How to Get Them off?-Assessment of Innovative Techniques for Generation and Detachment of Mature Osteoclasts for Biomaterial Resorption Studies.

The fusion process of mononuclear monocytes into multinuclear osteoclasts in vitro is an essential process for the study of osteoclastic resorption of biomaterials. Thereby biomaterials offer many influencing factors such as sample shape, material composition, and surface topography, which can have a decisive influence on the fusion and thus the entire investigation. For the specific investigation of resorption, it can therefore be advantageous to skip the fusion on samples and use mature, predifferentiated osteoclasts directly. However, most conventional detachment methods (cell scraper, accutase), lead to a poor survival rate of osteoclasts or to a loss of function of the cells after their reseeding. In the present study different conventional and novel methods of detachment in combination with different culture surfaces were investigated to obtain optimal osteoclast differentiation, yield, and vitality rates without loss of function. The innovative method-using thermoresponsive surfaces for cultivation and detachment-was found to be best suited. This is in particular due to its ability to maintain osteoclast activity, as proven by TRAP 5b-, CTSK-activity and resorption pits on dentin discs and decellularized osteoblast-derived matrix plates. In conclusion, it is shown, that osteoclasts can be predifferentiated on cell culture dishes and transferred to a reference biomaterial under preservation of osteoclastic resorption activity, providing biomaterial researchers with a novel tool for material characterization.

Artificial Extracellular Matrices Containing Bioactive Glass Nanoparticles Promote Osteogenic Differentiation in Human Mesenchymal Stem Cells.

The present study analyzes the capacity of collagen (coll)/sulfated glycosaminoglycan (sGAG)-based surface coatings containing bioactive glass nanoparticles (BGN) in promoting the osteogenic differentiation of human mesenchymal stroma cells (hMSC). Physicochemical characteristics of these coatings and their effects on proliferation and osteogenic differentiation of hMSC were investigated. BGN were stably incorporated into the artificial extracellular matrices (aECM). Oscillatory rheology showed predominantly elastic, gel-like properties of the coatings. The complex viscosity increased depending on the GAG component and was further elevated by adding BGN. BGN-containing aECM showed a release of silicon ions as well as an uptake of calcium ions. hMSC were able to proliferate on coll and coll/sGAG coatings, while cellular growth was delayed on aECM containing BGN. However, a stimulating effect of BGN on ALP activity and calcium deposition was shown. Furthermore, a synergistic effect of sGAG and BGN was found for some donors. Our findings demonstrated the promising potential of aECM and BGN combinations in promoting bone regeneration. Still, future work is required to further optimize the BGN/aECM combination for increasing its combined osteogenic effect.

Hemocyanin Modification of Chitosan Scaffolds with Calcium Phosphate Phases Increase the Osteoblast/Osteoclast Activity Ratio-A Co-Culture Study.

The ongoing research on biomaterials that support bone regeneration led to the quest for materials or material modifications that can actively influence the activity or balance of bone tissue cells. The bone biocompatibility of porous chitosan scaffolds was modified in the present study by the addition of calcium phosphates or hemocyanin. The first strategy comprised the incorporation of calcium phosphates into chitosan to create a biomimetic chitosan-mineral phase composite. The second strategy comprised dip-coating of chitosan scaffolds with hemocyanin extracted from crayfish hemolymph. The cytocompatibility was assessed in a mono-culture of human bone marrow stromal cells (hBMSCs) and their differentiation to osteoblasts; in a mono-culture of human monocytes (hMs) and their maturation to osteoclasts; and in a co-culture of hBMSC/osteoblasts-hM/osteoclasts. Mineral incorporation caused an increase in scaffold bioactivity, as shown by reduced calcium concentration in the cell culture medium, delayed differentiation of hBMSCs, and reduced osteoclastic maturation of hMs in mono-culture. Dip-coating with hemocyanin led to increased proliferation of hBMSCs and equivalent osteoclast maturation in mono-culture, while in co-culture, both an inhibitory effect of mineral incorporation on osteoblastogenesis and stimulatory effects of hemocyanin were observed. It was concluded that highly bioactive scaffolds (containing mineral phases) restrain osteoblast and osteoclast development, while hemocyanin coating significantly supports osteoblastogenesis. These influences on the osteoblasts/osteoclasts activity ratio may support scaffold-driven bone healing in the future.

