Florian Despang

Proliferation and osteogenic differentiation of human bone marrow stromal cells on alginate–gelatine–hydroxyapatite scaffolds with anisotropic pore structure

Porous mineralized scaffolds are required for various applications in bone engineering. In particular, tube‐like pores with controlled orientation inside the scaffold may support homogeneous cell seeding as well as sufficient nutrient supply and may facilitate blood vessel ingrowth. Scaffolds with parallely orientated tube‐like pores were generated by diffusion‐controlled ionotropic gelation of alginate. Incorporation of hydroxyapatite (HA) during the gelation process yielded stable scaffolds with an average pore diameter of approximately 90 µm. To evaluate the potential use of alginate–gelatine–HA scaffolds for bone tissue engineering, in vitro tests with human bone marrow stromal cells (hBMSCs) were carried out. We analysed biocompatibility and cell penetration into the capillary pores by microscopic methods. hBMSCs were also cultivated on alginate–gelatine–HA scaffolds for 3 weeks in the presence and absence of osteogenic supplements. We studied proliferation and osteogenic differentiation in terms of total lactate dehydrogenase (LDH) activity, DNA content and alkaline phosphatase (ALP) activity and found a 10–14‐fold increase of cell number after 2 weeks of cultivation, as well as an increase of specific ALP activity for osteogenic‐induced hBMSCs. Furthermore, the expression of bone‐related genes [ALP, bone sialoprotein II (BSPII)] was analysed. We found an increase of ALP as well as BSPII expression for osteogenic‐induced hBMSCs on alginate–gelatin–HA scaffolds. Copyright

Fabrication of porous scaffolds by three‐dimensional plotting of a pasty calcium phosphate bone cement under mild conditions

The major advantage of hydroxyapatite (HA)‐forming calcium phosphate cements (CPCs) used as bone replacement materials is their setting under physiological conditions without the necessity for thermal treatment that allows the incorporation of biological factors. In the present study, we have combined the biocompatible consolidation of CPCs with the potential of rapid prototyping (RP) techniques to generate calcium phosphate‐based scaffolds with defined inner and outer morphology. We demonstrate the application of the RP technique three‐dimensional (3D) plotting for the fabrication of HA cement scaffolds. This was realized by utilizing a paste‐like CPC (P‐CPC) which is stable as a malleable paste and whose setting reaction is initiated only after contact with aqueous solutions. The P‐CPC showed good processability in the 3D plotting process and allowed the fabrication of stable 3D structures of different geometries with adequate mechanical stability and compressive strength. The cytocompatibility of the plotted P‐CPC scaffolds was demonstrated in a cell culture experiment with human mesenchymal stem cells. The mild conditions during 3D plotting and post‐processing and the realization of the whole procedure under sterile conditions make this approach highly attractive for fabrication of individualized implants with respect to patient‐specific requirements by simultaneous plotting of biological components. Copyright

Biphasic, but monolithic scaffolds for the therapy of osteochondral defects

Abstract For the regenerative therapy of osteochondral defects – deep lesions of the articular cartilage in which the underlying bone tissue is already affected too – special implant materials and scaffolds are needed. In this study, two new approaches will be presented, leading to biphasic, but monolithic scaffold materials. Both consist of a mineralised layer for filling of the bony part of the defect and a non-mineralised one for the chondral part. Due to the preparation methods, both layers are fused together to give a unified whole without need of any artificial joining. The resulting materials, either based on collagen, hyaluronic acid and hydroxyapatite or calcium alginate gels and hydroxyapatite, seem to be suitable scaffolds for cultivating chondrocytes and osteoblasts – and therefore can act as matrices for tissue engineering of osteochondral grafts.

Synthesis and physicochemical, in vitro and in vivo evaluation of an anisotropic, nanocrystalline hydroxyapatite bisque scaffold with parallel‐aligned pores mimicking the microstructure of cortical bone

Scaffolds for bone regeneration are mostly prepared with an isotropic, sponge‐like structure mimicking the architecture of trabecular bone. We have developed an anisotropic bioceramic with parallel aligned pores resembling the honeycomb arrangement of Haversian canals of cortical bone and investigated its potential as a scaffold for tissue engineering. Parallel channel‐like pores were generated by ionotropic gelation of an alginate–hydroxyapatite (HA) slurry, followed by ceramic processing. Organic components were thermally removed at 650 °C, whereas the pore system was preserved in the obtained HA bioceramic in the processing stage of a bisque. Even without further sintering at higher temperatures, the anisotropic HA bisque (AHAB) became mechanically stable with a compressive strength (4.3 MPa) comparable to that of native trabecular bone. Owing to the low‐temperature treatment, a nanocrystalline microstructure with high porosity (82%) and surface area (24.9 m2/g) was achieved that kept the material dissolvable in acidic conditions, similar to osteoclastic degradation of bone. Human mesenchymal stem cells (hMSCs) adhered, proliferated and differentiated into osteoblasts when osteogenically induced, indicating the cytocompatibility of the bisque scaffold. Furthermore, we demonstrated fusion of human monocytes to osteoclast‐like cells in vitro on this substrate, similar to the natural pathway. Biocompatibility was demonstrated in vivo by implantation of the bisque ceramic into cortical rabbit femur defects, followed by histological analysis, where new bone formation inside the channel‐like pores and generation of an osteon‐like tissue morphology was observed. Copyright

