTT) were performed. SEM investigation showed that osteoblasts adhered to the surface of both versions of Osteochondral Plugs and demonstrated good spreading. Fluorescence microscopy and standard tests for biocompatibility (WST, LDH, MTT) showed superior biocompatibility of Version 2 to Version 1. It can be concluded that the incorporation of CaSO₄ into Plugs is disadvantageous for biocompatibility in vitro. Further tests are needed to assess biocompatibility in vivo.

22) Ceramics with Bone-like Morphology

This work reports about the preparation of ceramics with bone-like morphology using a powder based 3D printing process and micro tomography (μCT).

Data acquired with a μCT (Scanco μCT40) was transferred into a 3D printing device (Prometal R1), which used a ceramic powder (Ca10[K/Na](PO₄)₇). To compensate for sintering induced shrinkages and to prevent blocking of small pores the data was upscaled by a factor of 1.7. After drying the green parts at 105 °C for 24 h the excessive powder was tried to be removed by repeatedly tapping the samples against a glass plate. After sintering the resulting parts were analyzed with μCT for pore and strut size distribution.

It was found that overall porosity was lower in the resulting parts than in the original bone sample. This was attributed to insufficient removal of excessive powder and limited resolution. However, the remaining pores in the printed parts showed a comparable pore size distribution compared to the original bone sample. The strut size distribution of the copy was blunted and shifted to a higher peak value.

This work showed that in principal μCT derived data can be used to generate copies with 3D printing. Problems encountered were: removal of excessive powder in the green part, appropriate compensation of sintering induced shrinkages and limited resolution of the powder based 3D printing process.

23) Tissue Engineering of the Intervertebral Disc: Influence of Donor Pathology on the Regenerative Capacity of Intervertebral Disc Cell Cultures

Aim of the presented study was to analyse the influence of donor pathology on cell culture results for intervertebral disc tissue engineering.

Tissue samples from patients undergoing surgery because of degenerative spine diseases or kyphotic/scoliotic changes of the spine were collected with informed consent and analyzed. Cells were harvested by tissue digestion in 0.15% collagenase type II for 48 hours and cultured in DMEM supplemented with 10% FCS and 0.05 mg/ml Ascorbic Acid.

Average cell yield was 13.77 × 10⁴ per g tissue after isolation of tissue deriving from patients under 18 years old and 35.150 × 10⁴ per g when isolating cells from older donors (p < 0.008). Mean viability was 96% in both groups. Cells deriving from degenerative spines started to proliferate immediately after seeding while cells deriving from scoliotic spines needed a longer recovery period, but showed good proliferation features afterwards. Viability remained stable during monolayer culture and was above 95% during monolayer expansion. Histological analysis of native tissue did not reveal any significant pathology related differences. In contrast, cell cultures deriving from degenerative discs showed higher levels of Alcian Blue staining, Safranine O staining (p < 0.006) and Azan staining (p < 0.001) than samples deriving from scoliotic or kyphotic spines.

Culture viabilities and proliferation rates are similar and seem independent from patient age or pathology, but histological characteristics of cell cultures are different. Donor pathology should therefore be taken into account when planning intervertebral disc regeneration using tissue engineering techniques.

24) Clinical Tissue Engineering: Evaluation by Health Technology Assessment

The authors propose the usage and present own results of international Health Technology Assessment (HTA) methods and guidelines for the evaluation of clinical cartilage Tissue Engineering: quality control and risk/ benefit/ cost-analysis.

At the moment we are working on a HTA assessment evaluating publications (randomized controlled trials, no case studies) on different types of Matrix Associated Chondrocyte Implantation (MACI) techniques/biomaterials. The report will include published data until September 2008. Outcomes of the usage of varying scaffolds are discussed in detail.

HTA specifies a process of systematically evaluating medical procedures and technologies with relation to public health care to support scientific decision making: high quality research information on the costs, effectiveness and broader impact of health technologies is produced. Most importance is given to the traceability of results: systematic work and the disclosure of methods makes results open to scrutiny. HTA is committed to a qualitative concept of progress.

25) Demineralized Bone Matrix for Bone Formation in Immunocompetent and Nude Animal Models

Human demineralized bone matrix (DBM) is an osteoinductive material used as a bone graft substitute. The purpose of this work was to demonstrate the importance of immunological response against DBM after implantation in small and large immunocompetent animal models in comparison to “nude” mice and rats.

Three types of human DBM were tested: 2 commercial (renamed A and B) and “home made” DBM (renamed C, demineralized in 0.6N HCl) supplied by our tissue bank and compared to fully mineralized bone matrix (FMBM). Each experimental DBM was intramuscularly implanted and assessed after 4/8 weeks, 2/4 weeks, 1 week post-implantation in Nude Rats, Wistar Rats and Pig recipients, respectively. After explantation, DBM was studied by immunohistochemistry for immunological (CD3, CD68), graft resorption/fibrosis and osteoformation responses.

A significant higher degree of implants recovering was observed, for DBM B/C in contrast to DBM A and FMBM (>80% vs. <20%, p < 0.05). Inflammation, graft resorption and no osteo-formation was observed for FMBM in each animal model. Although newly bone formation was found, in nude rats implanted with DBM B and C (between 4 and 8 weeks post-transplantation), no osteo-induction and severe immunological responses were already found after 1 and 2 weeks post-implantation in Pig and Wistar rats, respectively. The commercial DBM A did not induced bone-formation and leads to severe implant destruction in both non-/immunocompetent animals.

This study demonstrates that the early immunological response against human matrix can precede and inhibit the newly-bone formation induced by the DBM.

26) Impact of Physical and Chemical Treatments on In Vitro and In Vivo Biocompatibility of Human Bone Tissues

Chemical and physical treatments were developed to reduce the immunogenicity and risk of virus/bacteria/prion transmission. This work studies the impact of treatments on in vitro and in vivo biocompatibility properties of tissues.

Three types of tissues were tested: i) fresh-frozen bone (FFB) as control, ii) freeze-dried treated irradiated bone (FDTB) and iii) frozen treated irradiated bone (FTB).

In vitro testing was performed by indirect (MTS, LDH) and direct (cell attachment, cell adhesion) toxicity assays with Saos₂ cell line. For in vivo testing, each group was subcutaneously implanted in immunocompetent Wistar rats to assess the immunological (CD3, CD68), recolonization (Masson's Trichrom) and revascularization (vWF) processes at 2/4 weeks post implantation.

For FFB as well as FDTB, no modification of cell viability (MTS/LDH) was observed for Saos₂ in contact with tissue extract. In contrast, a significant reduction of cell viability (MTS) was observed for FTB (p < 0.05). After 3 hours of direct contact between Saos₂ and bone tissues, a significant decrease of adhesion was demonstrated in case of FTB in comparison to FFB and FDTB. At 24h of contact, cellular adhesion was significantly decreased for FDTB and FTB (p < 0.05). A significant higher immunological reaction (lymphocytes/ macrophages recruitment and bone resorption) was found for FFB, after subcutaneous implantation in rats, in comparison to FDTB and FTB. In contrast, decellularized (FDTB/FTB) tissues significantly improved neo-vascularization in connective matrix after 15 days of implantation.

Human bone processing with defatting and decellularization promotes in vitro biocompatibility and in vivo tissue tolerance.

27) Photozymes: A Powerful Tool for Light-Controllable Biomineralization into Biomimetic Scaffold

Bone is a hierarchically structured composite material and a bone regenerative material usually should mimic the nanostructure and chemical composition of natural bone tissue. Biomimetic scaffolds, consisting of hydroxyapatite/bioactive glasses/gelatin (HA/CEL2/G) were produced. The content of the inorganic phase was selected to be 70 wt.%, close to bone composition. A chemical approach using a not-cytotoxic crosslinking agent (genipin) was applied to increase the mechanical properties and the water stability of the scaffold. Three-dimensional sponge-like porous HA/CEL2/G composites were fabricated by freeze-drying technique at −25°C. Improvements of the biomimetic properties of the scaffolds were tested by the incorporation of photozymes, macromolecules able to promote a light-controlled biomineralization (photodynamic biomineralization). Photozymes are amphiphilic water-soluble copolymers consisting of hydrophobic chromophoric and hydrophilic monomer units forming nanosized pseudo-micelles. Hydrophobic core allows to transfer the solar energy into chemical energy due to the antenna effect. Photozymes containing primary amino groups, such as chitosan-fluorescein, were introduced into matrices.

The scaffolds were characterized in terms of their water stability (swelling and dissolution test), chemical (infrared spectroscopy) and thermal properties (thermogravimetric analysis, differential scanning calorimetry), mechanical behavior and cell compatibility. Crosslinking treatment stabilized the sponges and decreased swelling and weight loss in water media. Cultures of MG63 osteoblasts-like cells were performed after UV irradiation of the sterilised photozyme-containing scaffolds and showed good adhesion and proliferation, good viability (evaluated by MTT test, 100-150% of the control) and good differentiation (alkaline phosphatase, up to 12 UI/l compared to 8.6 UI/l for control).

28)

ABSTRACT WITHDRAWN

29) Effect of the Incorporation of Simvastatin into a Radiopaque and Injectable Bone Cement Acrylic-glass Composite

The aim of this work is to formulate a radiopaque and bioactive bone acrylic cement (ABC) for specific use in vertebroplasty, by incorporating a glass in the system SiO₂-CaO-P₂O₅-SrO and to study the effect of the addition of simvastatin (SV) on some properties of the cement, for instance, polymerization kinetics, injectability of the curing cements, mechanical properties and bioactivity. Finally, release of SV was also studied.

The bioactive glasses were prepared by a sol-gel method. In order to improve the compatibility of the filler with the matrix, a surface treatment with MMA was carried out.

The cements were formulated using a solid: liquid ratio of 1.7:1. The liquid component consisted of MMA monomer and N, N′, dimethyl-4-toluidine (1 wt %) as activator. The solid component consisted of PMMA beads, BPO (1.5 wt %), 40% of bioactive glass: SiO₂ (57.75%)-P₂O₅ (4%)-CaO (28.25%)-SrO (10%) and SV (10 mg/g). The reference didn't contain simvastatin.

It was observed that the addition of the used quantities of SV did not affect significantly to the curing parameters, the Compressive Strength (σc) and the Young's Modulus (Ec). The presence of SV improved the injection time of the formulation containing bioactive glass, although it was no significant.