Gelatin-Modified Calcium/Strontium Hydrogen Phosphates Stimulate Bone Regeneration in Osteoblast/Osteoclast Co-Culture and in Osteoporotic Rat Femur Defects-In Vitro to In Vivo Translation.

The development and characterization of biomaterials for bone replacement in case of large defects in preconditioned bone (e.g., osteoporosis) require close cooperation of various disciplines. Of particular interest are effects observed in vitro at the cellular level and their in vivo representation in animal experiments. In the present case, the material-based alteration of the ratio of osteoblasts to osteoclasts in vitro in the context of their co-cultivation was examined and showed equivalence to the material-based stimulation of bone regeneration in a bone defect of osteoporotic rats. Gelatin-modified calcium/strontium phosphates with a Ca:Sr ratio in their precipitation solutions of 5:5 and 3:7 caused a pro-osteogenic reaction on both levels in vitro and in vivo. Stimulation of osteoblasts and inhibition of osteoclast activity were proven during culture on materials with higher strontium content. The same material caused a decrease in osteoclast activity in vitro. In vivo, a positive effect of the material with increased strontium content was observed by immunohistochemistry, e.g., by significantly increased bone volume to tissue volume ratio, increased bone morphogenetic protein-2 (BMP2) expression, and significantly reduced receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin (OPG) ratio. In addition, material degradation and bone regeneration were examined after 6 weeks using stage scans with ToF-SIMS and µ-CT imaging. The remaining material in the defects and strontium signals, which originate from areas exceeding the defect area, indicate the incorporation of strontium ions into the surrounding mineralized tissue. Thus, the material inherent properties (release of biologically active ions, solubility and degradability, mechanical strength) directly influenced the cellular reaction in vitro and also bone regeneration in vivo. Based on this, in the future, materials might be synthesized and specifically adapted to patient-specific needs and their bone status.

Strontium ions promote in vitro human bone marrow stromal cell proliferation and differentiation in calcium-lacking media.

In order to investigate the influence of calcium and strontium ion concentration on human bone marrow stromal cells and their differentiation to osteoblasts, different cell culture media have been used. Even though this study does not contain a bone substitute material, the reason for this study was the decrease of cation concentration by many biomaterials, due to induced apatite precipitation. As a consequence, the reduced calcium ion concentration is known to affect osteoblastic development. Therefore, the main focus was put on the question, whether an increased strontium concentration (in the range of mM) might be suitable to compensate the lack of calcium ions. The effect of solely strontium ions-with only calcium in the media resulting from fetal calf serum-was investigated. Commercially available calcium-free medium (modified α-MEM) was tested in comparison with media with varied calcium ion concentrations (0.9, 1.8, and 3.6 mM), or strontium ion concentration (0.4, 0.9, 1.8, and 3.6 mM). In case of calcium, higher concentrations cause increased proliferation, while differentiation was shifted to earlier points of time. Differentiation was increased by solely strontium ions only at 0.4-0.9 mM, while proliferation was highest for 0.9-1.8 mM. From these results, it can be concluded that strontium is able to compensate a lack of calcium to a certain degree. Thus, in contrast to calcium ion release, a strontium ion release from bone substitute materials might be applicable for stimulation of bone regeneration without influencing the media saturation.

Class I Biocompatible DLP-Printed Acrylate Impairs Adhesion and Proliferation of Human Mesenchymal Stromal Cells in Indirect Cytotoxicity Assay.

The popular method of digital light processing 3D printing (DLP) for complex and individual laboratory equipment requires materials that are as inert as possible for use in contact with cells for subsequent investigations. However, the per se incomplete curing of acrylate resins by UV light leaves residuals that are not suitable for cell culture application. Therefore, we evaluated the cytotoxicity of four commercially available acrylate resins with bone marrow-derived human mesenchymal stromal cells (BM-hMSC) in an indirect cytotoxicity test. This involved incubating the printed cylinders in Transwell™ inserts for 7 days. While the degree of crosslinking did not increase significantly between freshly printed and stored samples (3 weeks in ambient conditions), the storage improved the material's performance in terms of cytocompatibility. The DNA amount and LDH activity showed a direct influence of the resin residuals on cell adhesion. The class I acrylate Surgical Guide™ left no adherent cells after 7 days, regardless of previous storage. In comparison, the Basic Ivory™ resin after storage allowed same amount of adherent cells after 7 days as the polystyrene reference. We conclude that resin residuals of certain materials are released, which allows the use of the resins in indirect contact with cells thereafter.

