I. Manjubala

Bone Material Quality in Transiliac Bone Biopsies of Postmenopausal Osteoporotic Women After 3 Years of Strontium Ranelate Treatment

Strontium ranelate (SrR) is a relatively new treatment for osteoporosis. In this study we investigated its potential impact on human bone material quality in transiliac bone biopsies from postmenopausal osteoporotic women treated 3 years with calcium and vitamin D plus either 2 g SrR per day or placebo. Bone mineralization density distribution (BMDD), strontium (Sr) concentration, collagen cross‐link ratio, and indentation modulus were analyzed by quantitative backscattered electron imaging, electron‐induced X‐ray fluorescence analysis, synchrotron radiation induced micro X‐ray fluorescence elemental mapping, Fourier transform infrared imaging, and nanoindentation, respectively. The BMDD of SrR‐treated patients was shifted to higher atomic numbers (Zmean +1.5%, p < .05 versus placebo). We observed Sr being preferentially incorporated in bone packets formed during SrR treatment up to 6% atom fraction [Sr/(Sr + Ca)] depending on the SrR serum levels of the individuals (correlation r = 0.84, p = .018). Collagen cross‐link ratio was preserved in SR‐treated bone. The indentation modulus was significantly decreased in younger versus older bone packets for both placebo‐ (−20.5%, p < .0001) and SrR‐treated individuals (−24.3%, p < .001), whereas no differences were found between the treatment groups. In conclusion, our findings indicate that after SrR treatment, Sr is heterogeneously distributed in bone and preferentially present in bone packets formed during treatment. The effect of SrR on BMDD seems to be due mainly to the uptake of Sr and not to changes in bone calcium content. Taken together, these data provide evidence that the investigated bone quality determinants at tissue level were preserved in postmenopausal osteoporotic women after 3‐year treatment with 2 g SrR per day plus calcium and vitamin D.

Lathyrism-induced alterations in collagen cross-links influence the mechanical properties of bone material without affecting the mineral.

In the present study a rat animal model of lathyrism was employed to decipher whether anatomically confined alterations in collagen cross-links are sufficient to influence the mechanical properties of whole bone. Animal experiments were performed under an ethics committee approved protocol. Sixty-four female (47 day old) rats of equivalent weights were divided into four groups (16 per group): Controls were fed a semi-synthetic diet containing 0.6% calcium and 0.6% phosphorus for 2 or 4 weeks and β-APN treated animals were fed additionally with β-aminopropionitrile (0.1% dry weight). At the end of this period the rats in the four groups were sacrificed, and L2–L6 vertebra were collected. Collagen cross-links were determined by both biochemical and spectroscopic (Fourier transform infrared imaging (FTIRI)) analyses. Mineral content and distribution (BMDD) were determined by quantitative backscattered electron imaging (qBEI), and mineral maturity/crystallinity by FTIRI techniques. Micro-CT was used to describe the architectural properties. Mechanical performance of whole bone as well as of bone matrix material was tested by vertebral compression tests and by nano-indentation, respectively. The data of the present study indicate that β-APN treatment changed whole vertebra properties compared to non-treated rats, including collagen cross-links pattern, trabecular bone volume to tissue ratio and trabecular thickness, which were all decreased (p < 0.05). Further, compression tests revealed a significant negative impact of β-APN treatment on maximal force to failure and energy to failure, while stiffness was not influenced. Bone mineral density distribution (BMDD) was not altered either. At the material level, β-APN treated rats exhibited increased Pyd/Divalent cross-link ratios in areas confined to a newly formed bone. Moreover, nano-indentation experiments showed that the E-modulus and hardness were reduced only in newly formed bone areas under the influence of β-APN, despite a similar mineral content. In conclusion the results emphasize the pivotal role of collagen cross-links in the determination of bone quality and mechanical integrity. However, in this rat animal model of lathyrism, the coupled alterations of tissue structural properties make it difficult to weigh the contribution of the anatomically confined material changes to the overall mechanical performance of whole bone. Interestingly, the collagen cross-link ratio in bone forming areas had the same profile as seen in actively bone forming trabecular surfaces in human iliac crest biopsies of osteoporotic patients.