In vitro bioactivity was not affected by the addition of SV. After 3 days in SBF, an apatite like layer started to appear on the cement surface and after 7 days it covered the entire surface. Release of SV was also appropriate.

30) Heparin-Functionalized Chitosan Scaffolds: Investigation of Osteoblastic Activity

Heparin is widely used as an injectable anticoagulant, additionally, due to the similarities between ECM and heparin, it can be also used as an artificial ECM in tissue engineering studies. However, there are contradictory studies in the literature about the effect of heparin on cell proliferation and differentiation. The aim of this study was to investigate the effects of heparin-functionalized chitosan scaffolds on the activity of osteoblast-like cells. For this purpose chitosan scaffolds having the pore size of ∼100 μm were prepared by freeze-drying method. Since osteoblasts are 10-30 μm long, this pore size allows cells to migrate into chitosan sponges. Heparin was bound to the scaffolds both electrostatically and covalently. Morphology, proliferation and differentiation of MC3T3-E1 preosteoblasts on chitosan scaffolds were investigated in vitro.

Proliferation of osteoblast-like MC3T3-E1 cells on chitosan scaffolds was evaluated by reduction of MTT to formazan by mitochondrial succinate dehydrogenase. The MTT results of MC3T3-E1 cells on different types of chitosan scaffolds i.e. chitosan, activated chitosan, covalently heparin-bound chitosan and electrostaticaly heparin-bound chitosan scaffolds, indicated that covalently-bound heparin stimulates osteoblast proliferation significantly compared to other scaffolds. SEM images showed that cells on chitosans attached well to the scaffold and started to form extracellular matrix. SEM results also proved the stimulative effect of covalently-bound heparin on the osteoblast-like cell proliferation. ALP and osteocalcin levels of cells proliferated on covalently heparin-bound scaffolds are also higher than that of other scaffolds. In vitro studies have demonstrated chitosan scaffolds increase viability and proliferation of osteoblast-like MC3T3-E1 cells especially in the existence of covalently-bound heparin.

31) Characterization and In Vitro Evaluation of Chitosan-Carbonate Apatite-Citric Acid Nanocomposite Scaffolds

Nanocomposites composed of carbonate apatite and chitosan in the presence of citric acid were synthesized.

The morphological and componential properties of composites were characterised by FTIR, XRD and SEM methods. The developed scaffolds have a total porosity up to 90%. The carbonate apatite particulates, in sizes of about 50–100 nm, were distributed within the network chitosan homogeneously, moreover, inorganic particles could be controlled by the size of networks of organic matrix which was mediated with crosslink degree of chitosan.

The resorbability test showed that the dissolution kinetic of scaffolds is a multistage process. In the in vitro experiments, the properties of composite scaffolds were studied on the human fibroblast model. The adhesive properties, the acute cytotoxicity, and the dynamics (from 1 to 28 days) of the numerical population of the fibroblasts were estimated (a standard MTT test). It was found that all investigated samples do not induce acute cytotoxicity and have an adhesive capacity. In particular, the population of the fibroblasts increased from 5 to 10 times during the experiment.

These results provided an efficient approach toward new biomimetic tissue scaffold for the biomedical applications with enhanced intensity/bioactivity and controlled resorption rates.

32) In Vivo Evaluation of Carbonated Hydroxyapatite Scaffolds for Bone Tissue Engineering

Scaffolds with carbonate content about 6 wt.% were divided into 2 groups: 1st group – surface micropores <10 μm and inner cavities about 400 μm, 2nd group – surface macropores about 150 μm and inner cavities 300 μm.

X-ray diffraction, Fourier transform infrared spectroscopy (FTIR), evaluation of solubility in isotonic solution of 0, 9% NaCl for 3 weeks, scanning electron microscopy (SEM), histology evaluation (15 days) in hole bone defect on Wistar rats, statistic morphometric analysis of bone matrix formation was performed by one-way analysis of variance (ANOVA) (a significance level α = 0.05) with the MicrocalTM OriginTM Version 5.0 software package.

X-ray diffraction results demonstrated that the broadening peaks of the CHA scaffolds are similar to the pattern of natural human bone. FTIR (FTIR-4200, Shimadzu Co Ltd) have shown the broad dublet bands at 1420 1455 cm⁻¹ and the band at 875 cm⁻¹, which were characteristics of carbonate, were clearly observed in the CHA. The resorption rate in 0, 9% NaOH was higher in CHA group 2. SEM showed difference in structure of sintered CHA: scaffolds in the 1st group had surface micropores <10 μm, on the cross section we observed inner cavities about 200 μm in diameter; scaffolds in 2nd group had surface macropores about 150 μm and inner cavities about 300 μm. Histological evaluation showed an active bone formation on scaffolds in 2nd group on day 15.

Therefore, it is reasonable to use CHA scaffolds with surface macropores about 150 μm as a bone substitute for filling bone defects. Porous scaffolds with micropores <10 μm have large inner cavities which could be used as a delivery carrier for stem cells and antibiotics.

33) A Technique for Stabilisation of Critical Size Defects in Sheep Tibia for Translational Research on Large Bone Defects as a Possible Standard Model

For testing of new bone substitutes for human orthopaedics animal models are required. Despite a lot of experiments in large animals no standard model has been established. Experimental setups vary in defect size, stabilisation technique, location of the defect, and the preservation of the periosteum. This situation makes comparability of different studies difficult and causes the need of more experimental animals.

In this study 35 sheep were operated for the evaluation of different treatments of a critical size defect. The whole duration of the in vivo study was three months (2 animals out of 7 per group) and six months (5 animals out of 7). In all animals a mid diaphyseal, cylindrical ostectomy including the periosteum of 3 cm length was created in the tibia. The defect was stabilised using a novel CF PEEK (Carbonfibre Polyether-ether-keton) plate and a LCP.

The surgical procedure was easy and fast to perform. All sheep recovered well. In total 35 sheep were operated for this study. Two had to be excluded from evaluation criteria. Continuous radiological evaluation was possible during the entire study.

The animal model used in this study provides excellent premises for research on critical size defects in large animals which is often associated with high rates of complications and which is lacking standardised models. As well it offers a variety of post-mortem analyses which makes it an ideal model for translational research.

34) Human Osteoblasts Induce Proliferation and Neo-Vessel Formation of Human Umbilical Vein Endothelial Cells in a 3D-Coculture

Angiogenesis is a cue element in the early wound healing being considered most important for tissue regeneration and for the direction of inflammatory cells. Particularly, the survival of tissue engineered bone constructs strictly depends on appropriate oxygen and nutrient supply which initially depends on diffusion after the implantation. The improvement of vascularization by implementation of endothelial cells or angiogenic growth factors may unveil a key solution for engineering bone constructs of considerable sizes. In the present study, we describe a long-term culture environment that supports the survival, proliferation, and in vitro vasculogenesis of human umbilical vein endothelial cells (HUVEC).

This condition could be achieved in a co-culture of HUVEC and primary human osteoblasts (hOB) employing polyurethane scaffolds and platelet rich plasma in a static microenvironment. Cell proliferation and differentiation was analyzed using histological, immunohistological, and confocal laser microscopic methods and quantitative PCR.

We could clearly show that hOB support cell proliferation and spontaneous formation of multiple tube-like structures by HUVEC which were positive for endothelial cell markers CD31 and vWF. By contrast, most of the HUVEC did not proliferate and did not form any apparent vessel-like structures in a monoculture. Immunohistochemical and qPCR analyses of gene expression revealed that cell differentiation of hOB and HUVEC was stable in the long-term co-culture. The three dimensional, FCS-free co-culture system could provide a new basis for the development of complex tissue engineered constructs with a high regeneration and vascularization capacity.

35) Enhancing Bone Regeneration with Stem Cell Seeded Scaffolds Cultured in a Mechano-Bioreactor

Mesenchymal stem cells are exposed to perfusion and mechanical shear stress during fracture healing and bone remodeling. The aim of this study was to investigate bone marrow stromal cell proliferation and differentiation under the influence of perfusion and mechanical stimulation.

HBMSC's of 7 individuals were harvested, grown in vitro, and combined. 106 hBMSC's were seeded on a bovine spongiosa disc and incubated in a bioreactor system. Cell culture was continued using 3 different conditions: Continuous perfusion (group A), 10% cyclic compression at 0.5 Hz (group B) and static controls (group C). After 24 h, one, two, and three weeks, we determined cell proliferation (MTS-assay) and osteogenic differentiation (osteocalcin ELISA, Runx2 mRNA). Tenascin-C mRNA was quantified to exclude fibroblast differentiation.

In groups A and B, proliferation was enhanced after 2 weeks (48.6 ± 19.6 × 1000 (A) and 44.6 ± 14.3 × 1000 cells (B)) and after 3 weeks (46.6 ± 15.1 × 1000 (A) and 44.8 ± 10.2 × 1000 cells (B)) compared with controls (26.3 ± 10.8 × 1000 (2 weeks) and 17.1 ± 6.5 × 1000 cells (3 weeks), p < 0.03). Runx2 mRNA was upregulated in both stimulated groups after 1, 2, and 3 weeks compared to control (group A, 1 week: 5.2 ± 0.7-fold; p < 0.01, 2 weeks: 4.4 ± 1.9-fold; p < 0.01, 3 weeks: 3.8 ± 1.7-fold; p = 0.013; group B, 1 week: 3.6 ± 1.1-fold, p < 0.01, 2 weeks: 4.2 ± 2.2-fold, p < 0.01; 3 weeks: 5.3 ± 2.7-fold, p < 0.01). hBMSC's stimulated by cyclic compression expressed the highest amount of osteocalcin at all time points (1 week: 294.5 ± 88.4 mg/g protein, 2 weeks: 294.4 ± 73.3 mg/g protein, 3 weeks: 293.1 ± 83.6 mg/g protein, p ≤ 0.03).

The main stimulus for cell proliferation in a 3-dimensional culture of hBMSC's is continuous perfusion whereas mechanical stimulation fosters osteogenic commitment of hBMSC's. This study thereby contributes to the understanding of physical stimuli that influence hBMSC's in a 3-dimensional cell culture system.

36) Alveolar Ridge Augmentation: Bone Regeneration Instead of Reparation

For the elder patient risk minimization in surgical treatment is required. In the demographic development we see more primary diseases in these patients.