Effects of Gamma Irradiation and Supercritical Carbon Dioxide Sterilization on Methacrylated Gelatin/Hyaluronan Hydrogels.

Biopolymer hydrogels have become an important group of biomaterials in experimental and clinical use. However, unlike metallic or mineral materials, they are quite sensitive to sterilization. The aim of this study was to compare the effects of gamma irradiation and supercritical carbon dioxide (scCO2) treatment on the physicochemical properties of different hyaluronan (HA)- and/or gelatin (GEL)-based hydrogels and the cellular response of human bone marrow-derived mesenchymal stem cells (hBMSC). Hydrogels were photo-polymerized from methacrylated HA, methacrylated GEL, or a mixture of GEL/HA. The composition and sterilization methods altered the dissolution behavior of the biopolymeric hydrogels. There were no significant differences in methacrylated GEL release but increased methacrylated HA degradation of gamma-irradiated samples. Pore size/form remained unchanged, while gamma irradiation decreased the elastic modulus from about 29 kPa to 19 kPa compared to aseptic samples. HBMSC proliferated and increased alkaline phosphatase activity (ALP) particularly in aseptic and gamma-irradiated methacrylated GEL/HA hydrogels alike, while scCO2 treatment had a negative effect on both proliferation and osteogenic differentiation. Thus, gamma-irradiated methacrylated GEL/HA hydrogels are a promising base for multi-component bone substitute materials.

Cellular adhesion and chondrogenic differentiation inside an alginate-based bioink in response to tailorable artificial matrices and tannic acid treatment.

Many established bioinks fulfill important requirements regarding fabrication standards and cytocompatibility. Current research focuses on development of functionalized bioinks with an improved support of tissue-specific cell differentiation. Many approaches primarily depend on decellularized extracellular matrices or blood components. In this study, we investigated the combination of a highly viscous alginate-methylcellulose (algMC) bioink with collagen-based artificial extracellular matrix (aECM) as a finely controllable and tailorable system composed of collagen type I (col) with and without chondroitin sulfate (CS) or sulfated hyaluronan (sHA). As an additional stabilizer, the polyphenol tannic acid (TA) was integrated into the inks. The assessment of rheological properties and printability as well as hydrogel microstructure revealed no adverse effect of the integrated components on the inks. Viability, adhesion, and proliferation of bioprinted immortalized human mesenchymal stem cells (hTERT-MSC) was improved indicating enhanced interaction with the designed microenvironment. Furthermore, chondrogenic matrix production (collagen type II and sulfated glycosaminoglycans) by primary human chondrocytes (hChon) was enhanced by aECM. Supplementing the inks with TA was required for these positive effects but caused cytotoxicity as soon as TA concentrations exceeded a certain amount. Thus, combining tailorable aECM with algMC and balanced TA addition proved to be a promising approach for promoting adhesion of immortalized stem cells and differentiation of chondrocytes in bioprinted scaffolds.

Ketoprofen-Based Polymer-Drug Nanoparticles Provide Anti-Inflammatory Properties to HA/Collagen Hydrogels.

Current limitations of wound dressings for treating chronic wounds require the development of novel approaches. One of these is the immune-centered approach, which aims to restore the pro-regenerative and anti-inflammatory properties of macrophages. Under inflammatory conditions, ketoprofen nanoparticles (KT NPs) can reduce pro-inflammatory markers of macrophages and increase anti-inflammatory cytokines. To assess their suitability as part of wound dressings, these NPs were combined with hyaluronan (HA)/collagen-based hydro- (HGs) and cryogels (CGs). Different HA and NP concentrations and loading techniques for NP incorporation were used. The NP release, gel morphology, and mechanical properties were studied. Generally, colonialization of the gels with macrophages resulted in high cell viability and proliferation. Furthermore, direct contact of the NPs to the cells reduced the level of nitric oxide (NO). The formation of multinucleated cells on the gels was low and further decreased by the NPs. For the HGs that produced the highest reduction in NO, extended ELISA studies showed reduced levels of the pro-inflammatory markers PGE2, IL-12 p40, TNF-α, and IL-6. Thus, HA/collagen-based gels containing KT NPs may represent a novel therapeutic approach for treating chronic wounds. Whether effects observed in vitro translate into a favorable profile on skin regeneration in vivo will require rigorous testing.

