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.

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.

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.

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.

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.

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.

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.

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.

Influence of different modifications of a calcium phosphate bone cement on adhesion, proliferation, and osteogenic differentiation of human bone marrow stromal cells.

Collagen and noncollagenous proteins of the extracellular bone matrix are able to stimulate bone cell activities and bone healing. The modification of calcium phosphate bone cements used as temporary bone replacement materials with these proteins seems to be a promising approach to accelerate new bone formation. In this study, we investigated adhesion, proliferation, and osteogenic differentiation of human bone marrow stromal cells (hBMSC) on Biocement D/collagen composites which have been modified with osteocalcin and O-phospho-L-serine. Modification with osteocalcin was carried out by its addition to the cement precursor before setting as well as by functionalization of the cement samples after setting and sterilization. hBMSC were cultured on these samples for 28 days with and without osteogenic supplements. We found a positive impact especially of the phosphoserine-modifications but also of both osteocalcin-modifications on differentiation of hBMSC indicated by higher expression of the osteoblastic markers matrix metalloproteinase-13 and bone sialo protein II. For hBMSC cultured on phosphoserine-containing composites, an increased proliferation has been observed. However, in case of the osteocalcin-modified samples, only osteocalcin adsorbed after setting and sterilization of the cement samples was able to promote initial adhesion and proliferation of hBMSC. The addition of osteocalcin before setting results in a finer microstructure but the biological activity of osteocalcin might be impaired due to the sterilization process. Thus, our data indicate that the initial adhesion and proliferation of hBMSC is enhanced rather by the biological activity of osteocalcin than by the finer microstructure.

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.

A bioactive triphasic ceramic-coated hydroxyapatite promotes proliferation and osteogenic differentiation of human bone marrow stromal cells.

Hydroxyapatite (HA) ceramics are widely used as bone graft substitutes because of their biocompatibility and osteoconductivity. However, to enhance the success of therapeutic application, many efforts are undertaken to improve the bioactivity of HA. We have developed a triphasic, silica-containing ceramic-coated hydroxyapatite (HASi) and evaluated its performance as a scaffold for cell-based tissue engineering applications. Human bone marrow stromal cells (hBMSCs) were seeded on both HASi and HA scaffolds and cultured with and without osteogenic supplements for a period of 4 weeks. Cellular responses were determined in vitro in terms of cell adhesion, viability, proliferation, and osteogenic differentiation, where both materials exhibited excellent cytocompatibility. Nevertheless, an enhanced rate of cell proliferation and higher levels of both alkaline phosphatase expression and activity were observed for cells cultured on HASi with osteogenic supplements. These findings indicate that the bioactivity of HA endowed with a silica-containing coating has definitely influenced the cellular activity, projecting HASi as a suitable candidate material for bone regenerative therapy.