Jan Schrooten

Normocalcemia is maintained in mice under conditions of calcium malabsorption by vitamin D-induced inhibition of bone mineralization

Serum calcium levels are tightly controlled by an integrated hormone-controlled system that involves active vitamin D [1,25(OH)(2)D], which can elicit calcium mobilization from bone when intestinal calcium absorption is decreased. The skeletal adaptations, however, are still poorly characterized. To gain insight into these issues, we analyzed the consequences of specific vitamin D receptor (Vdr) inactivation in the intestine and in mature osteoblasts on calcium and bone homeostasis. We report here that decreased intestinal calcium absorption in intestine-specific Vdr knockout mice resulted in severely reduced skeletal calcium levels so as to ensure normal levels of calcium in the serum. Furthermore, increased 1,25(OH)(2)D levels not only stimulated bone turnover, leading to osteopenia, but also suppressed bone matrix mineralization. This resulted in extensive hyperosteoidosis, also surrounding the osteocytes, and hypomineralization of the entire bone cortex, which may have contributed to the increase in bone fractures. Mechanistically, osteoblastic VDR signaling suppressed calcium incorporation in bone by directly stimulating the transcription of genes encoding mineralization inhibitors. Ablation of skeletal Vdr signaling precluded this calcium transfer from bone to serum, leading to better preservation of bone mass and mineralization. These findings indicate that in mice, maintaining normocalcemia has priority over skeletal integrity, and that to minimize skeletal calcium storage, 1,25(OH)(2)D not only increases calcium release from bone, but also inhibits calcium incorporation in bone.

The effect of pore geometry on the in vitro biological behavior of human periosteum-derived cells seeded on selective laser-melted Ti6Al4V bone scaffolds

The specific aim of this study was to gain insight into the influence of scaffold pore size, pore shape and permeability on the in vitro proliferation and differentiation of three-dimensional (3-D) human periosteum-derived cell (hPDC) cultures. Selective laser melting (SLM) was used to produce six distinct designed geometries of Ti6Al4V scaffolds in three different pore shapes (triangular, hexagonal and rectangular) and two different pore sizes (500 μm and 1000 μm). All scaffolds were characterized by means of two-dimensional optical microscopy, 3-D microfocus X-ray computed tomography (micro-CT) image analysis, mechanical compression testing and computational fluid dynamical analysis. The results showed that SLM was capable of producing Ti6Al4V scaffolds with a broad range of morphological and mechanical properties. The in vitro study showed that scaffolds with a lower permeability gave rise to a significantly higher number of cells attached to the scaffolds after seeding. Qualitative analysis by means of live/dead staining and scanning electron micrography showed a circular cell growth pattern which was independent of the pore size and shape. This resulted in pore occlusion which was found to be the highest on scaffolds with 500 μm hexagonal pores. Interestingly, pore size but not pore shape was found to significantly influence the growth of hPDC on the scaffolds, whereas the differentiation of hPDC was dependent on both pore shape and pore size. The results showed that, for SLM-produced Ti6Al4V scaffolds with specific morphological and mechanical properties, a functional graded scaffold will contribute to enhanced cell seeding and at the same time can maintain nutrient transport throughout the whole scaffold during in vitro culturing by avoiding pore occlusion.

A calcium-induced signaling cascade leading to osteogenic differentiation of human bone marrow-derived mesenchymal stromal cells

The response of osteoprogenitors to calcium (Ca(2+)) is of primary interest for both normal bone homeostasis and the clinical field of bone regeneration. The latter makes use of calcium phosphate-based bone void fillers to heal bone defects, but it is currently not known how Ca(2+) released from these ceramic materials influences cells in situ. Here, we have created an in vitro environment with high extracellular Ca(2+) concentration and investigated the response of human bone marrow-derived mesenchymal stromal cells (hMSCs) to it. Ca(2+) enhanced proliferation and morphological changes in hMSCs. Moreover, the expression of osteogenic genes is highly increased. A 3-fold up-regulation of BMP-2 is observed after only 6h and pharmaceutical interference with a number of proteins involved in Ca(2+) sensing showed that not the calcium sensing receptor, but rather type L voltage-gated calcium channels are involved in mediating the signaling pathway between extracellular Ca(2+) and BMP-2 expression. MEK1/2 activity is essential for the effect of Ca(2+) and using microarray analysis, we have identified c-Fos as an early Ca(2+) response gene. We have demonstrated that hMSC osteogenesis can be induced via extracellular Ca(2+), a simple and economic way of priming hMSCs for bone tissue engineering applications.

