Zeinab Tahmasebi Birgani

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.

Biological evaluation of porous aliphatic polyurethane/hydroxyapatite composite scaffolds for bone tissue engineering.

Biomaterial scaffolds meant to function as supporting structures to osteogenic cells play a pivotal role in bone tissue engineering. Recently, we synthesized an aliphatic polyurethane (PU) scaffold via a foaming method using non-toxic components. Through this procedure a uniform interconnected porous structure was created. Furthermore, hydroxyapatite (HA) particles were introduced into this process to increase the bioactivity of the PU matrix. To evaluate the biological performances of these PU-based scaffolds, their influence on in vitro cellular behavior and in vivo bone forming capacity of the engineered cell-scaffold constructs was investigated in this study. A simulated body fluid test demonstrated that the incorporation of 40 wt % HA particles significantly promoted the biomineralization ability of the PU scaffolds. Enhanced in vitro proliferation and osteogenic differentiation of the seeded mesenchymal stem cells were also observed on the PU/HA composite. Next, the cell-scaffold constructs were implanted subcutaneously in a nude mice model. After 8 weeks, a considerable amount of vascularized bone tissue with initial marrow stroma development was generated in both PU and PU/HA40 scaffold. In conclusion, the PU/HA composite is a potential scaffold for bone regeneration applications.

Substrate geometry directs the in vitro mineralization of calcium phosphate ceramics

Repetitive concavities on the surface of bone implants have recently been demonstrated to foster bone formation when implanted at ectopic locations in vivo. The current study aimed to evaluate the effect of surface concavities on the surface mineralization of hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) ceramics in vitro. Hemispherical concavities with different diameters were prepared at the surface of HA and β-TCP sintered disks: 1.8mm (large concavity), 0.8mm (medium concavity) and 0.4mm (small concavity). HA and β-TCP disks were sintered at 1100 or 1200°C and soaked in simulated body fluid for 28 days at 37°C; the mineralization process was followed by scanning electron microscopy, energy-dispersive spectroscopy, X-ray diffraction and calcium quantification analyses. The results showed that massive mineralization occurred exclusively at the surface of HA disks treated at 1200°C and that nucleation of large aggregates of calcium phosphate started specifically inside small concavities instead of on the planar surface of the disks. Regarding the effect of concavity diameter size on surface mineralization, it was observed that small concavities induce 124- and 10-fold increased mineralization compared to concavities of large or medium size, respectively. The results of this study demonstrated that (i) in vitro surface mineralization of calcium phosphate ceramics with surface concavities starts preferentially within the concavities and not on the planar surface, and (ii) concavity size is an effective parameter to control the spatial position and extent of mineralization in vitro.

A theranostic agent to enhance osteogenic and magnetic resonance imaging properties of calcium phosphate cements.

With biomimetic biomaterials, like calcium phosphate cements (CPCs), non-invasive assessment of tissue regeneration is challenging. This study describes a theranostic agent (TA) to simultaneously enhance both imaging and osteogenic properties of such a bone substitute material. For this purpose, mesoporous silica beads were produced containing an iron oxide core to enhance bone magnetic resonance (MR) contrast. The same beads were functionalized with silane linkers to immobilize the osteoinductive protein BMP-2, and finally received a calcium phosphate coating, before being embedded in the CPC. Both in vitro and in vivo tests were performed. In vitro testing showed that the TA beads did not interfere with essential material properties like cement setting. Furthermore, bioactive BMP-2 could be efficiently released from the carrier-beads. In vivo testing in a femoral condyle defect rat model showed long-term MR contrast enhancement, as well as improved osteogenic capacity. Moreover, the TA was released during CPC degradation and was not incorporated into the newly formed bone. In conclusion, the described TA was shown to be suitable for longitudinal material degradation and bone healing studies.

