The physicochemical properties of synthetically produced bone substitute materials (BSM) have a major impact on biocompatibility. This affects bony tissue integration, osteoconduction, as well as the degradation pattern and the correlated inflammatory tissue responses including macrophages and multinucleated giant cells (MNGCs). Thus, influencing factors such as size, special surface morphologies, porosity, and interconnectivity have been the subject of extensive research. In the present publication, the influence of the granule size of three identically manufactured bone substitute granules based on the technology of hydroxyapatite (HA)-forming calcium phosphate cements were investigated, which includes the inflammatory response in the surrounding tissue and especially the induction of MNGCs (as a parameter of the material degradation). For the in vivo study, granules of three different size ranges (small = 0.355–0.5 mm; medium = 0.5–1 mm; big = 1–2 mm) were implanted in the subcutaneous connective tissue of 45 male BALB/c mice. At 10, 30, and 60 days post implantationem, the materials were explanted and histologically processed. The defect areas were initially examined histopathologically. Furthermore, pro- and anti-inflammatory macrophages were quantified histomorphometrically after their immunohistochemical detection. The number of MNGCs was quantified as well using a histomorphometrical approach. The results showed a granule size-dependent integration behavior. The surrounding granulation tissue has passivated in the groups of the two bigger granules at 60 days post implantationem including a fibrotic encapsulation, while a granulation tissue was still present in the group of the small granules indicating an ongoing cell-based degradation process. The histomorphometrical analysis showed that the number of proinflammatory macrophages was significantly increased in the small granules at 60 days post implantationem. Similarly, a significant increase of MNGCs was detected in this group at 30 and 60 days post implantationem. Based on these data, it can be concluded that the integration and/or degradation behavior of synthetic bone substitutes can be influenced by granule size.

The aim of this study was to predefine the pore structure of β-tricalcium phosphate (β-TCP) scaffolds with different macro pore sizes (500, 750, and 1000 µm), to characterize β-TCP scaffolds, and to investigate the growth behavior of cells within these scaffolds. The lead structures for directional bone growth (sacrificial structures) were produced from polylactide (PLA) using the fused deposition modeling techniques. The molds were then filled with β-TCP slurry and sintered at 1250 °C, whereby the lead structures (voids) were burnt out. The scaffolds were mechanically characterized (native and after incubation in simulated body fluid (SBF) for 28 d). In addition, biocompatibility was investigated by live/dead, cell proliferation and lactate dehydrogenase assays. The scaffolds with a strand spacing of 500 µm showed the highest compressive strength, both untreated (3.4 ± 0.2 MPa) and treated with simulated body fluid (2.8 ± 0.2 MPa). The simulated body fluid reduced the stability of the samples to 82% (500), 62% (750) and 56% (1000). The strand spacing and the powder properties of the samples were decisive factors for stability. The fact that β-TCP is a biocompatible material is confirmed by the experiments. No lactate dehydrogenase activity of the cells was measured, which means that no cytotoxicity of the material could be detected. In addition, the proliferation rate of all three sizes increased steadily over the test days until saturation. The cells were largely adhered to or within the scaffolds and did not migrate through the scaffolds to the bottom of the cell culture plate. The cells showed increased growth, not only on the outer surface (e.g., 500: 36 ± 33 vital cells/mm² after three days, 180 ± 33 cells/mm² after seven days, and 308 ± 69 cells/mm² after 10 days), but also on the inner surface of the samples (e.g., 750: 49 ± 17 vital cells/mm² after three days, 200 ± 84 cells/mm² after seven days, and 218 ± 99 living cells/mm² after 10 days). This means that the inverse 3D printing method is very suitable for the presetting of the pore structure and for the ingrowth of the cells. The experiments on which this work is based have shown that the fused deposition modeling process with subsequent slip casting and sintering is well suited for the production of scaffolds for bone replacement.