We show the case of a 75 year old female patient with upper jaw atrophy after wearing a full denture for 20 years. Two primary diseases were known: hypertension and cardiac arrhythmia. Surgical treatment with bone transplantation from the iliac crest was relatively contraindicated. Under monitoring and sedation the alveolar ridge in the upper jaw was built up with TCP (tricalciumphosphat, Vitos-Strip), PRP (platelet rich plasma) and rhBMP-2 (Induct-Os), endoscopically assisted with bridge flap technique. No bone transplantation from the iliac crest was needed.

For safety reasons due to the primary diseases the patient was hospitalised for one night. Wound healing was complication free. X-rays pre and post operative (directly and after three months) showed de novo grown bone at last.

Using minimal invasive surgical treatment hospitalisation, risk of infection and complications were minimized. Cost-benefit analysis also shows advantages of bone regeneration compared to bone transplantation. The healing process was advanced.

Modern bone regeneration shows advantages in all sections compared to autogenous bone transplantation still declared as gold standard. Especially for ethical reasons it would be desirable that bone regeneration increasingly replaces bone transplantation.

37) Evaluation of Bioactive Glass Abrasion for Dental Implants to Improve Osseous Integration

Melt-derived Bioglass® 45S5 was produced in-house. Bioactivity was determined by FT-IR after immersion in simulated body fluid. Optimum abrasion conditions were determined including the particle size fractions used, nozzle pressure, disk feed rates and powder flow. The topography and roughness were investigated by atomic force microscopy and confocal microscopy. In vitro biocompatibility was evaluated using primary human osteoblast-like cells and both indirect and direct assays according to ISO 10993-5, were performed. The microbial responses using a staphylococcal model were investigated using direct plate counts, growth inhibition by agar diffusion and differential dye uptake methodologies. Samples were successfully air abraded with Bioglass® 45S5 and a Sa range between 0.3 μm and 1.2 μm was achieved under the conditions applied. Elution studies showed no indirect acute cytotoxicity as determined by tetrazolium dye precipitation. Direct contact tests using resazurin dye conversion (AlamarBlue) showed an increase in metabolic activity of cells for the study period of 14 days.

38) Blood Coagulum after Schulte: Effects on the Proliferation of Osteoblast-like Cells

The “Schulte coagulum” is a technique for bone regeneration of intraoral bony defects. This procedure has been successfully used in the clinical routine of oral surgery for decades. Its simple composition includes patient's own coagulated blood, gelatine sponge, penicillin G and thrombin. The benefit from the Schulte coagulum should not be restricted to its retraction stability.

Proliferation of MC3T3-E1 cells has been determined by a [3H]-thymidine assay under the influence of the different components.

Thrombin increased the proliferation rate of the osteoblast-like cells in a concentration-dependent manner suggesting that thrombin acts as a growth factor on these cells. Penicllin G on the other hand had a negative effect on the proliferation rate. The thrombin-induced proliferation is significantly restrained in presence of penicillin G and can not be completely reversed even with high thrombin concentrations. The investigation of various coagulum components revealed that thrombin is not the only factor, responsible for the cell proliferation. Inhibition of the PDGF receptor activity of the MC3T3-E1 cells decreased proliferation indicating that PDGF is released from the coagulum. Among the compounds excreted from activated platelets, PDGF BB increased the proliferation rate as expected, while ADP and serotonin had no effect.

The results indicate that thrombin and components of the coagulum increase the proliferation of the osteoblast-like cells and might contribute to bone regeneration in vivo. Therefore, the Schulte coagulum is a valid alternative to bone substitutes, bone transplantation or stimulation of bone regeneration with bone-specific growth factors.

39) The Impact of the Oxygen Environment on Mesenchymal Stem Cell Expansion and Chondrogenic Differentiation

In vitro expansion and differentiation of mesenchymal stem cells (MSCs) rely on specific environmental conditions. Experiments demonstrated that one crucial factor is the oxygen environment. In order to understand the impact of the oxygen tension on MSC expansion and chondrogenic differentiation in vitro we developed a mathematical model of these processes and applied it predicting optimised assays. We compared ovine MSCs under physiologically low and atmospheric oxygen tension. Low oxygen tension improved their in vitro expansion as demonstrated by monoclonal expansion and colony forming assays.

Moreover, it accelerated the induction of a chondrogenic phenotype in subsequent 3D differentiation cultures. We introduced a hybrid stochastic multi-scale model of MSC organisation in vitro. The model assumes that cell adaptation to un-physiological high oxygen tension reversibly changes the structure of MSC populations with respect to differentiation. In simulation series we demonstrated that these changes profoundly affect the chondrogenic potential of the populations. Our mathematical model gives a consistent explanation of our experimental findings. Our approach provides new insights into the organisation of MSC populations in vitro. The results suggest that MSC differentiation is largely reversible and that lineage plasticity is restricted to stem cells and early progenitors. The model predicts a significant impact of short term low oxygen treatment on MSC differentiation and an optimal chondrogenic differentiation at 10-11% pO₂.

40) Modified Pluronic F127 Hydrogel as a Cell Delivery System for Bone Tissue Engineering

The Tri-block copolymers of poly(ethylene oxide)- poly (propylene oxide)-poly(ethylene oxide) [PEO-PPO-PEO], Pluronic® F127 , are thermosensitive hydrogels. At body temperature, Pluronic® F127 forms a gel and can be injected in the body to release drugs/proteins/gene constructs. But by body fluids the Pluronic polymer is diluted below the critical gelation concentration and disintegrates easily. To increase the in vivo residence time of Pluronic F127®, the hydroxyl endgroups were chemically converted to N-methacryloyl-depsipeptides, without interfering with its thermosensitive characteristics. In order to tailor the degradation rate, the structure of the depsipeptide was altered by using L-lactide acid and alanine, forming the UV crosslinkable Pluronic ALA-L hydrogel. The use of this modified hydrogel for Bone Tissue Engineering was evaluated in vitro.

MC3T3-E1 cells loaded on macroporous gelatin CultiSpher-S® microcarriers are mixed with different (w/v)% Pluronic ALA-L. Cell viability was assessed by fluorescent live/death cell staining in the thermosensitive UV crosslinked hydrogel and the in vivo instable thermosensitive hydrogel, in this case without the addition of medium (to prevent disintegration).

Without UV cross-linking, the cell viability drops drastically within one week to 30%. After UV cross-linking medium can be added without sudden disintegration of the gel, and cell viability remains in the range of 90 to 75% one week after encapsulation.

This modified Pluronic® hydrogel combines the well known biological features of Pluronic®, with a slow degradation speed, and good viability percentages of encapsulated cells, making it a useful system to deliver cells to a bone defect.

41) Ionic Composition and Cell Viability in Primary Cultures of Fibrous and Hyaline Cartilage Chondrocytes

Autologous chondrocyte implantation is a well-established method for the treatment of several chondral defects. In the present study, we have compared the cell viability of primary cultures of fibrous and hyaline cartilage chondrocytes using electron-probe X-ray microanalysis.

In this work, we used 2 cartilage biopsies from the articular surface of the knee joint and 2 biopsies from the disc of the temporomandibular joint of Wistar rats. Samples were cut into small pieces and treated with collagenase at 37° for 3 h. Then, chondrocytes were harvested by centrifugation and cultured in DMEM medium supplemented with 10% fetal serum, antibiotics and several growth factors to generate primary cell cultures. Cell ionic composition and viability was determined by histochemical quantitative electron probe X-ray microanalysis using a Philips XL-30 scanning electron microscope equipped with an EDAX detector.

Electron probe X-ray microanalysis revealed that fibrous cartilage cells showed an intracellular K/Na ratio of 7.31 while hyaline cartilage cells showed an intracellular K/Na ratio of 3.45. The intracellular contents of chlorine, phosphorous and sulfur in fibrous chondrocytes were 194.47 ± 45.24, 294.22 ± 39.35 and 83.66 ± 27.99 mmol/kg of dry cell weight, respectively. For hyaline cartilage cells, the concentrations of the same elements were 208.77 ± 40.43, 243.43 ± 42.12 and 69.41 ± 17.44 mmol/kg of dry cell weight, respectively.

These results imply that fibrous cartilage cells could have higher viability than hyaline cartilage cells in primary culture. Therefore, fibrous chondrocytes could offer a better option for use in tissue engineering at this step.

42) Quality Control of Cultured Hyaline Cartilage Cells as Determined by Quantitative Histochemical Microanalysis

The use of chondrocytes in the treatment of different pathologies is highly dependent on the use of viable cells. Cell viability should be evaluated in cultured chondrocytes intended for clinical use. In this study, we have determined the cell viability of primary cell cultures of hialyne chondrocytes at different cell subcultures by using electron-probe X-ray microanalysis

10 cartilage biopsies obtained from the condilar surface of the femoro-tibial joints of 5 Wistar rats were digested with collagenase II at 37° for 3 h to isolate the cell population. Chondrocytes were cultured in culture media supplemented with several growth factors. Subconfluent chondrocytes were subcultured using trypsin-EDTA. Cell viability was determined in the first 3 cell passages by quantitative electron probe X-ray microanalisys using gold grids covered with pioloform and by quantification of the percentage of alive and dead cells after trypan blue staining for 5 minutes.

Electron-probe X-ray microanalysis revealed that the intracellular K/Na ratio progressively decreased from 3.45 for the first cell passage to 0.55 for the third one. Other element concentrations such as P, S, or Mg showed the same pattern.

On the other hand, trypan blue staining showed that cell viability was 67.3%, 97.4% and 97.5% of the cells at first, second and third passages, respectively.

Considering both results, we conclude that cells at the second cell passage could have the highest cell viability and should therefore be preferentially used in tissue engineering.

43) Vascularised Bone Substitutes

The aim of this work is to develop and optimize biomorphous ceramic constructs with polymodal hierarchical porosity via 3D printing. The construction shall offer beneficial conditions for vascularisation in the case of regeneration of sized bone defects. The scaffolds are applied for the preparation of a bone substitute. An arteriovenous loop acts as a seed for vascularisation. The existence of a capillary network at the time of transplantation facilitates the survival of tissue cells and ensures the physiological function of generated tissue. Hydroxyl apatite and a reactive inorganic binder phase based on calcium aluminate or calcium phosphate, respectively, were applied for 3D printing of scaffolds.