Response of human bone marrow stromal cells to a novel ultra-fine-grained and dispersion-strengthened titanium-based material

A novel titanium-based material, containing no toxic or expensive alloying elements, was compared to the established biomaterials: commercially pure titanium (c.p.Ti) and Ti6Al4V. This material (Ti/1.3HMDS) featured similar hardness, yield strength and better wear resistance than Ti6Al4V, as well as better electrochemical properties at physiological pH7.4 than c.p.Ti grade 1 of our study. These excellent properties were obtained by utilizing an alternative mechanism to produce a microstructure of very fine titanium silicides and carbides (<100 nm) embedded in an ultra-fine-grained Ti matrix (365 nm). The grain refinement was achieved by high-energy ball milling of Ti powder with 1.3 wt.% of hexamethyldisilane (HMDS). The powder was consolidated by spark plasma sintering at moderate temperatures of 700 degrees C. The microstructure was investigated by optical and scanning electron microscopy (SEM) and correlated to the mechanical properties. Fluorescence microscopy revealed good adhesion of human mesenchymal stem cells on Ti/1.3HMDS comparable to that on c.p.Ti or Ti6Al4V. Biochemical analysis of lactate dehydrogenase and specific alkaline phosphatase activities of osteogenically induced hMSC exhibited equal proliferation and differentiation rates in all three cases. Thus the new material Ti/1.3HMDS represents a promising alternative to the comparatively weak c.p.Ti and toxic elements containing Ti6Al4V.

Mineralized scaffolds for hard tissue engineering by ionotropic gelation of alginate

Alginates form gels with tube-like pores when covered with solutions of di- or trivalent cations. This phenomenon also referred to as ionotropic gelation has been known for more than 30 years. By mixing a calcium phosphate powder and an alginate as the starting material, the mineral phase of bone is incorporated. Such porous structures can be used for scaffolds in hard tissue engineering. The starting materials and stabilizing additives are dispersed in an aqueous solution. Then a solution of Ca-ions is deposited onto the surface of the slurry. The slurry can be gelled by ion exchange of Na-ions in the alginate with Ca-ions. A primary thin gel layer with the function of a membrane is immediately formed. By diffusional control of cation transport through the membrane, the slurry gradually transforms to the gel forming tube-like pores in direction of cation diffusion. Like the gelation of pure alginate the concentration of electrolyte and the kind of cations and anions influence the size (diameter and length) and size growth of the pores, but the tolerance in the preparation conditions is much smaller. The diameters of the pores can be adjusted between 50 and 500 m which fits the optimum size for cell seeding and blood capillary ingrowth very well. By selecting the proper drying method the inherent shrinkage can be controlled. Hydroxyapatite sintered at high temperatures loses the ability to be resorbed by osteoclasts in vivo. Therefore, we have developed scaffolds with channel-like pores from alginate/calcium phosphate composites without the necessity for heating them to higher temperatures.

Swelling and mechanical properties of alginate hydrogels with respect to promotion of neural growth.

Soft alginate hydrogels support robust neurite outgrowth, but their rapid disintegration in solutions of high ionic strength restricts them from long-term in vivo applications. Aiming to enhance the mechanical stability of soft alginate hydrogels, we investigated how changes in pH and ionic strength during gelation influence the swelling, stiffness, and disintegration of a three-dimensional (3D) alginate matrix and its ability to support neurite outgrowth. Hydrogels were generated from dry alginate layers through ionic crosslinks with Ca(2+) (≤ 10 mM) in solutions of low or high ionic strength and at pH 5.5 or 7.4. High- and low-viscosity alginates with different molecular compositions demonstrated pH and ionic strength-independent increases in hydrogel volume with decreases in Ca(2+) concentrations from 10 to 2 mM. Only soft hydrogels that were synthesized in the presence of 150 mM of NaCl (Ca-alginate NaCl) displayed long-term volume stability in buffered physiological saline, whereas analogous hydrogels generated in NaCl-free conditions (Ca-alginate) collapsed. The stiffnesses of Ca-alginate NaCl hydrogels elevated from 0.01 to 19 kPa as the Ca(2+)-concentration was raised from 2 to 10 mM; however, only Ca-alginate NaCl hydrogels with an elastic modulus ≤ 1.5 kPa that were generated with ≤ 4 mM of Ca(2+) supported robust neurite outgrowth in primary neuronal cultures. In conclusion, soft Ca-alginate NaCl hydrogels combine mechanical stability in solutions of high ionic strength with the ability to support neural growth and could be useful as 3D implants for neural regeneration in vivo.