Subcutaneous tissue response and osteogenic performance of calcium phosphate nanoparticle-enriched hydrogels in the tibial medullary cavity of guinea pigs

In the current study, oligo(poly(ethylene glycol) fumarate) (OPF)-based hydrogels were tested for the first time as injectable bone substitute materials. The primary feature of the material design was the incorporation of calcium phosphate (CaP) nanoparticles within the polymeric matrix in order to compare the soft tissue response and bone-forming capacity of plain OPF hydrogels with CaP-enriched OPF hydrogel composites. To that end, pre-set scaffolds were implanted subcutaneously, whereas flowable polymeric precursor solutions were injected in a tibial ablation model in guinea pigs. After 8 weeks of implantation, histological and histomorphometrical evaluation of the subcutaneous scaffolds confirmed the biocompatibility of both types of hydrogels. Nevertheless, OPF hydrogels presented a loose structure, massive cellular infiltration and extensive material degradation compared to OPF-CaP hydrogels that were more compact. Microcomputed tomography and histological and histomorphometrical analyses showed comparable amounts of new trabecular bone in all tibias and some material remnants in the medial and distal regions. Particularly, highly calcified areas were observed in the distal region of OPF-CaP-treated tibias, which indicate a heterogeneous distribution of the mineral phase throughout the hydrogel matrix. This phenomenon can be attributed to either hindered gelation under highly perfused in vivo conditions or a faster degradation rate of the polymeric hydrogel matrix compared to the nanostructured mineral phase, resulting in loss of entrapment of the CaP nanoparticles and subsequent sedimentation.

Enzymatic pH control for biomimetic deposition of calcium phosphate coatings

The current study examines the enzymatic decomposition of urea into carbon dioxide and ammonia as a means to increase the pH during biomimetic deposition of calcium phosphate (CaP) onto implant surfaces. The kinetics of the enzymatically induced pH increase were studied by monitoring pH, calcium concentration and conductivity of the aqueous solutions as a function of time, urease concentration and initial concentrations of calcium and phosphate ions. Cryogenic transmission electron microscopy was used to study the process of homogeneous CaP precipitation in solution, whereas CaP deposition on conventional acid-etched titanium and micropatterned polystyrene (PS) surfaces was studied using scanning electron microscopy. The data presented in this study confirm that the substrate-enzyme combination urea-urease offers strong control over the rate of pH increase by varying the concentrations of precursor salts and urease. Formation of biomimetic CaP coatings was shown to proceed via formation of ionic polymeric assemblies of prenucleation complexes. The process of deposition and corresponding coating morphology was strongly dependent on the concentration of calcium, phosphate and urease. Finally, it was shown that the substrate-enzyme combination urea-urease allowed for spatial distribution of CaP crystals along the grooves of micropatterned PS surfaces at low concentrations of calcium, phosphate and urease, stressing the sensitivity of the presented method.

Development of Highly Functional Biomaterials by Decoupling and Recombining Material Properties.

Development of functional biomaterials by a design-driven approach is described, whereby individual properties are first decoupled to investigate their sole effects on a biological process. Following this investigation, they are recombined in such a way that the overall performance and applicability of the biomaterial is improved. This is in contrast to classical, processing-driven biomaterials development where the properties of a material are mainly determined by the possibilities of the technique used to produce it.

Stimulatory effect of cobalt ions incorporated into calcium phosphate coatings on neovascularization in an in vivo intramuscular model in goats

UNLABELLED Rapid vascularization of bone graft substitutes upon implantation is one of the most important challenges to overcome in order to achieve successful regeneration of large, critical-size bone defects. One strategy for stimulating vascularization during the regeneration process is to create a hypoxic microenvironment by either directly lowering the local oxygen tension, or by applying hypoxia-mimicking factors. Cells compensate for the hypoxic condition by releasing angiogenic factors leading to new blood vessel formation. In the present study, we explored the potential of cobalt ions (Co(2+)), known chemical mimickers of hypoxia, to stimulate vascularization within a bone graft substitute in vivo. To this end, Co(2+) ions were incorporated into calcium phosphate (CaPs) coatings deposited on poly(lactic acid) (PLA) particles with their effect on the formation of new blood vessels studied upon intramuscular implantation in goats. PLA particles and CaP-coated particles without Co(2+) ions served as controls. Pathological scoring of the inflammatory response following a 12-week implantation period showed no significant differences between the four types of materials. Based on histological and immunohistochemical analyses, both blood vessel area and number of blood vessels in CaP-coated PLA particles containing Co(2+) were higher than in the uncoated PLA particles and CaP-coated PLA particles without Co(2+). Analysis of blood vessel size distribution indicated abundant formation of small blood vessels in all the samples, while large blood vessels were predominantly found in PLA particles coated with CaP containing Co(2+) ions. The results of this study support the use of CaPs containing Co(2+) ions to enhance vascularization in vivo. STATEMENT OF SIGNIFICANCE In this work, we have investigated the potential of cobalt ions, incorporated into thin calcium phosphate (CaP) coatings that were deposited on particles of poly(lactic acid) (PLA), to induce neovascularization in vivo. Qualitative and quantitative histological and immunohistochemical analyses showed that both the number of blood vessels and the total blood vessel area were higher in CaP-coated PLA particles containing cobalt ions as compared to the uncoated PLA particles and CaP-coated PLA particles without the metallic additive. Furthermore, a wider distribution of blood vessel sizes, varying from very small to large vessels was specifically observed in samples containing cobalt ions. This in vivo study will significantly contribute to the existing knowledge on the use of bioinorganics, which are simple and inexpensive inorganic factors that can be used to control relevant biological process during tissue regeneration, such as vascularization. As such, we are convinced that this manuscript will be of interest to the readers of Acta Biomaterialia.