Cytocompatibility analyses of new implant materials or biomaterials are not only prescribed by the Medical Device Regulation (MDR), as defined in the DIN ISO Norm 10993-5 and -12, but are also increasingly replacing animal testing. In this context, jellyfish collagen has already been established as an alternative to mammalian collagen in different cell culture conditions, but a lack of knowledge exists about its applicability for cytocompatibility analyses of biomaterials. Thus, the present study was conducted to compare well plates coated with collagen type 0 derived from Rhizostoma pulmo with plates coated with bovine and porcine collagen. The coated well plates were analysed in vitro for their cytocompatibility, according to EN ISO 10993-5/−12, using both L929 fibroblasts and MC3T3 pre-osteoblasts. Thereby, the coated well plates were compared, using established materials as positive controls and a cytotoxic material, RM-A, as a negative control. L929 cells exhibited a significantly higher viability (#### p < 0.0001), proliferation (## p < 0.01), and a lower cytotoxicity (## p < 0.01 and # p < 0.05)) in the Jellagen® group compared to the bovine and porcine collagen groups. MC3T3 cells showed similar viability and acceptable proliferation and cytotoxicity in all collagen groups. The results of the present study revealed that the coating of well plates with collagen Type 0 derived from R. pulmo leads to comparable results to the case of well plates coated with mammalian collagens. Therefore, it is fully suitable for the in vitro analyses of the cytocompatibility of biomaterials or medical devices.

Polymeric micelle-like nanoparticles have demonstrated effectiveness for the delivery of some poorly soluble or hydrophobic anticancer drugs. In this study, a hydrophobic moiety, deoxycholic acid (DCA) was first bonded on a polysaccharide, chitosan (CS), for the preparation of amphiphilic chitosan (CS-DCA), which was further modified with a cationic glycidyltrimethylammounium chloride (GTMAC) to form a novel soluble chitosan derivative (HT-CS-DCA). The cationic amphiphilic HT-CS-DCA was easily self-assembled to micelle-like nanoparticles about 200 nm with narrow size distribution (PDI 0.08–0.18). The zeta potential of nanoparticles was in the range of 14 to 24 mV, indicating higher positive charges. Then, doxorubicin (DOX), an anticancer drug with poor solubility, was entrapped into HT-CS-DCA nanoparticles. The DOX release test was performed in PBS (pH 7.4) at 37 °C, and the results showed that there was no significant burst release in the first two hours, and the cumulative release increased steadily and slowly in the following hours. HT-CS-DCA nanoparticles loaded with DOX could easily enter into MCF-7 cells, as observed by a confocal microscope. As a result, DOX-loaded HT-CS-DCA nanoparticles demonstrated a significant inhibition activity on MCF-7 growth without obvious cellular toxicity in comparison with blank nanoparticles. Therefore, the anticancer efficacy of these cationic HT-CS-DCA nanoparticles showed great promise for the delivery of DOX in cancer therapy.

The present publication reports the purification effort of two natural bone blocks, that is, an allogeneic bone block (maxgraft^®, botiss biomaterials GmbH, Zossen, Germany) and a xenogeneic block (SMARTBONE^®, IBI S.A., Mezzovico-Vira, Switzerland) in addition to previously published results based on histology. Furthermore, specialized scanning electron microscopy (SEM) and in vitro analyses (XTT, BrdU, LDH) for testing of the cytocompatibility based on ISO 10993-5/-12 have been conducted. The microscopic analyses showed that both bone blocks possess a trabecular structure with a lamellar subarrangement. In the case of the xenogeneic bone block, only minor remnants of collagenous structures were found, while in contrast high amounts of collagen were found associated with the allogeneic bone matrix. Furthermore, only island-like remnants of the polymer coating in case of the xenogeneic bone substitute seemed to be detectable. Finally, no remaining cells or cellular remnants were found in both bone blocks. The in vitro analyses showed that both bone blocks are biocompatible. Altogether, the purification level of both bone blocks seems to be favorable for bone tissue regeneration without the risk for inflammatory responses or graft rejection. Moreover, the analysis of the maxgraft^® bone block showed that the underlying purification process allows for preserving not only the calcified bone matrix but also high amounts of the intertrabecular collagen matrix.