The interplay of collagen/bioactive glass nanoparticle coatings and electrical stimulation regimes distinctly enhanced osteogenic differentiation of human mesenchymal stem cells.

Increasing research has incorporated bioactive glass nanoparticles (BGN) and electric field (EF) stimulation for bone tissue engineering and regeneration applications. However, their interplay and the effects of different EF stimulation regimes on osteogenic differentiation of human mesenchymal stem cells (hMSC) are less investigated. In this study, we introduced EF with negligible magnetic field strength through a well-characterized transformer-like coupling (TLC) system, and applied EF disrupted (4/4) or consecutive (12/12) regime on type I collagen (Col) coatings with/without BGN over 28 days. Additionally, dexamethasone was excluded to enable an accurate interpretation of BGN and EF in supporting osteogenic differentiation. Here, we demonstrated the influences of BGN and EF on collagen topography and maintaining coating stability. Coupled with the release profile of Si ions from the BGN, cell proliferation and calcium deposition were enhanced in the Col-BGN samples after 28 days. Further, osteogenic differentiation was initiated as early as d 7, and each EF regime was shown to activate distinct pathways. The disrupted (4/4) regime was associated with the BMP/Smad4 pathways that up-regulate Runx2/OCN gene expression on d 7, with a lesser effect on ALP activity. In contrast, the canonical Wnt/ß-Catenin signaling pathway activated through mechanotransduction cues is associated with the consecutive (12/12) regime, with significantly elevated ALP activity and Sp7 gene expression reported on d 7. In summary, our results illustrated the synergistic effects of BGN and EF in different stimulation regimes on osteogenic differentiation that can be further exploited to enhance current bone tissue engineering and regeneration approaches. STATEMENT OF SIGNIFICANCE: The unique release mechanisms of silica from bioactive glass nanoparticles (BGN) were coupled with pulsatile electric field (EF) stimulation to support hMSC osteogenic differentiation, in the absence of dexamethasone. Furthermore, the interplay with consecutive (12/12) and disrupted (4/4) stimulation regimes was investigated. The reported physical, mechanical and topographical effects of BGN and EF on the collagen coating, hMSC and the distinct progression of osteogenic differentiation (canonical Wnt/ß-Catenin and BMP/Smad) triggered by respective stimulation regime were not explicitly reported previously. These results provide the fundamentals for further exploitations on BGN composites with metal ions and rotation of EF regimes to enhance osteogenic differentiation. The goal is sustaining continual osteogenic differentiation and achieving a more physiologically-relevant state and bone constructs in vitro.

Strontium substitution of gelatin modified calcium hydrogen phosphates as porous hard tissue substitutes.

Aiming at the generation of a high strontium-containing degradable bone substitute, the exchange of calcium with strontium in gelatin-modified brushite was investigated. The ion substitution showed two mineral groups, the high-calcium containing minerals with a maximum measured molar Ca/Sr ratio of 80%/20% (mass ratio 63%/37%) and the high-strontium containing ones with a maximum measured molar Ca/Sr ratio of 21%/79% (mass ratio 10%/90%). In contrast to the high-strontium mineral phases, a high mass loss was observed for the calcium-based minerals during incubation in cell culture medium (alpha-MEM), but also an increase in strength owing to dissolution and re-precipitation. This resulted for the former in a decrease of cation concentration (Ca + Sr) in the medium, while the pH value decreased and the phosphate ion concentration rose significantly. The latter group of materials, the high-strontium containing ones, showed only a moderate change in mass and a decrease in strength, but the Ca + Sr concentration remained permanently above the initial calcium concentration in the medium. This might be advantageous for a future planned application by supporting bone regeneration on the cellular level.

Novel degradation flow-through chamber for in vitro biomaterial characterization.

