Artificial inorganic biohybrids: The functional combination of microorganisms and cells with inorganic materials.

Biohybrids can be defined as the functional combination of proteins, viable cells or microorganisms with non-biological materials. This article reviews recent findings on the encapsulation of microorganisms and eukaryotic cells in inorganic matrices such as silica gels or cements. The entrapment of biological entities into a support material is of great benefit for processing since the encapsulation matrix protects sensitive cells from shear forces, unfavourable pH changes, or cytotoxic solvents, avoids culture-washout, and simplifies the separation of formed products. After reflecting general aspects of such an immobilization as well as the chemistry of the inorganic matrices, we focused on manufacturing aspects and the application of such biohybrids in biotechnology, medicine as well as in environmental science and for civil engineering purpose. STATEMENT OF SIGNIFICANCE: The encapsulation of living cells and microorganisms became an intensively studied and rapidly expanding research field with manifold applications in medicine, bio- and environmental technology, or civil engineering. Here, the use of silica or cements as encapsulation matrices have the advantage of a higher chemical and mechanical resistance towards harsh environmental conditions during processing compared to their polymeric counterparts. In this perspective, the article gives an overview about the inorganic material systems used for cell encapsulation, followed by reviewing the most important applications. The future may lay in a combination of the currently achieved biohybrid systems with additive manufacturing techniques. In a longer perspective, this would enable the direct printing of cell loaded bioreactor components.

Bone regeneration capacity of magnesium phosphate cements in a large animal model.

Magnesium phosphate minerals have captured increasing attention during the past years as suitable alternatives for calcium phosphate bone replacement materials. Here, we investigated the degradation and bone regeneration capacity of experimental struvite (MgNH4PO4·6H2O) forming magnesium phosphate cements in two different orthotopic ovine implantation models. Cements formed at powder to liquid ratios (PLR) of 2.0 and 3.0 g ml-1 were implanted into trabecular bone using a non-load-bearing femoral drill-hole model and a load-bearing tibial defect model. After 4, 7 and 10 months the implants were retrieved and cement degradation and new bone formation was analyzed by micro-computed tomography (µCT) and histomorphometry. The results showed cement degradation in concert with new bone formation at both defect locations. Both cements were almost completely degraded after 10 months. The struvite cement formed with a PLR of 2.0 g ml-1 exhibited a slightly accelerated degradation kinetics compared to the cement with a PLR of 3.0 g ml-1. Tartrat-resistant acid phosphatase (TRAP) staining indicated osteoclastic resorption at the cement surface. Energy dispersive X-ray analysis (EDX) revealed that small residual cement particles were mostly accumulated in the bone marrow in between newly formed bone trabeculae. Mechanical loading did not significantly increase bone formation associated with cement degradation. Concluding, struvite-forming cements might be promising bone replacement materials due to their good degradation which is coupled with new bone formation. STATEMENT OF SIGNIFICANCE: Recently, the interest in magnesium phosphate cements (MPC) for bone substitution increased, as they exhibit high initial strength, comparably elevated degradation potential and the release of valuable magnesium ions. However, only few in vivo studies, mostly including non-load-bearing defects in small animals, have been performed to analyze the degradation and regeneration capability of MPC derived compounds. The present study examined the in vivo behavior of magnesiumammoniumphosphate hexahydrate (struvite) implants with different porosity in both mechanically loaded and non-loaded defects of merino sheep. For the first time, the effect of mechanical stimuli on the biological outcome of this clinically relevant replacement material is shown and directly compared to the conventional unloaded defect situation in a large animal model.

Trivalent chromium incorporated in a crystalline calcium phosphate matrix accelerates materials degradation and bone formation in vivo.