Fatigue behavior of porous biomaterials manufactured using selective laser melting

Porous titanium alloys are considered promising bone-mimicking biomaterials. Additive manufacturing techniques such as selective laser melting allow for manufacturing of porous titanium structures with a precise design of micro-architecture. The mechanical properties of selective laser melted porous titanium alloys with different designs of micro-architecture have been already studied and are shown to be in the range of mechanical properties of bone. However, the fatigue behavior of this biomaterial is not yet well understood. We studied the fatigue behavior of porous structures made of Ti6Al4V ELI powder using selective laser melting. Four different porous structures were manufactured with porosities between 68 and 84% and the fatigue S-N curves of these four porous structures were determined. The three-stage mechanism of fatigue failure of these porous structures is described and studied in detail. It was found that the absolute S-N curves of these four porous structures are very different. In general, given the same absolute stress level, the fatigue life is much shorter for more porous structures. However, the normalized fatigue S-N curves of these four structures were found to be very similar. A power law was fitted to all data points of the normalized S-N curves. It is shown that the measured data points conform to the fitted power law very well, R(2)=0.94. This power law may therefore help in estimating the fatigue life of porous structures for which no fatigue test data is available. It is also observed that the normalized endurance limit of all tested porous structures (<0.2) is lower than that of corresponding solid material (c.a. 0.4).

Mechanical behavior of regular open-cell porous biomaterials made of diamond lattice unit cells.

Cellular structures with highly controlled micro-architectures are promising materials for orthopedic applications that require bone-substituting biomaterials or implants. The availability of additive manufacturing techniques has enabled manufacturing of biomaterials made of one or multiple types of unit cells. The diamond lattice unit cell is one of the relatively new types of unit cells that are used in manufacturing of regular porous biomaterials. As opposed to many other types of unit cells, there is currently no analytical solution that could be used for prediction of the mechanical properties of cellular structures made of the diamond lattice unit cells. In this paper, we present new analytical solutions and closed-form relationships for predicting the elastic modulus, Poisson׳s ratio, critical buckling load, and yield (plateau) stress of cellular structures made of the diamond lattice unit cell. The mechanical properties predicted using the analytical solutions are compared with those obtained using finite element models. A number of solid and porous titanium (Ti6Al4V) specimens were manufactured using selective laser melting. A series of experiments were then performed to determine the mechanical properties of the matrix material and cellular structures. The experimentally measured mechanical properties were compared with those obtained using analytical solutions and finite element (FE) models. It has been shown that, for small apparent density values, the mechanical properties obtained using analytical and numerical solutions are in agreement with each other and with experimental observations. The properties estimated using an analytical solution based on the Euler-Bernoulli theory markedly deviated from experimental results for large apparent density values. The mechanical properties estimated using FE models and another analytical solution based on the Timoshenko beam theory better matched the experimental observations.

Bone regeneration performance of surface-treated porous titanium

The large surface area of highly porous titanium structures produced by additive manufacturing can be modified using biofunctionalizing surface treatments to improve the bone regeneration performance of these otherwise bioinert biomaterials. In this longitudinal study, we applied and compared three types of biofunctionalizing surface treatments, namely acid-alkali (AcAl), alkali-acid-heat treatment (AlAcH), and anodizing-heat treatment (AnH). The effects of treatments on apatite forming ability, cell attachment, cell proliferation, osteogenic gene expression, bone regeneration, biomechanical stability, and bone-biomaterial contact were evaluated using apatite forming ability test, cell culture assays, and animal experiments. It was found that AcAl and AnH work through completely different routes. While AcAl improved the apatite forming ability of as-manufactured (AsM) specimens, it did not have any positive effect on cell attachment, cell proliferation, and osteogenic gene expression. In contrast, AnH did not improve the apatite forming ability of AsM specimens but showed significantly better cell attachment, cell proliferation, and expression of osteogenic markers. The performance of AlAcH in terms of apatite forming ability and cell response was in between both extremes of AnH and AsM. AcAl resulted in significantly larger volumes of newly formed bone within the pores of the scaffold as compared to AnH. Interestingly, larger volumes of regenerated bone did not translate into improved biomechanical stability as AnH exhibited significantly better biomechanical stability as compared to AcAl suggesting that the beneficial effects of cell-nanotopography modulations somehow surpassed the benefits of improved apatite forming ability. In conclusion, the applied surface treatments have considerable effects on apatite forming ability, cell attachment, cell proliferation, and bone ingrowth of the studied biomaterials. The relationship between these properties and the bone-implant biomechanics is, however, not trivial.