A Z^TM Printer 310 was used (4D Concepts GmbH) for printing the three dimensional objects. Mixtures from CaO (PO₄)₃/OH (hydroxyl apatite) and Ca₃(PO₄)₂ (β-tricalciumphosphate) are applied as biomorphous compounds. These are bound by a hydraulic reaction in the system CaO-Al₂O₃-H₂O or CaO-P₂O₅-H₂O, respectively, at room temperature under ambient conditions.

Porous scaffolds of cylindrical shape with a diameter of 9 mm were successfully printed. During printing the reactive inorganic binder phase is hydrated via injection of an aqueous solution. Since the binding reaction takes place at room temperature under ambient conditions, the fabrication process offers the possibility to integrate functional proteins such as growth factors. Scaffolds of variable size and shape adopted to the bone defect morphology can be designed by CAD with great flexibility. While macropore channels are formed by 3D printing, the microporosity is created by varying the grain size of the granulated powders.

44) Development of a Biotechnological Proceeding for the Extensive Expansion of Mesenchymal Stem Cells Obtained from Human Umbilical Cord

Searching for new and more easily accessible sources of human mesenchymal stem cells (hMSC), other than bone marrow, is a timely subject that is attracting more and more attention. Research over the last years shows that hMSC can be successfully isolated from extra-embryonic tissues discarded after birth, such as placenta, amnion or umbilical cord (UC), where the frequency of MSC is substantially higher than in bone marrow.

We optimized culture conditions for extensive expansion of hMSC obtained from UC perivascular tissue or whole UC. Cells were seeded in culture flasks in different densities varying from 62.5 to 4000 cells/cm² and cultured in four slightly different αMEM containing 10% human serum and 50 μg/ml gentamycin. Cell growth and vitality, consumption of glucose, glutamine as well as cell surface epitopes were analyzed by microscopic, fluorescencespectroscopic, HPLC and flow cytometric methods.

Cells were highly positive for typical stem cell surface markers CD73, CD90, CD105 and CD44. They exhibited high proliferative activity particularly when supported by αMEM containing 2 mM L-glutamine or αMEM additionally supplemented with osteogenic agents (dexamethasone, ascorbate-2-phosphate, and β-glycerophosphate). In any case cell long term progeny or use of cell seeding densities lower then 500 cells/cm² were associated with non-uniform colonization of the cultivation area as well as with a loss of cell viability and proliferative activity. However cell seeding in the density of 500 cells/cm² allows avoiding frequent disruption of cell-cell contacts and displays the best expansion strategy.

45) Porous Nano-Hydroxyapatite/Alginate Scaffolds for Bone Regeneration

Major target for prosthetic constructs of bone is the ability to elicit osteoinduction, osteoconduction and osteointegration. Promising constructs are represented by composite scaffolds made of biodegradable natural polysaccharides endowed with biocompatibility and mechanical properties. Alginate is a good candidate when its limits - weak mechanical properties and lack of cellular interactions - are overcome by reinforcing with inorganic components. In the present study an innovative strategy was developed to obtain three-dimensional scaffolds made up of alginate and nano-Hydroxyapatite. Biocompatibility and bioactivity of the scaffolds were tested in vitro and in vivo.

Nano-sized HAp was synthesized by wet chemical method using CaCl₂ and (NH₄)2HPO₄. Nano-HAp was characterized by RAMAN, X-RD and SEM analysis. 3D porous scaffolds were prepared from a mixture of Alginate and nano-HAp powder. The gelification was obtained by addition of Glucono-δ-Lactone. Structural characterization was performed by SEM, confocal microscopy and micro-CT analysis. Scaffolds were loaded with osteosarcoma cell lines and primary osteoblast to evaluate proliferation and bone markers expression. Bone formation and osteointegration was studied in rabbit models.

Cells displayed a high rate of proliferation in the scaffolds and RT-PCR and ELISA assays confirmed conservation of osteoblast phenotype and bone matrix synthesis. SEM images showed spherical cells embedded in newly synthesized ECM.

Histomorphometric analysis on implants inserted in rabbit femoral diaphisis, revealed ingrowth of newly deposited lamellar bone inside scaffold with a high Bone to Implant Contact index. The results obtained seem promising for a future use of scaffolds for in vivo bone regeneration protocols.

46) Nanocomposite Systems Based on Silver Nanoparticles for Antimicrobial Applications

Infections associated with orthopedic implants are life-threatening complications that often occur in medical device insertions. Presently, bacteria are becoming remarkably resistant to antibiotics so there is a new interest on silver as a promising antimicrobial material mainly in the form of nanoparticles. It is known that silver particles must be nanosized because their aggregation leads to the loss of properties; an efficient way to prevent their aggregation is by using polymers.

In this work solutions and gels containing silver nanoparticles were prepared by reducing silver ions in the presence of polysaccharides and their antibacterial activity was assessed. Biological tests were performed with four strains of bacteria. Bacterial growth kinetics tests and colony count tests were performed using solutions with various concentration of polymer and silver. A mixed polysaccharide gel was prepared and the antimicrobial properties of this gel were studied by bacterial adhesion tests. Cytotoxicity assays (LDH) of the gels were performed with osteoblasts (MG63), fibroblast-like (NIH3T3) and hepatocyte-like cell lines (HepG2). Bacterial growth kinetics of the system proved that the silver nanoparticles solutions do possess antimicrobial effect. Colony Count tests revealed the system to be bactericidal. A 3D hydrogel was prepared and it displayed as well a powerful antimicrobial activity, as shown by bacterial adhesion tests. Cytotoxicity assays proved that this gel was not toxic to the cell lines used. They display a powerful bactericidal activity either in solutions or as gels which are not toxic to cells.

47) Off-label Use of rhBMP-2 (InductOs) in Orthopaedic Surgery

Dibotermin-α, recombinant human Bone Morphogenetic Protein, is a powerful tool in orthopaedic surgery by binding to the receptors of mesenchymal cells and triggering them to differentiate into bone-forming cells.

Although the drug is only approved for very narrow indications (Single level (L4-S1) anterior lumbar spine fusion for DDD in adults - Adjunct to standard care using open tibial fracture reduction and intramedullary nail fixation), we used it in critical situations, such as pseudarthrosis, defective bone situation or difficult surgical access, where it proved to be most effective. The need for sound fusion within an acceptable time period of a few weeks was the main indication for the off-label use even in children, in tumorous disease or in post-infecteous surgery.

In every case, rhBMP-2 (InductOs) was applied following the instructions by the pharmaceutical company on an acs (absorbable collagen sponge)-carrier. Only one package of Dibotermin-α per case was used. In every case the local application was carried out in a reliable stable area, with the acs applied either in a cage or along the standard implants, eventually in combination with cancellous bone grafts. In the early results, local adverse effects as inflammation, ödema, heterotopic ossification or nerve or root compression syndromes were not dealt with.

Our individual results underline the surgical possibilities of rhBMP-2 (InductOs) application in orthopaedic surgery. Nevertheless, as an off-label indication its use should be limited for very demanding and challenging procedures requiring a “single shot” strategy. As long as the on-label indications remain restricted, every off-label use requires proper information of the patient on possible risks before obtaining his informed consent according to the Declaration of Helsinki.

48) The Development of Novel NF-kB Decoy ODN Coated Interconnected Porous Hydroxyapatite

It was demonstrated that interconnected porous calcium hydroxyapatite (IP-CHA: NEOBONE®) showed superior osteoinduction because of uniform pore size and >10 μm in diameter interconnected pore size. In inflammatory disease such as rheumatoid arthritis, it was demonstrated that bone resorption was accelerated and bone fragility of periarticular was demonstrated. In vitro and in vivo, we demonstrated that the administration of NF-κB decoy oligonucleotide (ODN) attenuated the function of osteoclast and inflammatory cytokines. It was demonstrated that inflammatory cytokines such as TNF-α suppressed the function of osteoblast. So we developed NF-κB decoy ODN coated IP-CHA.

In this study, we evaluate in vivo function of NF-κB decoy ODN coated IP-CHA.

Normal (control) and NF-κB decoy ODN coated IP-CHA (NF-κB decoy) was implanted in rabbit femoral condyle. Implanted IP-CHA was subdivided into three zones. At 1,2,4 weeks after implantation, mean ratio of bone formation area and anti cathepsin K positive area was measured by software. Compression strength of IP-CHA was evaluated with samples at 4 weeks after implantation.

In zone 1 and 2, mean ratio of bone formation area at 1,2 and 4 weeks after implantation were significantly higher in NF-κB decoy group than those in control group. In zone1, mean ratio of cathepsinK positive area at 2 and 4 weeks were significantly higher in control group than NF-κB group. Compression strength of IP-CHA showed no significance between control and NF-κB group.

NF-κB decoy ODN coated IP-CHA indicated the superior osteoinduction. In future, this novel IP-CHA will be beneficial tool for arthritis patients.

49) IGF-1 Release from a Collagen-GAG Scaffold for Bone and Cartilage Repair

The local release of biomolecules and cells that accompanies the spontaneous repair process in these defects can result in a limited degree of repair; the use of scaffolds, cells and active molecules to assist this spontaneous repair, however, has been shown to be a promising method of improving the quality of this repair in a number of clinical applications. Pore size, specific surface area, pore interconnectivity and a number of other key parameters of this scaffold can be controlled to facilitate optimal cell migration, waste and nutrient transport, and vascular invasion in vivo. The research described in this paper focuses on the incorporation of a chondrogenic growth factor, IGF-1, into this scaffold to enhance further the repair of osteochondral defects. Elution of IGF-1 adsorbed onto the struts of the scaffold was monitored using a radiolabeling technique.

A well characterised collagen - GAG scaffold, ¹²⁵I-IGF-1 (185KBq): IGF-1 1:10000

Adsorption: A 1.5mg collagen-GAG scaffold was placed in a plastic tube. Radioiodinated IGF-1 solution (0.5ng ¹²⁵I-IGF-1 + 5 μg IGF-1, or 1.3ng ¹²⁵I-IGF-1 + 13 μg IGF-1) in 0.5 ml PBS was added and the tube was placed on a shaker and incubated at 37°C. The scaffold was removed after 24 hrs and counted.