Remodeling of calcium phosphate bone cements is a crucial prerequisite for their application in the treatment of large bone defects. In the present study trivalent chromium ions were incorporated into a brushite forming calcium phosphate cement in two concentrations (10 and 50 mmol/mol ß-tricalcium phosphate) and implanted into a femoral defect in rats for 3 and 6 month, non-modified brushite was used as reference. Based on our previous in vitro findings indicating both an enhanced osteoclastic activity and cytocompatibility towards osteoprogenitor cells we hypothesized a higher in vivo remodeling rate of the Cr3+ doped cements compared to the reference. A significantly enhanced degradation of the modified cements was evidenced by micro computed tomography, X-ray and histological examinations. Furthermore the formation of new bone tissue after 6 month of implantation was significantly increased from 29% to 46% during remodeling of cements, doped with the higher Cr3+ amount. Time of flight secondary ion mass spectrometry (ToF-SIMS) of histological sections was applied to investigate the release of Cr3+ ions from the cement after implantation and to image their distribution in the implant region and the surrounding bone tissue. The relatively weak incorporation of chromium into the newly formed bone tissue is in agreement to the low chromium concentrations which were released from the cements in vitro. The faster degradation of the Cr3+ doped cements was also verified by ToF-SIMS. The positive effect of Cr3+ doping on both degradation and new bone formation is discussed as a synergistic effect of Cr3+ bioactivity on osteoclastic resorption on one hand and improvement of cytocompatibility and solubility by structural changes in the calcium phosphate matrix on the other hand. STATEMENT OF SIGNIFICANCE: While biologically active metal ions like strontium, magnesium and zinc are increasingly applied for the modification of ceramic bone graft materials, the present study is the first report on the incorporation of low doses of trivalent chromium ions into a calcium phosphate based biomaterial and testing of its performance in bone defect regeneration in vivo. Chromium(III)-doped calcium phosphate bone cements show improved cytocompatibility and both degradation rate and new bone formation in vivo are significantly increased compared to the reference cement. This important discovery might be the starting point for the application of trivalent chromium salts for the modification of bone graft materials to increase their remodelling rate.

Osteoclastic differentiation and resorption is modulated by bioactive metal ions Co2+, Cu2+ and Cr3+ incorporated into calcium phosphate bone cements.

Biologically active metal ions in low doses have the potential to accelerate bone defect healing. For successful remodelling the interaction of bone graft materials with both bone-forming osteoblasts and bone resorbing osteoclasts is crucial. In the present study brushite forming calcium phosphate cements (CPC) were doped with Co2+, Cu2+ and Cr3+ and the influence of these materials on osteoclast differentiation and activity was examined. Human osteoclasts were differentiated from human peripheral blood mononuclear cells (PBMC) both on the surface and in indirect contact to the materials on dentin discs. Release of calcium, phosphate and bioactive metal ions was determined using ICP-MS both in the presence and absence of the cells. While Co2+ and Cu2+ showed a burst release, Cr3+ was released steadily at very low concentrations (below 1 µM) and both calcium and phosphate release of the cements was considerably changed in the Cr3+ modified samples. Direct cultivation of PBMC/osteoclasts on Co2+ cements showed lower attached cell number compared to the reference but high activity of osteoclast specific enzymes tartrate resistant acid phosphatase (TRAP), carbonic anhydrase II (CAII) and cathepsin K (CTSK) and significantly increased gene expression of vitronectin receptor. Indirect cultivation with diluted Co2+ cement extracts revealed highest resorbed area compared to all other modifications and the reference. Cu2+ cements had cytotoxic effect on PBMC/osteoclasts during direct cultivation, while indirect cultivation with diluted extracts from Cu2+ cements did not provoke cytotoxic effects but a strictly inhibited resorption. Cr3+ doped cements did not show cytotoxic effects at all. Gene expression and enzyme activity of CTSK was significantly increased in direct culture. Indirect cultivation with Cr3+ doped cements revealed significantly higher resorbed area compared to the reference. In conclusion Cr3+ doped calcium phosphate cements are an innovative cement modification because of their high cytocompatibility and support of active resorption by osteoclasts.