Combinatorial incorporation of fluoride and cobalt ions into calcium phosphates to stimulate osteogenesis and angiogenesis.

Bone healing requires two critical mechanisms, angiogenesis and osteogenesis. In order to improve bone graft substitutes, both mechanisms should be addressed simultaneously. While the individual effects of various bioinorganics have been studied, an understanding of the combinatorial effects is lacking. Cobalt and fluoride ions, in appropriate concentrations, are known to individually favor the vascularization and mineralization processes, respectively. This study investigated the potential of using a combination of fluoride and cobalt ions to simultaneously promote osteogenesis and angiogenesis in human mesenchymal stromal cells (hMSCs). Using a two-step biomimetic method, wells of tissue culture plates were coated with a calcium phosphate (CaP) layer without or with the incorporation of cobalt, fluoride, or both. In parallel, hMSCs were cultured on uncoated well plates, and cultured with cobalt and/or fluoride ions within the media. The results revealed that cobalt ions increased the expression of angiogenic markers, with the effects being stronger when the ions were added as a dissolved salt in cell medium as compared to incorporation into CaP. Cobalt ions generally suppressed the ALP activity, the expression of osteogenic genes, and the level of mineralization, regardless of delivery method. Fluoride ions, individually or in combination with cobalt, significantly increased the expression of many of the selected osteogenic markers, as well as mineral deposition. This study demonstrates an approach to simultaneously target the two essential mechanisms in bone healing: angiogenesis and osteogenesis. The incorporation of cobalt and fluoride into CaPs is a promising method to improve the biological performance of fully synthetic bone graft substitutes.

Monolithic calcium phosphate/poly(lactic acid) composite versus calcium phosphate-coated poly(lactic acid) for support of osteogenic differentiation of human mesenchymal stromal cells

Calcium phosphates (CaPs), extensively used synthetic bone graft substitutes, are often combined with other materials with the aim to overcome issues related to poor mechanical properties of most CaP ceramics. Thin ceramic coatings on metallic implants and polymer-ceramic composites are examples of such hybrid materials. Both the properties of the CaP used and the method of incorporation into a hybrid structure are determinant for the bioactivity of the final construct. In the present study, a monolithic composite comprising nano-sized CaP and poly(lactic acid) (PLA) and a CaP-coated PLA were comparatively investigated for their ability to support proliferation and osteogenic differentiation of bone marrow-derived human mesenchymal stromal cells (hMSCs). Both, the PLA/CaP composite, produced using physical mixing and extrusion and CaP-coated PLA, resulting from a biomimetic coating process at near-physiological conditions, supported proliferation of hMSCs with highest rates at PLA/CaP composite. Enzymatic alkaline phosphatase activity as well as the mRNA expression of bone morphogenetic protein-2, osteopontin and osteocalcin were higher on the composite and coated polymer as compared to the PLA control, while no significant differences were observed between the two methods of combining CaP and PLA. The results of this study confirmed the importance of CaP in osteogenic differentiation while the exact properties and the method of incorporation into the hybrid material played a less prominent role.