Elution: The same scaffold was then placed in 1 ml PBS and shaken at 37°C. The solution was sampled, counted periodically and replaced with fresh PBS.

The scaffold has an interconnected pore structure with high porosity (85 ± 3%), and a mean pore size of 95.4 ± 19 μm. Initial burst release was followed by sustained release.

50) Fabrication of Open Porous Calcium Phosphate/ Protein Scaffolds Resembling Human Bone Structure

Human bone features a complex open porous structure including dense regions referred to as Compacta as well as sponge-like regions called Spongiosa. At the same time, the specific chemical composition of bone, consisting of a mineral phase (bone apatite) and an organic phase (collagen), provides its biological functionality.

The aim of this research project is the fabrication of open porous calcium phosphate/protein scaffolds with bone-like properties regarding bioactivity, controlled biodegradation and sufficient mechanical stability.

Two different fabrication methods are applied to allow the simulation of both human bone structures. Freeze Cast Processing is used to fabricate a Compacta-like structure while a Polymer Replica Method is applied to adapt the structure of the Spongiosa.

Protein functionalisation is done via two different fabrication routes. For the first method a calcium phosphate/protein green body is manufactured by directly mixing the proteins into the slurry. A thus fabricated composite has a high resorbability as an advantage, but mechanical properties must mainly arise from cross-linking of fibrous proteins (e. g. collagen) as sintering is not possible. For the second method the protein is infiltrated into the sintered calcium phosphate composites. The initial mechanical stability is higher, but the composites are less resorbable.

Protein adsorption is quantified by zeta potential and VIS spectroscopy measurements. In biofunctionalized scaffolds the proteins in the structure are dyed for visualization with fluorescence microscopy. In order to assess the bioactivity the Calcium phosphate/protein composites are cell tested. To estimate the degradation of the scaffolds Ca₂⁺ -solubility is measured as a function of pH.

51) Caveolin in Human Mesenchymal Stem Cells and Its Interactions with Biomaterials

Proliferation and osteogenic differentiation of human mesenchymal stem cells (hMSC) can be influenced by surface roughness and chemical modification. Therefore the cellular organization and expression of caveolin-1 was studied in hMSC on titanium discs with different surface roughness and functionalization.

Polished titanium discs were coated with thin layer of plasma polymerized allylamine (PAA). On that surface Collagen I (Col) was immobilized via bifunctional linker molecules PEG or GDA. In addition, pure titanium discs with different topographies were used. hMSC were cultivated on the modified titanium surfaces. Immunofluorescence analyses were performed for the expression of caveolin, ph-caveolin, vinculin, ph-FAK, integrin ß1 and GM1 using confocal laser scanning microscopy. Protein and mRNA expression of caveolin and ph-caveolin were analysed by quantitative RT-PCR and Western blot.

In cytoplasm caveolin was detectable in association with the actin cytoskeleton. Activated ph-caveolin, but not caveolin was localized in focal contacts together with integrin ß1 and vinculin. On chemically modified titanium surfaces no obvious differences of cell shapes were detectable. Expression of caveolin and ph-caveolin on PAA-surface was decreased compared to PAA-PEG-Col, PAA-GDA-Col, TCPS or polished titanium. The analysis of titanium with different surface roughness showed decreased expression of caveolin on rough surfaces. Concerning mRNA expression of caveolin in hMSC no distinct effects were found.

The cellular organization and expression of caveolin can be affected by material surface characteristics. We suggest that biomaterial surfaces control the biology of hMSC by affecting both the cellular organization and expression of caveolin.

52) Availability of Human Mesenchymal Stem Cells for Non-Autologous Bone Regeneration: An In Vivo Study for Regeneration of Critical Size Bone Defects in Sheep

Mesenchymal stem cells (MSC) are an attractive cell source for regeneration of bone. Due to a lack of expression of immunological relevant surface antigens, this cell type might be available for non-autologous cell transplantation. Although immunosuppressive properties have been demonstrated and xenogenic MSC show an engraftment after xenogenic transplantation without immunosuppression, it remains unclear if non-autologous MSC equal autologous MSC concerning their regeneration capacity.

After isolation and cultivation on mineralized collagen, xenogenic human and autologous ovine MSC were transplanted into a 3.0 cm diaphyseal tibial defect of 7 sheep. Animals were sacrificed after 3 and 6 months. Unloaded scaffolds served as a control. Radiography was performed every 2 weeks, histological evaluation was performed after euthanasia (including in-situ hybridization for detection of human MSC). Bone regeneration was analyzed using (semi-) quantitative scoring systems on radiographic and histological levels.

MSC lead to significant increase in bone formation after 8 weeks compared to unloaded controls (p < 0.05). This difference could also be detected in histological evaluation of the specimen after euthanasia (p < 0.01). Compared to the autologous MSC, xenogenic MSC lead to inferior bone formation (p < 0.05). Nevertheless, an engraftment of xenogenic MSC was detected using human specific in situ hybridization, while no severe systemic or local immune response was detected.

Although xenogenic MSC were detected and no severe immune response was detected, the xenogenic transplantation of MSC lead to inferior bone formation compared to autologous MSC. To identify the biological principle beyond this observation will be part of further studies.

53) Immediate Implant Placement and Provisionalization after Long Axis Root Fracture and Complete Loss of the Facial Bony Lamella

To explore the performance of a new flapless surgical approach for immediate implant placement, simultaneous alveolar ridge augmentation and immediate provisionalization in case of total loss of the facial bony lamella due to long-axis root fracture.

Overall 21 Nobel Perfect™ implants were placed in 19 patients (follow up 13 to 36 months). All patients had sustained complete loss of the facial bony lamellae. In a flapless technique, implants were inserted simultaneous to subperiostal bone augmentation with autogenous bone chips and underwent immediate provisionalization. Outcome variables were implant success rates, marginal bone levels and Pink Esthetic Score (PES).

All implants achieved excellent primary stability, there were no implant losses. Marginal bone levels averaged 1.3 mm (oder 1.5 mm ohne die Verlustfall) above the first thread inter-proximally and remained stable within the observational period exept one case of bone loss below the first thread. Post-operative cone beam tomographies (CBT) were available for 16 implants sites und revealed regeneration of the facial lamella to the level of the first thread. Overall the “Pink Esthetic Score” remained unchanged with the surgical intervention. A mean PES of 12.5 was achieved at the final examination. Oral hygiene (SBI) was highly predictive for the esthetic result.

Survival rates, marginal bone levels and esthetic results suggest proof of principle for the new flapless immediate implant placement technique in case of total loss of the facial bony lamella. Oral hygiene status should be considered for patient allocation to single stage or multiple stage treatment.

54) Simulation In Silico of Bone Tissue Regeneration within Polimeric Scaffolds

This work is concerned with the mathematical modelling and computer implementation of bone growth within polymeric (short-term bioreabsorbable scaffolds). It is therefore intended the prediction of bone tissue regeneration and scaffold evolution mass by means of computer simulation.

A multiscale mathematical model is developed. The model account for two different spatial scales, i.e., the pore scaffold level where the apposition of new bone tissue and biodegradation occurs and the bone organ/tissue level where the scaffold is implanted. The apparent properties are derived by invoking the asymptotic homogenization theory. At the pore level, a model of bone growth (given by the strain energy as a mechanical stimulus and the cell population concentration) and scaffold degradation (hydrolysis) are posed. As an example of application, a femur of a New Zealand rabbit implanted with a poly(caprolactone) (PCL) scaffold was analyzed. The bone geometry was reconstructed by computer tomography, whereas the scaffold microstructure (pore level) was idealised following a face cubic centred arrangement of the empty pores along a unit cell of the scaffold microstructure.

The model allows obtaining the bone organ density distribution as well as the microscopic bone distribution and scaffold degradation. For this specific application, the model predicts 27.5% and 51.3% of overall bone regeneration and scaffold degradation, respectively, versus the overall scaffold volume.

Requiring further experimental validation, the model may be useful in the finding of the optimal scaffold in the framework of bone tissue engineering.

55) Bioactivity of Collagen/Nano-B-Tricalcium Phosphate Crosslinked Scaffolds Used in Bone Tissue Engineering

Stability and biocompatibility of scaffolds used in tissue engineering play an important role in cell adhesion and growth. New composite materials were prepared by sonicating a collagen (COL) solution (0.8 %, w/w) and nano-β-tricalcium phosphate (β-nTCP) powder, in weight ratios of 1:1 and 1:2.6. These mixtures were conditioned as porous scaffolds by freeze-drying at two different freezing temperatures, −80 °C and −30 °C. In order to improve their biostability, COL/β-nTCP scaffolds were crosslinked by treatment with 200 mM 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC). Porosity and density were physico-chemically determined and pore morphology was observed at an environmental scanning electron microscopy (ESEM). Biochemical properties of the crosslinked COL/β-nTCP scaffold was analyzed by assaying the calcium amounts released from the scaffold in natural mimicking conditions and their degradation in the presence of bacterial collagenase. It was observed a better biostability of the chemical crosslinked scaffolds over non-crosslinked ones.

Human mesenchymal stem cells (hMSC) were obtained from bone marrow aspirate of the iliac crest by density gradient centrifugation. hMSC were characterized by fibroblast colony forming unit capacity, immunophenotypic (CD45, CD90 and CD105) by flow cytometry and osteoblast differentiation potential by Alizarin Red assay.

The effect of COL/β-nTCP scaffolds on the viability and differentiation of hMSC was assessed by MTT assay and osteoblast marker identification. All scaffolds presented a good cytocompatibility and supported cell proliferation at 72 h after cell seeding and differentiation.

All these results demonstrate that chemically cross-linked COL/β-nTCP scaffolds are potential candidates for bone substitutes with enhanced biostability and good biocompatibility.

56) Towards an In-Vitro 3D Multi-Cell Co-Culture Model of Bone Tissue

The ultimate goal of this work is to generate a 3D osteoblastic-osteoclastic-endothelial multi-cell co-culture system, as an in-vitro model to mimic the process of bone matrix deposition and remodelling. Here, we investigate the feasibility to generate the three cell lineages using human adipose tissue derived cells(AT) and cd14+ monocytes from peripheral blood (PBM).