Data on TOF-SIMS analysis of Cu2+, Co2+ and Cr3+ doped calcium phosphate cements.

This article contains data of time of flight secondary ion mass spectrometry (TOF-SIMS) analysis of brushite-forming calcium phosphate cements doped with biologically active metal ions. This data are related to the research article "Cu2+, Co2+ and Cr3+ doping of a calcium phosphate cement influences materials properties and response of human mesenchymal stromal cells" (Schamel et al., 2017) [1]. Cu2+, Co2+ and Cr3+ doped ß-tricalcium phosphate precursor powders were used to prepare cement samples. The incorporation and distribution of the metal ions in the cement matrix was visualized by imaging mass spectrometry.

Intrinsic 3D Prestressing: A New Route for Increasing Strength and Improving Toughness of Hybrid Inorganic Biocements.

Cement is the most consumed resource and is the most widely used material globally. The ability to extrinsically prestress cementitious materials with tendons usually made from steel allows the creation of high-strength bridges and floors from this otherwise brittle material. Here, a dual setting cement system based on the combination of hydraulic cement powder with an aqueous silk fibroin solution that intrinsically generates a 3D prestressing during setting, dramatically toughening the cement to the point it can be cut with scissors, is reported. Changes of both ionic concentration and pH during cement setting are shown to create an interpenetrating silk fibroin inorganic composite with the combined properties of the elastic polymer and the rigid cement. These hybrid cements are self-densifying and show typical ductile fracture behavior when dry and a high elasticity under wet conditions with mechanical properties (bending and compressive strength) nearly an order of magnitude higher than the fibroin-free cement reference.

In vitro ion adsorption and cytocompatibility of dicalcium phosphate ceramics.

BACKGROUND: In vitro cell testing of degradable bioceramics such as brushite or monetite is often challenging due to the ion release into or adsorption from the culture medium. These ionic changes are then mostly responsible for cell proliferation and activity, which prohibits the investigation of effects originating from surface topography or further material modifications. METHODS: Here, we aimed to solve this problem by developing a pre-conditioning regime following the repeated immersion of brushite and monetite samples in various Ca2+, Mg2+ and PO43- containing electrolytes, followed by studying ion adsorption / release as well as changes in phase composition and in vitro cytocompatibility with MG63 cells. RESULTS: The results demonstrated that by using DMEM cell culture medium in a ratio of 10 ml/sample was sufficient to minimize changes of ionic composition after 7 d with a daily change of the medium. This leads to changes of the surface composition with dissolution of the brushite phase. In turn, this also positively influences the in vitro cytocompatibility with a 2-3 fold higher cell number and cell activity on the DMEM pretreated surfaces. CONCLUSIONS: Controlled sample washing prior to cell testing using DMEM medium seems to be a valuable procedure not only to stabilize the pH during cell culture but also to maintain ion concentrations within a cell friendly range.

Phytic acid as alternative setting retarder enhanced biological performance of dicalcium phosphate cement in vitro.

Dicalcium phosphate cement preparation requires the addition of setting retarders to meet clinical requirements regarding handling time and processability. Previous studies have focused on the influence of different setting modifiers on material properties such as mechanical performance or injectability, while ignoring their influence on biological cement properties as they are used in low concentrations in the cement pastes and the occurrence of most compounds in human tissues. Here, analyses of both material and biological behavior were carried out on samples with common setting retardants (citric acid, sodium pyrophosphate, sulfuric acid) and novel (phytic acid). Cytocompatibility was evaluated by in vitro tests with osteoblastic (hFOB 1.19) and osteoclastic (RAW 264.7) cells. We found cytocompatibility was better for sodium pyrophosphate and phytic acid with a three-fold cell metabolic activity by WST-1 test, whereas samples set with citric acid showed reduced cell number as well as cell activity. The compressive strength (CS) of cements formed with phytic acid (CS = 13 MPa) were nearly equal to those formed with citric acid (CS = 15 MPa) and approximately threefold higher than for other setting retardants. Due to a proven cytocompatibility and high mechanical strength, phytic acid seems to be a candidate replacement setting retardant for dicalcium phosphate cements.