The characterization of degradation of biodegradable materials for tissue regeneration is classically carried out in three steps: in vitro degradation analysis, in vitro cell culture, and in vivo animal experiments. Each step involves an increasing complexity and should serve a more sophisticated material selection, which serves as an orientation to clinical studies and the final application in patients. Recently, the usefulness of degradation analyses is being discussed. In this context, the aim of this work is to increase the importance of in vitro degradation analysis by using flowing media to move closer to the in vivo situation. In the long term, this should lead to a more sensitive biomaterial characterization as well as to a replacement of time-consuming static or quasi-dynamic incubation experiments. The practicability of the novel chamber is demonstrated in context of a degradation study of silica/collagen/calcium phosphate composites in flowing media with physiological (2.4 mM) and lowered (0.5 mM) calcium ion concentrations. This is done by comparison with static and quasi-dynamic incubation experiments. In order to keep all media regimes comparable to each other, for the dynamic experiment, a flow rate was chosen equivalent to the medium exchange in quasi-dynamic incubation. Under flow-through conditions, there is a clearly decreased tendency to lower the calcium concentration, so that a concentration close to the physiological initial situation can be continuously maintained.

In vitro evaluation of textile chitosan scaffolds for tissue engineering using human bone marrow stromal cells.

Textile chitosan fiber scaffolds were developed and tested in terms of biocompatibility for human bone marrow stromal cells (hBMSCs). A part of the scaffolds was further modified by coating with fibrillar collagen type I in order to biologize the surface. hBMSCs of two donors were used for cell culture experiments in vitro. Confocal laser scanning microscopy (CLSM) as well as scanning electron microscopy (SEM) revealed fast attachment and morphological adaptation of the cells on both the raw chitosan fibers and the collagen-coated scaffolds. Cells were osteogenically induced after 3 days and cultivated for up to 28 days on the scaffolds. Activity of lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) was analyzed to evaluate proliferation as well as osteogenic differentiation. We found a 3.5-6-fold increase in the cell number, whereas the collagen coating did not noticeably influence these factors. Osteogenic differentiation was confirmed by the course of ALP activity and immunostaining of osteocalcin. The feature of the collagen-coated as well as the raw chitosan fiber scaffolds to support attachment, proliferation, and differentiation of hBMSCs suggests a potential application of chitosan fibers and textile chitosan scaffolds for the tissue engineering of bone.

Organically modified hydroxyapatite (ormoHAP) nanospheres stimulate the differentiation of osteoblast and osteoclast precursors: a co-culture study.

Isolated nanospheres consisting of organically modified hydroxyapatite (ormoHAP), prepared by an electric field-assisted ion double migration process, were embedded in foamed gelatin to form a composite scaffold. Degradation rates have been demonstrated to correlate with the crosslinking degree (40%, 80%) as well as with the mineral content of the scaffolds (0%, 20%, 40%). A human co-culture model of osteoblasts and osteoclasts, derived from bone marrow stromal cells and monocytes, respectively, without external addition of the factors RANKL and M-CSF, was run for up to 42 d in order to characterize the action of the ormoHAP-gelatin scaffolds on the co-culture. Examination was performed by quantitative biochemical methods (DNA, LDH, ALP, TRAP5b), gene expression analysis (ALP, BSP II, RANKL, IL-6, VTNR, CTSK, TRAP, OSCAR, CALCR) and confocal laser scanning microscopy (cell nuclei, actin, CD68, TRAP). Results confirm that ormoHAP embedded in the gelatin matrix enhanced TRAP 5b activity. As a feedback, ALP activity and gene expression of BSP II of osteoblasts increased. Finally, a sequence of cell cross-talk actions is suggested, which can explain the behavior of the formed vital co-culture and moreover the influence of the presence and concentration of ormoHAP.

Biomaterial based treatment of osteoclastic/osteoblastic cell imbalance - Gelatin-modified calcium/strontium phosphates.