Improving the osteointegration and bone–implant interface by incorporation of bioactive particles in sol–gel coatings of stainless steel implants

In this study, we report a hybrid organic-inorganic TEOS-MTES (tetraethylorthosilicate-methyltriethoxysilane) sol-gel-made coating as a potential solution to improve the in vivo performance of AISI 316L stainless steel, which is used as permanent bone implant material. These coatings act as barriers for ion migration, promoting the bioactivity of the implant surface. The addition of SiO(2) colloidal particles to the TEOS-MTES sol (10 or 30 mol.%) leads to thicker films and also acts as a film reinforcement. Also, the addition of bioactive glass-ceramic particles is considered responsible for enhancing osseointegration. In vitro assays for bioactivity in simulated body fluid showed the presence of crystalline hydroxyapatite (HA) crystals on the surface of the double coating with 10mol.% SiO(2) samples on stainless steel after 30 days of immersion. The HA crystal lattice parameters are slightly different from stoichiometric HA. In vivo implantation experiments were carried out in a rat model to observe the osteointegration of the coated implants. The coatings promote the development of newly formed bone in the periphery of the implant, in both the remodellation zone and the marrow zone. The quality of the newly formed bone was assessed for mechanical and structural integrity by nanoindentation and small-angle X-ray scattering experiments. The different amount of colloidal silica present in the inner layer of the coating slightly affects the material quality of the newly formed bone but the nanoindentation results reveal that the lower amount of silica in the coating leads to mechanical properties similar to cortical bone.

In vitro bioactivity of bioresorbable porous polymeric scaffolds incorporating hydroxyapatite microspheres

Biomimetic composites consisting of polymer and mineral components, resembling bone in structure and composition, were produced using a rapid prototyping technique for bone tissue engineering applications. Solid freeform fabrication, known as rapid prototyping (RP) technology, allows scaffolds to be designed with pre-defined and controlled external and internal architecture. Using the indirect RP technique, a three-component scaffold with a woodpile structure, consisting of poly-L-lactic acid (PLLA), chitosan and hydroxyapatite (HA) microspheres, was produced that had a macroporosity of more than 50% together with micropores induced by lyophilization. X-ray diffraction analysis indicated that the preparation and construction of the composite scaffold did not affect the phase composition of the HA. The compressive strength and elastic modulus (E) for the PLLA composites are 0.42 and 1.46 MPa, respectively, which are much higher than those of chitosan/HA composites and resemble the properties of cellular structure. These scaffolds showed excellent biocompatibility and ability for three-dimensional tissue growth of MC3T3-E1 pre-osteoblastic cells. The pre-osteoblastic cells cultured on these scaffolds formed a network on the HA microspheres and proliferated not only in the macropore channels but also in the micropores, as seen from the histological analysis and electron microscopy. The proliferating cells formed an extracellular matrix network and also differentiated into mature osteoblasts, as indicated by alkaline phosphatase enzyme activity. The properties of these scaffolds indicate that they can be used for non-load-bearing applications.

Size and habit of mineral particles in bone and mineralized callus during bone healing in sheep.