Simultaneous formation and mineralization of star-P(EO-stat-PO) hydrogels.

Natural bone is an organic-inorganic composite of highly ordered collagen fibrils and ~60-70% nanocrystalline hydroxyapatite (HA) crystals resulting in a high fracture resistance for various mechanical loading situations. This study aimed to synthesize highly mineralized hydrogels to mimic the mechanical properties of cancellous bone. A six armed star molecule functionalized with isocyanate groups as reactive termini (NCO-sP(EO-stat-PO)) was used to build up a hydrogel matrix, which was then subsequently mineralized with hydroxyapatite nanocrystals following the hydrolysis of incorporated α-tricalcium phosphate particles. The advantage of this dual setting approach in comparison to simply adding unreactive filler particles to the hydrogel was demonstrated to be a strength improvement by the factor of 30. After 1-28d setting, the mechanical properties of a composite with 30wt% NCO-sP(EO-stat-PO) such as elasticity (5.3-1.4%), compression strength (11-23MPa) and E-modulus (211-811MPa) were found to be similar to the properties of cancellous bone.

Cu2+, Co2+ and Cr3+ doping of a calcium phosphate cement influences materials properties and response of human mesenchymal stromal cells.

The application of biologically active metal ions to stimulate cellular reactions is a promising strategy to accelerate bone defect healing. Brushite-forming calcium phosphate cements were modified with low doses of Cu2+, Co2+ and Cr3+. The modified cements released the metal ions in vitro in concentrations which were shown to be non-toxic for cells. The release kinetics correlated with the solubility of the respective metal phosphates: 17-45 wt.-% of Co2+ and Cu2+, but <1 wt.-% of Cr3+ were released within 28days. Moreover, metal ion doping led to alterations in the exchange of calcium and phosphate ions with cell culture medium. In case of cements modified with 50mmol Cr3+/mol ß-tricalcium phosphate (ß-TCP), XRD and SEM analyses revealed a significant amount of monetite and a changed morphology of the cement matrix. Cell culture experiments with human mesenchymal stromal cells indicated that the observed cell response is not only influenced by the released metal ions but also by changed cement properties. A positive effect of modifications with 50mmol Cr3+ or 10mmol Cu2+ per mol ß-TCP on cell behaviour was observed in indirect and direct culture. Modification with Co2+ resulted in a clear suppression of cell proliferation and osteogenic differentiation. In conclusion, metal ion doping of the cement influences cellular activities in addition to the effect of released metal ions by changing properties of the ceramic matrix.

Thiol-ene Clickable Poly(glycidol) Hydrogels for Biofabrication.

In this study we introduce linear poly(glycidol) (PG), a structural analog of poly(ethylene glycol) bearing side chains at each repeating unit, as polymer basis for bioink development. We prepare allyl- and thiol-functional linear PG that can rapidly be polymerized to a three-dimensionally cross-linked hydrogel network via UV mediated thiol-ene click reaction. Influence of polymer concentration and UV irradiation on mechanical properties and swelling behavior was examined. Thiol-functional PG was synthesized in two structural variations, one containing ester groups that are susceptible to hydrolytic cleavage, and the other one ester-free and stable against hydrolysis. This allowed the preparation of degradable and non-degradable hydrogels. Cytocompatibility of the hydrogel was demonstrated by encapsulation of human bone marrow-derived mesenchymal stem cells (hBMSCs). Rheological properties of the hydrogels were adjusted for dispense plotting by addition of high molecular weight hyaluronic acid. The optimized formulation enabled highly reproducible plotting of constructs composed of 20 layers with an overall height of 3.90 mm.

A Bone Glue with Sustained Adhesion under Wet Conditions.