Osteoporotic bone represents - particularly in case of fractures - difficult conditions for its regeneration. In the present study, the focus was put on a degradable bone substitute material of gelatin-modified calcium and strontium phosphates facing the special demands of osteoporotic bone. The release of strontium ions from the material ought to stimulate osteoblastogenesis either direct by ion release or indirect after material resorption by increased presence and activity of osteoclasts, which subsequently stimulate osteoblasts. A new porous material was produced from calcium phosphate, strontium phosphate and a mixed phase of calcium/strontium phosphate precipitated in presence of gelatin. Initially, ion release was analyzed in standard­calcium containing (2.0 mM) and low-calcium (0.4 mM) minimum essential medium. The cultivation of human peripheral blood mononuclear cells next to the material led to formation of osteoclast-like cells, able to migrate, fuse, and differentiate. Especially, the mixed gelatin-modified calcium/strontium phosphate allowed osteoclastogenesis as proven morphologically and by real-time quantitative polymerase chain reaction (RT-qPCR). It was precisely this material that led to the best osteoblastic reaction of human bone marrow stromal cells cultured on the material. The investigations of the bone substitute material indicate active involvement in the balance of cells of the bone morphogenetic unit.

Increased pore size of scaffolds improves coating efficiency with sulfated hyaluronan and mineralization capacity of osteoblasts.

BACKGROUND: Delayed bone regeneration of fractures in osteoporosis patients or of critical-size bone defects after tumor resection are a major medical and socio-economic challenge. Therefore, the development of more effective and osteoinductive biomaterials is crucial. METHODS: We examined the osteogenic potential of macroporous scaffolds with varying pore sizes after biofunctionalization with a collagen/high-sulfated hyaluronan (sHA3) coating in vitro. The three-dimensional scaffolds were made up from a biodegradable three-armed lactic acid-based macromer (TriLA) by cross-polymerization. Templating with solid lipid particles that melt during fabrication generates a continuous pore network. Human mesenchymal stem cells (hMSC) cultivated on the functionalized scaffolds in vitro were investigated for cell viability, production of alkaline phosphatase (ALP) and bone matrix formation. Statistical analysis was performed using student's t-test or two-way ANOVA. RESULTS: We succeeded in generating scaffolds that feature a significantly higher average pore size and a broader distribution of individual pore sizes (HiPo) by modifying composition and relative amount of lipid particles, macromer concentration and temperature for cross-polymerization during scaffold fabrication. Overall porosity was retained, while the scaffolds showed a 25% decrease in compressive modulus compared to the initial TriLA scaffolds with a lower pore size (LoPo). These HiPo scaffolds were more readily coated as shown by higher amounts of immobilized collagen (+ 44%) and sHA3 (+ 25%) compared to LoPo scaffolds. In vitro, culture of hMSCs on collagen and/or sHA3-coated HiPo scaffolds demonstrated unaltered cell viability. Furthermore, the production of ALP, an early marker of osteogenesis (+ 3-fold), and formation of new bone matrix (+ 2.5-fold) was enhanced by the functionalization with sHA3 of both scaffold types. Nevertheless, effects were more pronounced on HiPo scaffolds about 112%. CONCLUSION: In summary, we showed that the improvement of scaffold pore sizes enhanced the coating efficiency with collagen and sHA3, which had a significant positive effect on bone formation markers, underlining the promise of using this material approach for in vivo studies.

Novel textile chitosan scaffolds promote spreading, proliferation, and differentiation of osteoblasts.

Two novel scaffold models made of chitosan fibers were designed, fabricated, and investigated. Raw chitosan fibers were either tightened between plastic rings or were processed into stand-alone scaffolds. Chitosan fiber scaffolds were further modified by coating with a thin layer of fibrillar collagen type I to biologize the surface. Cell culture experiments were carried out using murine osteoblast-like cells (7F2). Confocal laser scanning microscopy (cLSM) as well as scanning electron microscopy (SEM) revealed fast attachment and morphological adaptation of the cells on both the raw chitosan fibers and the collagen-coated scaffolds. Cells were cultivated for up to 4 weeks on the materials and proliferation as well as osteogenic differentiation was quantitatively analyzed in terms of lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) activity. We found a 14-16-fold increase of cell number and the typical pattern of ALP activity, whereas the collagen coating does not remarkably influence these parameters. The maintenance of osteogenic phenotype on the novel materials was furthermore confirmed by immunostaining of osteocalcin and study of matrix mineralization. The feature of the collagen-coated but also the raw chitosan fiber scaffolds to support the attachment, proliferation, and differentiation of osteoblast-like cells suggest a potential application of chitosan fibers and textile chitosan scaffolds for the tissue engineering of bone.