AT with or without PBM were seeded and cultured on 3D ceramic scaffolds using a perfusion bioreactor in presence of Machrophage Colony Stimulating Factor and Receptor Activator for Nuclear Factor κB-Ligand for 19 days. Cell phenotypes were assessed by cytofluorimetry and Tartrate Resistant Acid Phosphatase (TRAP) staining. To assess the functionality of the cells, constructs were subcutaneously implanted in nude mice and the formation of bone tissue and human blood vessels was histologically and immunohistochemically assessed.

Cytofluorimetry analysis demonstrated the presence of endothelial (1.2% of CD34+/CD31+), osteoclastic (13% cd14+/cd31+) and osteoblastic (22% STRO1-/ALP+) cell lineages in constructs generated by AT and PBM. Osteoclastic cells (TRAP+) attached to the scaffolds were detected only in the presence of PBM. In-vivo, human cell-derived blood vessel and sporadic bone formation were found in both groups. The presence of human-derived osteoclasts is currently underway.

The study indicates that AT and PBM cells, in a 3D dynamic environment, can be used to establish an in-vitro co-culture model of osteoblastic, osteoclastic and endothelial lineage cells, retaining their functionality following in-vivo implantation. Ongoing experiments will investigate the potential use of this model for testing drugs regulating bone homeostasis.

57) COOL – An Innovative Bone Cement

A newly developed bone cement, named COOL, should be assessed concerning its mechanical properties and biocompatibility in comparison to conventional PMMA based bone cement.

COOL is a powder/liquid two component system. The powder is a composition of different bioceramics as well as metal oxides for improving mechanical strength and radiopacity. The liquid is a solute acid-modified polymethylmethacrylate (PMMA). By mixing these components a creamy, adhesive compound is formed. The cure of the material occurs by a cementation reaction at body temperature. Mechanical properties of COOL were determined according to ISO 5833. Biocompatibility was evaluated in a model of primary human osteoblasts. Cell morphology was analyzed by light microscopy and cell viability by alamarblue™ assay. As a reference Refobacinâ Bone Cement R was used.

Mechanical properties like bending and compression strength are depending on the composition of COOL. By a 70/30 ratio between powder and liquid, a bending strength of 19,2 ± 2,5 MPa and a compressive strength of 16 ± 3,7 MPa were estimated. Pore sizes up to 150 μm could be obtained. This allows bone cells to grow into the cement. Osteoblasts incubated with COOL showed a significant higher density as well as higher viability in comparison to reference material. Furthermore, alkaline phosphatase (ALP) activity was higher in osteoblasts cultured with COOL compared to reference material or control cultures, which indicates bioinductive capacity of the new material.

COOL could be a promising alternative to conventional PMMA bone cements in orthopaedic surgery.

58) Differential Expression of OTX Genes during Osteogenic Diffferentiation

An efficient fusion is still the goal of most spine procedures and recent results point to Mesenchymal Stem Cells (MSCs) as a promising therapeutical tool, however several aspects of their biology still need to be clarified. Developmental cues are candidate regulatory mechanisms controlling adult stem cells. We previously reported that Otx1, a key factor in brain morphogenesis and sense organs development, plays important roles also in blood cell production. Here we investigated whether Otx genes may be involved also in the regulation of MSCs, particularly during osteogenic differentiation, as blood and bone share several common features.

We first analysed the expression of Otx genes and a panel of candidate upstream and downstream genes in MC3T3-E1 cells, derived from newborn mouse calvaria, which can be induced to differentiate and provide a model currently used in literature. Using semiquantitative and quantitative PCR (Real Time qPCR), a similar study was carried out also in human MSCs, maintained in vitro in expansion conditions or after osteogenic induction.

Otx1 is active both in murine and human cells and its expression is modulated during osteogenic differentiation. Furthermore, the expression pattern of Otx1 correlates with the activity of early osteogenic markers, suggesting an involvement of the gene at early stages of osteogenesis. The identification of new molecular cues, such as Otx1, regulating bone cells may allow the development of more efficient strategies for tissue regeneration.

59) 3D Microenvironments and Live Monitoring of Osteogenesis in Single Cells

Connection between cell shape and regulation of cellular processes such as proliferation, differen-tiation and extra cellular matrix (ECM) production is obvious and correlate with changes in expression of genes involved in these processes. The gradual process of osteogenesis can be followed by different proteins being expressed at various time points, comprising early (e.g. runx2, bone alkaline phosphatase) and late (e.g. osteocalcin) genes. The aim of our project is to develop new tools for live monitoring of single cell differentiation and cell – surface interactions in engineered quasi three-dimensional (3D) microenvironments.

Reporter-gene constructs upon which expression in cells the green fluorescent protein (GFP) is expressed under the control of osteogenesis specific promoters have been generated. Transfer of plasmid DNA directly into the nucleus of human bone marrow cells (HBMC) and human bone cells (HBC) was enabled by nucleofection. In addition to monitor differentiation by expression and fluorescence of GFP, cells were immunohistochemically stained against bone-specific proteins.

The 3D microwells were produced by standard microfabrication techniques in silicon followed by thin film replica molding in PDMS. Passivation of the surface was followed by functionalization of the wells with fibronectin. Cells seeded in microwells were monitored by fluorescence microscopy.

Primary results show attachment and growth of human cells in functionalized microwells as well as an efficient transfection with osteogenic reporter-gene constructs. Immunohistochemical staining as well as the GFP reporter strategy allows monitoring of osteogenic differentiation in 3D microenvironments on a single cell level.

60) Manufacturing and Characterization of Biodegradable Nanocomposites Based on Wollastonite and Polycaprolactone

The aim of this work was to manufacture bioactive and biodegradable nanocomposites. The effects of phase composition and morphology on physicochemical, bioactive and biocompatible features were investigated.

Wollastonite powders were obtained by the controlled pyrolysis of poly(methylphenylsiloxane) resin with active fillers as previously described. Obtained ceramics was milled to nano-grain size. Nanocomposites with various content of ceramic phase were obtained by adding wollastonite powders to 10% wt solution of polycaprolactone (Sigma-Aldrich, Mn = 80,000g /mol) in dichloromethane (Fluka). Chemical composition, physicochemical properties and morphology of prepared nanocomposites have been studied. To investigate bioactivity, test in the simulated body fluid (SBF) and studies with cell lines were carried out.

Our data demonstrate that addition of wollastonite leads to obtain a composite with bioactive features. Addition of nano-bioceramic particles changes mechanical and physicochemical properties of polycaprolactone.

Incorporation of bioactive nanowollastonite particles into biodegradable polymers opens new possibilities in hard tissue regeneration. Obtained materials are characterized by good combination of physicochemical, mechanical and bioactive features. Their osteogenic potential in contact with living cells is currently under investigation.

61) In Vitro Studies on Wollastonite Based Nanocomposite for Medical Application

The aim of the work was to prepare nanocomposites from PCL (ε-polycaprolactone)/nanowollastonite, evaluate their physicochemical properties and in vitro biocompatibility.

Formation of apatite layer on the implant surface is very important for strong binding to host bone tissue. Wollastonite (CaSiO₃) shows very good bone compatibility and conductivity. Also it demonstrates high bone inductivity property in marrow by inducting favorable proteins and osteocytes response and hence stimulating of new bone formation [1]. Nanowollastonite has been also reported to enhance the mechanical properties of polymer composites.

Ceramic wollastonite powders were obtained by the controlled pyrolysis of organocilicon precursors with active fillers as previously described [2] and milled to nano-grain size. The nanocomposite were prepared by incorporating 1,2,4wt.% of ceramic powders into PCL matrix using solution casting method. Chemical composition, physicochemical properties and morphology of prepared nanocomposites have been studied. For characterization of the in vitro bioactivity, samples were immersed in simulated body fluid (SBF) for different time periods. Also cell adhesion and proliferation in contact with nanocomposites were examined.

Our data demonstrate that nanocomposite with low content of ceramic phase (1,2,4%) are characterized by bioactive features. Addition of nano-bioceramic particles changes mechanical and physicochemical properties of polycaprolactone and therefore in vitro response in contact with simulated body fluid and the cell cultures.

All these physico - chemical properties and bioative nature of PCL/Woll nanocompostie makes them potential candidates in biomedical applications.

62) Tissue Engineering and Cryopreservation of Artificial Tissue Constructs Using a Synthetic ß-TCP Ceramic

Cryopreservation in vitro generated tissue constructs allow long term storage and assures disposability according to the surgeon's requirements if temporal flexibility is needed. While cryopreservation of single cell suspensions is well established, protocols and procedures to cryopreserve complete tissue constructs are not yet available.

Within an extensive screening process five different commercially available biomaterials have been tested regarding to their suitability for bone tissue engineering: Structural analyses were made to acquire basic material parameters such as surface condition, porosity, pore size, and interconnectivity. If geometrical properties referred to the suitability for use as scaffold material, the material behaviour under cell culture conditions was analysed and the stability under cryopreservation conditions was tested.

Identified as the most promising material in the test, a porous sintered ß-TCP ceramic (Cerasorb M®, Curasan AG, Kleinostheim) has been used for breeding tissue constructs by seeding the material with human osteoprogenitor cells and cultivating it for 5–7 weeks using osteogenic-supplemented medium. Bone formation and calcification were checked by measuring the ALP activity, the osteocalcin concentration and the calcium content of the culture medium, and by performing a polychromatic sequence labelling.

A cryoprotocol was developed down to −80°C including special equipment for the handling during cultivation, freezing and thawing of the lab-generated bone tissue constructs. Excellent preservation results could be achieved. The transfer of the protocol can take place to a tissue bank as a standard routine within next two years.

63) Functional Bone Tissue Engineering Based on Dynamic 3D-Cultivation

The principle of Tissue Engineering is to build artificial replacements by means of vital components. In order to achieve a functional graft, applicable cells were seeded on three-dimensional scaffolds and expanded in vitro.

The common used cell culture techniques generate cell layers, but it is not possible to create a three dimensional, functional multilayer cell structure on the surface of a cell culture dish. Therefore, three dimensional scaffolds are necessary, which provide a specific environment and architecture for the formation of the tissue.