Bone healing is known to occur through the successive formation and resorption of various tissues with different structural and mechanical properties. To get a better insight into this sequence of events, we used environmental scanning electron microscopy (ESEM) together with scanning small‐angle X‐ray scattering (sSAXS) to reveal the size and orientation of bone mineral particles within the regenerating callus tissues at different healing stages (2, 3, 6, and 9 weeks). Sections of 200 µm were cut from embedded blocks of midshaft tibial samples in a sheep osteotomy model with an external fixator. Regions of interest on the medial side of the proximal fragment were chosen to be the periosteal callus, middle callus, intercortical callus, and cortex. Mean thickness (T parameter), degree of alignment (ρ parameter), and predominant orientation (ψ parameter) of mineral particles were deduced from resulting sSAXS patterns with a spatial resolution of 200 µm. 2D maps of T and ρ overlapping with ESEM images revealed that the callus formation occurred in two waves of bone formation, whereby a highly disordered mineralized tissue was deposited first, followed by a bony tissue with more lamellar appearance in the ESEM and where the mineral particles were more aligned, as revealed by sSAXS. As a consequence, degree of alignment and mineral particle size within the callus increased with healing time, whereas at any given moment there were structural gradients, for example, from periosteal toward the middle callus.

Two stages in three-dimensional in vitro growth of tissue generated by osteoblastlike cells.

Bone regeneration is controlled by a variety of biochemical, biomechanical, cellular, and hormonal mechanisms. In particular, physical properties of the substrate such as stiffness and architecture highly influence the proliferation and differentiation of cells. The aim of this work is to understand the influence of scaffold stiffness and cell seeding densities on the formation of tissue by osteoblast cells within polyether urethane scaffolds containing pores of different sizes. MC3T3-E1 preosteoblast cells were seeded on the scaffold, and the amount of tissue formed within the pores was analyzed for culture times up to 49 days by phase contrast microscopy. The authors show that the kinetics of three-dimensional tissue growth in these scaffolds follows two stages and can be described by a universal growth law. The first stage is dominated by cell-material interactions with cell adherence and differentiation being strongly dependent on the polymer material. After a delay time of a few weeks, cells begin to grow within their own matrix, the delay being strongly dependent on substrate stiffness and seeding protocols. In this later stage of growth, three-dimensional tissue amplification is controlled rather by the pore geometry than the scaffold material properties. This emphasizes how geometric constraints may guide tissue formation in vitro and shows that optimizing scaffold architectures may improve tissue formation independent of the scaffold material used.

Fetal and postnatal mouse bone tissue contains more calcium than is present in hydroxyapatite

It has been shown for developing enamel and zebrafish fin that hydroxyapatite (HA) is preceded by an amorphous precursor, motivating us to examine the mineral development in mammalian bone, particularly femur and tibia of fetal and young mice. Mineral particle thickness and arrangement were characterized by (synchrotron) small-angle X-ray scattering (SAXS) combined with wide-angle X-ray diffraction (WAXD) and X-ray fluorescence (XRF) analysis. Simultaneous measurements of the local calcium content and the HA content via XRF and WAXD, respectively, revealed the total calcium contained in HA crystals. Interestingly, bones of fetal as well as newborn mice contained a certain fraction of calcium which is not part of the HA crystals. Mineral deposition could be first detected in fetal tibia at day 16.5 by environmental scanning electron microscopy (ESEM). SAXS revealed a complete lack of orientation in the mineral particles at this stage, whereas 1day after birth particles were predominantly aligned parallel to the longitudinal bone axis, with the highest degree of alignment in the midshaft. Moreover, we found that mineral particle length increased with age as well as the thickness, while fetal particles were thicker but much shorter. In summary, this study revealed strong differences in size and orientation of the mineral particles between fetal and postnatal bone, with bulkier, randomly oriented particles at the fetal stage, and highly aligned, much longer particles after birth. Moreover, a part of the calcium seems to be present in other form than HA at all stages of development.