Cell-laden biphasic scaffolds with anisotropic structure for the regeneration of osteochondral tissue

Sufficient treatment of chondral and osteochondral defects to restore function of the respective tissue remains challenging in regenerative medicine. Biphasic scaffolds that mimic properties of bone and cartilage are appropriate to regenerate both tissues at the same time. The present study describes the development of biphasic, but monolithic scaffolds based on alginate, which are suitable for embedding of living cells in the chondral part. Scaffolds are fabricated under sterile and cell‐compatible conditions according to the principle of diffusion‐controlled, directed ionotropic gelation, which leads to the formation of channel‐like, parallel aligned pores, running through the whole length of the biphasic constructs. The synthesis process leads to an anisotropic structure, as it is found in many natural tissues. The two different layers of the scaffolds are characterized by different microstructure and mechanical properties which provide a suitable environment for cells to form the respective tissue. Human chondrocytes and human mesenchymal stem cells were embedded within the chondral layer of the biphasic scaffolds during hydrogel formation and their chondrogenic (re)differentiation was successfully induced. Whereas viability of non‐induced human mesenchymal stem cells decreased during culture, cell viability of human chondrocytes and chondrogenically induced human mesenchymal stem cells remained high within the scaffolds over the whole culture period of 3 weeks, demonstrating successful fabrication of cell‐laden centimetre‐scaled constructs for potential application in regenerative treatment of osteochondral defects. Copyright

Novel Biomaterials with Parallel Aligned Pore Channels by Directed Ionotropic Gelation of Alginate: Mimicking the Anisotropic Structure of Bone Tissue

Regenerative medicine intends to restore lost functionality by healing tissues defects. For this novel types of biodegradable implants have to be used that first foster healing and later take part in the natural remodelling cycle of the body. In this way, patient’s cells can reconstruct and adapt the tissue according to the local situation and needs. Ideally, the implant should mimic the desired tissue. That means that the biomaterial should resemble the extracellular matrix (ECM) which is expressed by specific cells and acts as the biological scaffold of living tissues. The closer an artificial scaffold material mimics the pattern the easier it can be involved in the natural healing and remodelling processes, which is why more and more researchers try to establish biomimetic approaches for the development of tissue engineering scaffolds. Biological materials are seldom isotropic and for many tissue engineering applications distinct anisotropic materials are needed. E. g. compact bone exhibits a honeycomb-like structure with overlapping, cylindrical units (osteons) with the so-called Haversian canal in the centre. Scaffolds with parallel aligned pores, mimicking the osteon structure of compact bone can be synthesised by directed ionotropic gelation of the naturally occurring polysaccharide alginate. The parallel channels are formed via a sol-gel-process when dior multivalent cations diffuse into the sol in broad front, forming an alginate hydrogel. The pore size and pore alignment of such gels is influenced by the starting materials (e.g. concentrations, additives like powders or polymers) and the preparation process (e.g. temperature, drying process). The phenomenon was discovered already in the 50th of the last century but the biomedical potential of alginate scaffolds with parallel aligned pores structured by ionotropic gelation has been explored for osteoblasts, stem cell based tissue engineering, axon guiding or co-culture of vascular and muscle cells only in the past few years.

Comparative Biomechanical and Microstructural Analysis of Native versus Peracetic Acid-Ethanol Treated Cancellous Bone Graft

Bone transplantation is frequently used for the treatment of large osseous defects. The availability of autologous bone grafts as the current biological gold standard is limited and there is a risk of donor site morbidity. Allogenic bone grafts are an appealing alternative, but disinfection should be considered to reduce transmission of infection disorders. Peracetic acid-ethanol (PE) treatment has been proven reliable and effective for disinfection of human bone allografts. The purpose of this study was to evaluate the effects of PE treatment on the biomechanical properties and microstructure of cancellous bone grafts (CBG). Forty-eight human CBG cylinders were either treated by PE or frozen at −20°C and subjected to compression testing and histological and scanning electron microscopy (SEM) analysis. The levels of compressive strength, stiffness (Youngs modulus), and fracture energy were significantly decreased upon PE treatment by 54%, 59%, and 36%, respectively. Furthermore, PE-treated CBG demonstrated a 42% increase in ultimate strain. SEM revealed a modified microstructure of CBG with an exposed collagen fiber network after PE treatment. We conclude that the observed reduced compressive strength and reduced stiffness may be beneficial during tissue remodeling thereby explaining the excellent clinical performance of PE-treated CBG.