Relationship between unit cell type and porosity and the fatigue behavior of selective laser melted meta-biomaterials

Meta-materials are structures when their small-scale properties are considered, but behave as materials when their homogenized macroscopic properties are studied. There is an intimate relationship between the design of the small-scale structure and the homogenized properties of such materials. In this article, we studied that relationship for meta-biomaterials that are aimed for biomedical applications, otherwise known as meta-biomaterials. Selective laser melted porous titanium (Ti6Al4V ELI) structures were manufactured based on three different types of repeating unit cells, namely cube, diamond, and truncated cuboctahedron, and with different porosities. The morphological features, static mechanical properties, and fatigue behavior of the porous biomaterials were studied with a focus on their fatigue behavior. It was observed that, in addition to static mechanical properties, the fatigue properties of the porous biomaterials are highly dependent on the type of unit cell as well as on porosity. None of the porous structures based on the cube unit cell failed after 10(6) loading cycles even when the applied stress reached 80% of their yield strengths. For both other unit cells, higher porosities resulted in shorter fatigue lives for the same level of applied stress. When normalized with respect to their yield stresses, the S-N data points of structures with different porosities very well (R(2)>0.8) conformed to one single power law specific to the type of the unit cell. For the same level of normalized applied stress, the truncated cuboctahedron unit cell resulted in a longer fatigue life as compared to the diamond unit cell. In a similar comparison, the fatigue lives of the porous structures based on both truncated cuboctahedron and diamond unit cells were longer than that of the porous structures based on the rhombic dodecahedron unit cell (determined in a previous study). The data presented in this study could serve as a basis for design of porous biomaterials as well as for corroboration of relevant analytical and computational models.

Adhesion of bioactive glass coating to Ti6Al4V oral implant

Bioactive glass (BAG) is a bioactive material with a high potential as implant material. Reactive plasma spraying produces an economically feasible BAG-coating for Ti6A14V oral implants. This coating is only functional if it adheres well to the metal substrate and if it is strong enough to transfer all loads. To examine these two properties an appropriate mechanical adhesion test, the moment test, is developed. This test quantifies under a realistic loading condition the corresponding functional adhesion strength to be >84 MPa in tensile. To get a qualitative insight in the BAG-coating behavior during loading the mechanical test was combined with finite element analysis, acoustic emission and microscopic analysis. These analyses showed that the coating withstands without any damage an externally generated tensile stress of 47 MPa. Not only the initial adhesion is determining for the implant quality, but more important is the coating functionality after reaction of the BAG. Adhesion testing after two months of in vitro reaction in a simulated body fluid showed that coating adhesion strength decreased with 10%, but the implant system was still adequate for load-bearing applications.

Temperature memory effect of a nickel–titanium shape memory alloy

An incomplete transformation cycle induces a kinetic stop in the following complete transformation cycle in shape memory alloys. Therefore, the kinetic stop can be regarded as a memory of the previous arrest temperature. Herein, we show that the temperature memory effect of a nickel–titanium shape memory alloy can be expanded to be operational in a very wide temperature range by prestraining and constraining, which may be exploited for various practical applications.

Surface Roughness and Morphology Customization of Additive Manufactured Open Porous Ti6Al4V Structures

Additive manufacturing (AM) is a production method that enables the building of porous structures with a controlled geometry. However, there is a limited control over the final surface of the product. Hence, complementary surface engineering strategies are needed. In this work, design of experiments (DoE) was used to customize post AM surface treatment for 3D selective laser melted Ti6Al4V open porous structures for bone tissue engineering. A two-level three-factor full factorial design was employed to assess the individual and interactive effects of the surface treatment duration and the concentration of the chemical etching solution on the final surface roughness and beam thickness of the treated porous structures. It was observed that the concentration of the surface treatment solution was the most important factor influencing roughness reduction. The designed beam thickness decreased the effectiveness of the surface treatment. In this case study, the optimized processing conditions for AM production and the post-AM surface treatment were defined based on the DoE output and were validated experimentally. This allowed the production of customized 3D porous structures with controlled surface roughness and overall morphological properties, which can assist in more controlled evaluation of the effect of surface roughness on various functional properties.