The mechanical heterogeneity of the hard callus influences local tissue strains during bone healing: A finite element study based on sheep experiments

During secondary fracture healing, various tissue types including new bone are formed. The local mechanical strains play an important role in tissue proliferation and differentiation. To further our mechanobiological understanding of fracture healing, a precise assessment of local strains is mandatory. Until now, static analyses using Finite Elements (FE) have assumed homogenous material properties. With the recent quantification of both the spatial tissue patterns (Vetter et al., 2010) and the development of elastic modulus of newly formed bone during healing (Manjubala et al., 2009), it is now possible to incorporate this heterogeneity. Therefore, the aim of this study is to investigate the effect of this heterogeneity on the strain patterns at six successive healing stages. The input data of the present work stemmed from a comprehensive cross-sectional study of sheep with a tibial osteotomy (Epari et al., 2006). In our FE model, each element containing bone was described by a bulk elastic modulus, which depended on both the local area fraction and the local elastic modulus of the bone material. The obtained strains were compared with the results of hypothetical FE models assuming homogeneous material properties. The differences in the spatial distributions of the strains between the heterogeneous and homogeneous FE models were interpreted using a current mechanobiological theory (Isakson et al., 2006). This interpretation showed that considering the heterogeneity of the hard callus is most important at the intermediate stages of healing, when cartilage transforms to bone via endochondral ossification.

Mineral crystal alignment in mineralized fracture callus determined by 3D small-angle X-ray scattering

Callus tissue formed during bone fracture healing is a mixture of different tissue types as revealed by histological analysis. But the structural characteristics of mineral crystals within the healing callus are not well known. Since two-dimensional (2D) scanning small-angle X-ray scattering (sSAXS) patterns showed that the size and orientation of callus crystals vary both spatially and temporally [1] and 2D electron microscopic analysis implies an anisotropic property of the callus morphology, the mineral crystals within the callus are also expected to vary in size and orientation in 3D. Three-dimensional small-angle X-ray scattering (3D SAXS), which combines 2D SAXS patterns collected at different angles of sample tilting, has been previously applied to investigate bone minerals in horse radius [2] and oim/oim mouse femur/tibia [3]. We implement a similar 3D SAXS method but with a different way of data analysis to gather information on the mineral alignment in fracture callus. With the proposed accurate yet fast assessment of 3D SAXS information, it was shown that the plate shaped mineral particles in the healing callus were aligned in groups with their predominant orientations occurring as a fiber texture.

The influence of the thermal treatment of hydroxylapatite scaffolds on the physical properties and the bone cell in growth behaviour

The material bone consists of a biopolymer matrix (collagen) reinforced with mineral nanoparticles (carbonated hydroxylapatite), forming a natural composite which builds up a dense shell on the exterior and a network of struts with a mean diameter of 200µm in the core of many bones. The architecture of the foamy inner part of bones (spongiosa) is determined by loading conditions. The architecture strongly influences the mechanical properties of cellular solids together with the apparent density and the material it consists of. In addition, the ingrowth of bone cells into porous implants depends on pore size, size distribution and interconnectivity. From this it is clear that the possibility to design the architecture of a bone replacement material is beneficial from a biological as well as a mechanical point of view. Our approach uses rapid prototyping methods, ceramic gelcasting and sintering to produce cellular structures with designed architecture from hydroxylapatite and other bioceramics. The influence of sintering temperature and atmosphere on the physical properties of these scaffolds was investigated with x-ray diffraction and scanning electron microscopy. Furthermore, the cell ingrowth behaviour was determined in cell culture experiments, using the praeosteoblastic cell line MC3T3-E1, derived from mouse calvariae. The cell ingrowth behaviour was evaluated during a culture period of two and three weeks, by light microscopy and afterwards by histology after embedding and Giemsa-staining. The phase composition of the material was found to change with increasing sintering temperature and its surface characteristics was influenced by the sintering atmosphere. These changes also affected the cell ingrowth behaviour. In some experiments, the osteoblasts-like cells were found to cover the whole external and internal surface of the scaffold. The cells produced extracellular matrix consisting of collagen, which eventually filled nearly all the pores. In particular, the cells had the tendency to fill any crack or opening in the scaffolds, and to generally smooth the surfaces. In conclusion, rapid prototyping and ceramic gelcasting allows the freeform fabrication of porous bioceramics with controlled architecture. Such structures made of hydroxylapatit were found to support the growth of mouse osteoblasts.