Bone glues often suffer from low adhesion to bone under wet conditions. This study aims to improve wet adhesiveness of a bone glue based on a photocurable poly(ethylene glycol) dimethacrylate matrix through in situ interpenetrating network formation by addition of six-armed isocyanate functional star-shaped prepolymers (NCO-sP(EO-stat-PO)). Biodegradable ceramic fillers are added to adjust the paste workability. The 3-point bending strength of the bone glues is in the range of 3.5-5.5 MPa and not significantly affected by the addition of NCO-sP(EO-stat-PO). Storage in phosphate buffered saline (PBS) decreases the bending strength of all formulations to approximately 1 MPa but the adhesion to cortical bone increases from 0.15-0.2 to 0.3-0.5 MPa after adding 20-40 wt% NCO-sP(EO-stat-PO) to the matrix. Bone glues without the NCO-sP(EO-stat-PO) additive lose their adhesiveness to bone after aging in PBS for 7 days, whereas modified glues maintain a shear strength of 0.18-0.25 MPa demonstrating the efficacy of the approach. Scanning electron microscopy and energy-dispersive X-ray spectroscopy investigations of the fracture surfaces prove a high amount of residual adhesive on the bone surface indicating that adhesion to the bone under wet conditions is stronger than cohesion.

Novel bone wax based on poly(ethylene glycol)-calcium phosphate cement mixtures.

Classic bone wax is associated with drawbacks such as the risk of infection, inflammation and hindered osteogenesis. Here, we developed a novel self-setting bone wax on the basis of hydrophilic poly(ethylene glycol) (PEG) and hydroxyapatite (HA) forming calcium phosphate cement (CPC), to overcome the problems that are linked to the use of conventional beeswax systems. Amounts of up to 10 wt.% of pregelatinized starch were additionally supplemented as hemostatic agent. After exposure to a humid environment, the PEG phase dissolved and was exchanged by penetrating water that interacted with the HA precursor (tetracalcium phosphate (TTCP)/monetite) to form highly porous, nanocrystalline HA via a dissolution/precipitation reaction. Simultaneously, pregelatinized starch could gel and supply the bone wax with liquid sealing features. The novel bone wax formulation was found to be cohesive, malleable and after hardening under aqueous conditions, it had a mechanical performance (∼2.5 MPa compressive strength) that is comparable to that of cancellous bone. It withstood systolic blood pressure conditions for several days and showed antibacterial properties for almost one week, even though 60% of the incorporated drug vancomycin hydrochloride was already released after 8h of deposition by diffusion controlled processes. STATEMENT OF SIGNIFICANCE: The study investigated the development of alternative bone waxes on the basis of a hydroxyapatite (HA) forming calcium phosphate cement (CPC) system. Conventional bone waxes are composed of non-biodegradable beeswax/vaseline mixtures that are often linked to infection, inflammation and hindered osteogenesis. We combined the usage of bioresorbable polymers, the supplementation with hemostatic agents and the incorporation of a mineral component to overcome those drawbacks. Self-setting CPC precursors (tetracalcium phosphate (TTCP), monetite) were embedded in a resorbable matrix of poly(ethylene glycol) (PEG) and supplemented with pregelatinized starch. This formulation was found to be malleable and cohesive underwater. While immersion in an aqueous environment, CPC precursors formed highly porous, nanocrystalline HA via dissolution/precipitation reaction as water penetrated the novel wax formulation and PEG molecules simultaneously dissolved. The bone wax further withstood blood pressure conditions. After hardening, mechanical performance was comparable to that of cancellous bone and we also successfully provided the bone wax with antibacterial properties. In our opinion, the described bone wax formulation outmatches conventional bone waxes, as it circumvents the detriments being associated with the term "bone wax". Our wax has a novel composition and would broaden the application of CPC and besides, the general interest in bone waxes will increase, as they were long considered as a "first-line treatment" to avoid.