In this study, we cultivated osteoblasic precursor cells, mesenchymal stem cells and primary osteoblasts on a zirconium dioxide ceramic with regard to their osteogenic differentiation under static and dynamic conditions. The cell cultivation under dynamic conditions was performed in a complete platform for 3D cell cultivation consisting of a rotating bed system Z^®RP in a GMP compatible breeder and a control unit allowing a GMP conform documentation and evaluation. In summary, with dynamic cultivation we achieved extensive cell proliferation and differentiation of the mesenchymal stem cells along the bone lineage. Tissue like structures with fibrous (collagen) and globular (mineral) extracellular matrix components stained positive for calcium, the main bone characteristic.

64) Extracellular Matrix Synthesis in Large Animal and Human Derived Articular Chondrocytes: Implications for Cartilage Tissue Engineering

Despite large animal models play a pivotal role in the evaluation of cartilage repair strategies, there are only few studies available analyzing the interspecies differences of articular chondrocytes. The present study was to compare extracellular matrix synthesis of cultured large animal and human articular chondrocytes of the knee joint.

Primary chondrocytes were isolated from human, porcine, ovine and equine full thickness knee joint cartilage and investigated flow cytometrically for their proliferation rates. Cytoskeletal organization and extracellular matrix synthesis (collagen types II, I, cartilage proteoglycans) were analysed in freshly isolated or passaged chondrocytes using immunohistochemistry and westernblotting. Human and porcine chondrocytes were subsequently cultured on PGA constructs for 2 weeks and evaluated for extracellular cartilage matrix production.

Chondrocytes morphology and cytoskeletal assembly differed between species. Animal derived chondrocytes developed more rapidly F-actin stress fibres during culturing than human chondrocytes and their proliferation rate was higher. All chondrocytes expressed cartilage-specific extracellular matrix components such as collagen type II and proteoglycans. However, after monolayer expansion, cartilage proteoglycan expression was barely detectable in equine chondrocytes whereby collagen type I deposition increased compared with porcine and human chondrocytes. Cultured on PGA porcine chondrocytes revealed higher extracellular matrix synthesis capacities compared with human articular chondrocytes.

With respect to proliferation and extracellular matrix synthesis, human chondrocytes shared more similarity with porcine than with ovine or equine chondrocytes. Interspecies differences between human and large animal derived chondrocytes should be carefully considered when using animal models to develop cartilage repair strategies.

65) Mechanical Evaluation and In Vitro Analysis of a Porous Biomaterial (Polymethylmethacrylate and Hydroxyapatite) for Use in Cranioplasty through Rapid Prototyping

We investigated the possibility of application of the porous material composite of polymethylmethacrylate and hydroxyapatite as a biomaterial used in the treatment of craniofacial deformities using rapid prototyping as a support tool for this work. The main motivation of this work is to make this material readily available, disseminate and integrate systems, methods, and rapid prototyping, reducing costs and post-operative risks to the patient. Were conducted mechanical tests of compression and in vitro analysis. Were used 10 cylindrical test bodies of the porous material composed of PMMA / HA, 10 cylindrical test bodies of the only porous PMMA and 10 cylindrical test bodies of the PMMA (control). The process of incorporating the hydroxyapatite in the material that served as a matrix (PMMA) decreased its mechanical strength at levels considered satisfactory because this matrix shows 66.7% of porosity and the HAp will much contribute to the increase in the biocompatibility of polymethylmethacrylate. The results of in vitro tests indicated that the different samples of porous PMMA/HA and porous PMMA didn't present toxicity, could be this used in animal experiments (analysis in vivo), where they can be obtained the conclusive results regarding this material developed for bone implants.

66) Structural Implant Concept for Jaw Applications

The demands placed on alloplastic implants are high. Ideally, we strive to use a material that is cost effective, but not toxic, antigenic, or carcinogenic. Most importantly, the ideal implant would be permanently accepted. A variety of materials have been used for implants in facial aesthetic and reconstructive surgery, such as hydroxyapatite (HA), polymethylmethacrylate (PMMA), and metallic materials, some coming closer than others in approaching the qualities of an ideal implant material. No one material, however, is universally successful because few present satisfactory mechanical resistances associated with biological interaction.

Is proposed a bioactive material added with high strength mechanic, for such was designed a PMMA functional structure gradient, inner dense with porous and bioactivated surface. The inner dense part is responsibility for mechanical strength and the porous surface added hydroxyapatite is responsibility for the osseointegration.

The porous surface composite material was produced with hydroxyapatite–polymethylmethacrylate (HA–PMMA) by mixing HA granules (4.1 wt%), PMMA (14 wt%), MMA (42 wt%) and Carboxymethylcellulose powder (CMC) (1.9 wt%), Soken Chemical & Engineering Co., Ltd., Tokyo, Japan. The bulk material was prepared with PMMA powder (33.3 wt%) and MMA monomer (66.7 wt%).

The manufacturing allowed uniform hydroxyapatite distribution in the superficial and inside the scaffolds. It was observed 4% diametric shrinkage The results in vitro indicated that the different samples of porous PMMA/HA and porous PMMA didn't present toxicity, could be this used in animal experiments (analysis in vivo), where they can be obtained the conclusive results regarding this material developed for resurfacing implants.

67) Characterisation of Primary Human Tenocytes

Tendon ruptures are healing via formation of less mechanical resilient scar tissue which can lead to pain and re-ruptures. Causes for the functionally inferior extracellular matrix of tendon scars have been less investigated. The relation between surrounding cells, Collagen Type I-synthesising tenocytes and growth factors seems to play a relevant role. The aim of this work is to characterise tenocytes in vitro as a solid basis to study the influences of growth factors and develop strategies for rebuilding tendon tissue with more favourable biomechanical characteristics.

Tendon derived cells, isolated from human supraspinatus, biceps and achilles tendons were cultured in DMEM/HAM's F12 media (10% FCS, 1% Penicillin/Streptomycin) up to passage 3. mRNA expression of different markers (Scleraxis, Tenascin-C, Decorin, Biglycan, Aggrecan, Collagen Types I, II and III) was analysed in tenocytes and primary chondrocytes (negative control) using Real-Time-PCR.

Compared to chondrocytes the tenocytes showed higher expression rates of collagen types I, III and the tendon-specific marker Scleraxis as well as expressions of glycoproteins tends to be higher. This fits to the higher expression of the cartilage-specific markers Aggrecan and Collagen type II in chondrocytes.

The combined analysis of multiple marker expression by tenocytes in comparison with other musculoskeletal cells permits a more reliable characterisation of them. Most of the common tendon markers (e.g. Collagen type I) are also expressed by other musculoskeletal cells and are not sufficient to identify tenocytes. These well characterised tenocytes are suitable for further in vitro experiments to study the effects of growth factors.

68) Characterization of Composite Polysaccharide-Based Scaffolds

In this work two oppositely charged polysaccharides, alginate and a lactose-modified chitosan, mixed with hydroxyapatite have been used to prepare 3D porous biodegradable scaffolds which revealed high proliferation of osteoblasts.

The 3D biodegradable porous scaffold developed in this study has been prepared from mixture of polysaccharides and hydroxyapatite. MTS assay has been used to assess proliferation of cells. Micro Computed Tomography, Raman Spectroscopy and confocal microscopy have been performed.

The identity of hydroxyapatite after the gelification process was confirmed by direct comparison of the typical Raman peaks of hydroxyapatite powder with those obtained from the analysis of the hydrogel. Pore size detected with the SEM facility has been cross-checked in comparison with 3D reconstruction of μ-CT data. Custom produced MATLAB codes had been used to determinate the ideal path through the interconnected pores. Confocal microscopy allowed to observe that the green-fluorescent reswollen scaffold keeps the average pore size dimensions and interconnectivity of the freeze-dried ones. The morphological features of this construct have been investigated by several image analysis techniques revealing a correct scaffold architecture which allows cell-ingrowth and suitable diffusion of nutrients and catabolite exchange. Quantitative structural analysis revealed an adequate pore size, pore distribution and connectivity that effectively allow the cells to adhere and preserve their function. SEM and confocal transversal section analysis indicate that cells were able to penetrate deep into the scaffold core. MTS assay showed that cells not only were viable but also proliferate well in the porous network.

69) Human Skeletal Muscle-Derived Cells Undergo Matrix Mineralization in an Osteogenic Pellet Culture Assay

Objective was to investigate the osteogenic differentiation of human skeletal muscle-derived cells (hMDCs) using a 3D osteogenic culture system and micro-computed tomography (μCT) analysis.

Cells isolated from the skeletal muscle of 4 female and 4 male donors were obtained from Cook MyoSite Incorporated. Their expression of the CD56 was determined by flow cytometry. hMDCs were cultured as pellets (250 000 cells/pellet) in osteogenic medium for 28 days and nondestructively analysed at 7, 14, 21, and 28 days by μCT for mineralized matrix volume and density. Gene expression for osteocalcin (Ocn) and collagen type I (Col I) was determined using quantitative RT-PCR on day 0 and day 28.

Mineralization was evident in most hMDCs populations as early as 7 days and increased over time. A significant increase in bone volume was observed after 28 days, and density was significantly greater at 21 and 28 days. No significant difference in volume and density was observed between male and female cells. hMDCs with low CD56 expression did not mineralize as quickly as those expressing higher levels. There was no significant change in Ocn gene expression, but Col I expression was increased after 28 days.

hMDCs can produce bone matrix under specific culture conditions. CD56 may be a possible marker for the osteogenic potential of hMDCs. μCT analysis combined with 3D pellet culture is a valuable method to determine matrix mineralization. Hence, this method can be applied for the screening of potential cellular candidates for bone tissue engineering applications.

70) Endochondral Ossification: An Alternative Approach for Bone Repair?

Human Bone Marrow Stromal Cells (hBMSCs) can be used in the treatment of bone defects. In this study we test how in-vitro priming of hBMSCs influences the endochondral ossification process.

In vivo implantation

hBMSCs were seeded in collagen-GAG scaffolds and were cultured for 21 days on chondrogenic or osteogenic medium before they were subcutaneously implanted at in nude mice. After 28 days the scaffolds were obtained. Immunohistochemistry for Col-2 and Col-10 and Von Kossa/Thionin staining were performed.

In vitro pellet cultures

2.0*105 hBMSCs were pelleted and cultured in chondrogenic medium (including TGF-beta). After 21 days three different groups were made: 1 remained on chondrogenic medium; 2 Beta-Glycerol-Phosphate was added to allow mineralization; 3 TGF-beta was removed and Beta-Glycerol-Phosphate was added. At day 14, 21, 35 (immuno)histochemistry was performed. On the culture medium VEGFa and MMP-production were analyzed.