Gelatine modified monetite as a bone substitute material: An in vitro assessment of bone biocompatibility.

Calcium phosphate phases are increasingly used for bone tissue substitution, and the load bearing properties of these inherently brittle biomaterials are increased by inclusion of organic components. Monetite prepared using mineralization of gelatine pre-structured through phosphate leads to a significantly increased biaxial strength and indirect tensile strength compared to gelatine-free monetite. Besides the mechanical properties, degradation in physiological solutions and osteoblast and osteoclast cell response were investigated. Human bone marrow stromal cells (hBMSCs) showed considerably higher proliferation rates on the gelatine modified monetite than on polystyrene reference material in calcium-free as well as standard cell culture medium (α-MEM). Osteogenic differentiation on the material was comparable to polystyrene in both medium types. Osteoclast-like cells derived from monocytes were able to actively resorb the biomaterial. Osteoblastic differentiation and perhaps even more important the cellular resorption of the biomaterial indicate that it can be actively involved in the bone remodeling process. Thus the behavior of osteoblasts and osteoclasts as well as the adequate degradation and mechanical properties are strong indicators for bone biocompatibility, although in vivo studies are still required to prove this. STATEMENT OF SIGNIFICANCE: New and unique? A low temperature precipitationprocessforcalcium anhydrous hydrogen phosphateallows for the first time to produce monolithic compact composites of monetite and gelatine. The composite is degradable and resorbable. To prove that, the question arises: what is bone biocompatibility? The reaction of both mayor cell types of bone represents this biocompatibility. Therefore, human bone marrow stromal cells were seeded revealing the materials pro-osteogenic properties. Monocyte cultivation, becoming recently focus of interest, revealed the capability of the biomaterial to be actively resorbed by derived osteoclast-like cells. Not new but necessary ismechanical characterization, which is often only investigated as uniaxial property. Here, a biaxial method is applied, to characterize the materials properties closer to its application loads.

Bioactivity of xerogels as modulators of osteoclastogenesis mediated by connexin 43.

In order to investigate the effects of different degrees of bioactivity of xerogels on connexin 43 (cx43) signaling of osteoclasts a cell culture approach was developed. Cells isolated from peripheral blood mononuclear cells were cultured in combination with the xerogels and were harvested for further investigations on day 1, day 5, and day 10. By means of quantitative PCR increased cx43 mRNA levels and coincident decreasing mRNA levels of the calcium sensing receptor, TRAP, and Cathepsin K were detected with increasing bioactivity of the xerogel samples. Additionally, osteoclasts cultured on tissue culture plates were used to perform principle investigations on cell differentiation by means of transmission electron microscopy, life cell imaging, and immunofluorescence, and the results demonstrated that cx43-signaling could be attributed to migration and fusion of osteoclast precursors. Therefore, the positive correlation of cx43 expression with high xerogel bioactivity was caused by proceeding differentiation of the osteoclasts. Finally, the presently observed pattern of cx43 signaling refers to strong effects regarding bioactivity on cx43-associated cell differentiation of osteoclasts influenced by extracellular calcium ions.

Chitosan(PEO)/silica hybrid nanofibers as a potential biomaterial for bone regeneration.

New hybrid nanofibers prepared with chitosan (CTS), containing a total amount of polyethylene oxide (PEO) down to 3.6wt.%, and silica precursors were produced by electrospinning. The solution of modified sol-gel particles contained tetraethoxysilane (TEOS) and the organosilane 3-glycidyloxypropyltriethoxysilane (GPTEOS). This is rending stable solution toward gelation and contributing in covalent bonding with chitosan. The fibers encompass advantages of biocompatible polymer template silicate components to form self-assembled core-shell structure of the polymer CTS/PEO encapsulated by the silica. Potential applicability of this hybrid material to bone tissue engineering was studied examining its cellular compatibility and bioactivity. The nanofiber matrices were proved cytocompatible when seeded with bone-forming 7F2-cells, promoting attachment and proliferation over 7 days. These found to enhance a fast apatite formation by incorporation of Ca(2+) ions and subsequent immersion in modified simulated body fluid (m-SBF). The tunable properties of these hybrid nanofibers can find applications as active biomaterials in bone repair and regeneration.