In scaffolds chondrogenic pre-culturing resulted in Col-2 and Col-10 production, but no mineralization. Vessel ingrowth was present. Osteogenic pre-culturing showed mineralization but no Col-10 or vessel ingrowth.

Chondrogenic pellets showed Col-2 and Col-10. VEGF and MMPs were produced. Switching to BGP-containing medium diminished VEGF and MMP production.

In vivo implantation of in vitro chondrogenically primed hBMSCs results in vessel invasion. Chondrogenically differentiated hBMSCs produce VEGF and MMPs and are likely responsible for such vascular infiltration. These results suggest that chondrogenical priming of hBMSCs improves vessel invasion in bioengineered constructs, thereby optimizing its capacity to repair bone defects.

71) A Novel Meniscus Scaffold for the Treatment of Meniscal Tear or Meniscal Loss

To assess the safety and performance of a novel meniscus scaffold designed to restore the function of the meniscus after injury.

A novel meniscus scaffold, tailored towards the tissue regeneration requirements of the meniscus, has been developed. It has a low degradation rate, optimal pore size, high tear strength and compression modulus, with degraded end products that are biocompatible and safe for human use. The scaffold consists of a single biodegradable, aliphatic polyurethane, synthesised through a controlled production process. The polyurethane is produced as a honeycombed scaffold that consists of a 1,4 butanediol initiated poly(ε caprolactone) soft segment and hard segments containing two 1,4 butanediisocyanate and one 1,4 butanediol moieties. The scaffold is designed to be implanted during surgery into the area of removed meniscal tissue. When attached to the vascularized portion of the meniscus, it acts as a scaffold for blood vessel ingrowth and tissue regeneration. Safety (serious device- and procedure-related adverse events) and performance (vascularization measured by dynamic, contrast medium enhanced magnetic resonance imaging [MRI]) of the scaffold were assessed in a prospective feasibility study in which patients with an irreparable medial or lateral meniscal tear or partial meniscus loss, with intact rim, were treated with the scaffold.

No safety issues related to the scaffold were reported. Vascularization was demonstrated in 27 (>80%) of the 33 subjects with valid dynamic MRI assessments at 3 months.

These data demonstrate the successful use of the novel meniscus scaffold in the treatment of irreparable meniscus tears and loss of meniscus tissue.

72) Reinforcing Osteoporotic Sites Using an Innovative Injectable Calcium Phosphate-Based Drug Combined Device

Resorbable calcium phosphate (CaP) biomaterials have proved a noticeable efficacy in bone reconstruction surgery. Furthermore bisphosphonates (BPs) are well known antiresorptive agents largely used in systemic clinical treatments of osteoporosis. An injectable BP-combined CaP matrix has been developed in order to reinforce locally osteoporotic bone. The purpose of this study was to implant such a combined device in ewes' osteoporotic proximal femurs and to quantify bone structure modifications by three-dimensional microtomography (3D-μCT).

Calcium deficient apatite was loaded with zoledronate (7%w/w) and mixed with a sterile cellulosic-derived hydrogel that made it injectable. Eight ewes were ovariectomized in order to induce osteoporosis. Biomaterials were implanted for 12 weeks in proximal femur of osteoporotic ewes. 3D-μCT analysis was conducted on all implanted and control femurs. Bone volume density (BV/TV), trabecular thickness (TbTh), space between trabeculae (TbSp) and number of trabeculae (TbN) were measured. As control and treated femurs were paired, a non-parametric Wilcoxon test (± = 0.05) was applied for statistic analysis.

Osteoporosis induction is confirmed by a 40.0% decrease of BV/TV (iliac crest). Comparing treated versus control femurs for μ-CT histomorphometric measurements show significant increases (p < 0.05) for bone volume density (+32,3%), trabecular thickness (+15,8%) and trabecular number (+16,8%) and a significant decrease for trabecular space (−12,8%).

A local combined effect of calcium phosphate particles and bisphosphonate is evidenced on sheep osteoporotic proximal femurs. Reinforcing specific bone sites could be an alternative to current systemic injections.

73) An Original Approach for the Management of Post Operative Pain: Calcium Deficient Apatite-Loaded with a Local Anaesthetic

Synthetic calcium-deficient apatites (CDA) are chemical precursor for biphasic calcium phosphates that are widely used as bone substitutes in human surgery. The purpose of the study was to define in vitro release profiles of an analgesic which has been previously loaded onto CDA using isostatic compaction and to evaluate in vivo performance on pain.

CDA powder (Ca/P = 1.6) obtained from alkaline hydrolysis of dicalcium phosphate dihydrate was loaded with 1%, 4% and 16% w/w of bupivacaïn using isostatic compression. After incubation time of granules (30 min, 2 h 30, 5 h, 24 h, 48 h), 2 mL liquid were analyzed by UV spectrophotometry (270 nm). Wistar male rats were implanted in distal femur with 50mg of considered bupivacain-loaded CDA (N = 10). Analgesia was measured using electronic Von Frey monofilament electronic version, inflammatory response and neurological score.

Bupivacaïn was totally released after 48 hours of in vitro incubation.

During the first post-operative day, we observed a dose dependant analgesic effect with the bupivacaïn adsorbed quantity. Both inflammatory response and neurological score confirmed this result.

This drug-combined device has been shown to provide a release of local anaesthetic adapted to prevent short-term post operative pain. This approach could be integrated in the global management of pain needed for bone graft surgery. Although rat is considered as the validated model for evaluating pain, it seems not to be optimal to measure mid-term post op pain. Therefore additional in vivo experiments are under progress with rabbits and dogs.

74) Evaluation of Methacrylate-Encapped Poly(D,L-lactide-co-epsilon-Caprolactone) as Substrate for Bone Tissue Engineering in a Goat Tibial Unicortical Defect Model

An in situ, biodegradable, methacrylate-encapped poly (D,L-lactide-co-ε-caprolactone) (cross linkage achieved by photoinitiation) was developed for bone tissue regeneration. The biocompatibility and bone healing characteristics were evaluated in an in vivo goat model using serial radiographies, histology, histomorphometry and immunohistochemistry.

Three different polymer mixtures (pure polymer, plus 30% bioactive glass or α-TCP) were initially tested in a uni-cortical tibial defect model in 8 goats. Afterwards, different combinations of the polymer were mixed with cultivated mesenchymal stem cells (MSCs) (harvested from bone marrow and loaded on microcarriers) and α-TCP in another 8 goats.

The pure polymer clearly showed in the first series of goats an excellent biocompatibility but with only moderate osteoconductive properties which could be increased by the addition of α-TCP. Cell survival and proliferation were present in the polymer substituted bone defects in the second experiment. However, the addition of α-TCP was associated with less expansion and growth of the MSCs compared to other polymer composites. The polymer resorption rate and low porosity was considered to impede cell proliferation and ingrowth of bone so a faster resorption and higher porosity of the polymer seems imperative to develop an acceptable bone tissue engineering technique.

Although a further adaptation of the polymer is mandatory to improve its bone grafting properties, the cross-linkable properties of the polymer mixed with osteogenic differentiated MSCs offers interesting potentials for the restoration of complex orthopaedic situations.

75) Growing Bone Substitutes inside a Laminar Flow Reactor

Human adipose mesenchymal stem cells (haMSCs) were obtained from abdominal fat tissue of patients undergoing abdominoplasty by collagenase digestion. Cells were expanded under non-differentiating culture conditions (DMEM-F12 with supplements of amino acids, sodium pyruvate and penicillin/streptomycin) and seeded onto macroporous ceramic matrices (Sponceram^®). Cell-seeded matrices were cultured inside a laminar flow bioreactor for up to 3 months and analyzed by surface microscopy, grinding sectioning and electronmicroscopy. Particles released into the culture medium were analyzed for markers of bone differentiation by staining with Alizarin Red, von Kossa, Alcian Blue, Safranin-O and Trichrom after precipitation.

Cell-seeded matrices cultured inside the laminar flow reactor showed uniform distribution of cells within the porous matrices. From two weeks of culture onwards an accumulation of small white particles released from the matrix was noticed in the culture medium. These particles stained positive for minerals, calcium derivates, proteoglycans and collagen indicating begin of osteogenic differentiation inside the matrix. Grinding sections stained with toluidine blue demonstrated accumulation of cells inside the porous matrix with some deposition of metachromatic particles which may indicate mineral and calcium deposition.

Preliminary results demonstrate osteogenic differentiation of mesenchymal stem cells from adipose tissue grown in a three-dimensional porous ceramic matrix under laminar flow conditions without an osteogenic differentiation stimulans like dexamethasone present in the culture medium. These results suggest a potential for guiding cellular differentiation of mesenchymal stem cells by applying external controlled flow conditions. We propose that the laminar flow reactor has potential for growing bone substitutes in vitro for bone tissue engineering.

76)

ABSTRACT WITHDRAWN

77) Healing Potential of Mouse Calvarial Defect In Vitro

Mouse calvarial organ cultures have been widely used for investigating the biologic behaviour of intramembranous bones. However, little information is available regarding the healing potential of mouse calvarial defecta in vitro. The aim of this study was to investigate the healing potential of a mouse calvarial defect in vitro.

4-day old CD-1 mice from one litter were used. The parietal bones of the mouse calvaria were dissected out aseptically. The periosteum and dura mater were retained on the bone surfaces. Full thickness, circular defects (0.8 mm in diameter) were created through the parietal bones. The bones were cultured free-floating in a 12-well tissue culture plate in Dulbecco's Modified Eagle's Medium (DMEM). After the defects were filled with cells, some bones were still cultured in DMEM; others were cultured in an osteogenic medium. The calvaria were observed with phase contrast microscopy during this culture period. The bones were fixed and stained in bulk using Von Kossa staining method after one week and one month.

The cells migrated from the edge of the defect and fully filled the defect in one week. No mineral deposition in the defect was observed by Von Kossa staining method after one week culture. One month later, the defects cultured in the osteogenic medium were healed with bone tissue. However, the defects cultured in DMEM were healed with fibrous tissue.

The healing potential of mouse calvaria defects in vitro is dependent on the culture condition. A 0.8 mm diameter defect of mouse calvaria can heal in vitro.