Papers by Author: John A. Jansen

Abstract: The possibility to develop a bone implant with bioactive aspects and in situ drug-delivery properties, in order to provide local treatment in vivo, is a big challenge. Where conventional surface modifications for bone implants focused on the deposition of ceramic (mostly calcium phosphate, CaP) coatings, current surface engineering approaches attempt to incorporate active features to render bone implant surfaces capable to direct biological performance. Biomimetic apatite nanocrystals (nAp) represent, among the CaPs, an elective material for bone applications and their surface functionalization with drugs allows them to act as a drug-delivery vehicle. Since load-bearing bone implants are increasingly used in patients with compromised health conditions, surface engineering is important to warrant the performance of these implants under such conditions. In view of this, bisphosphonates (BPs) represent a treatment modality for a variety of disorders of bone metabolism associated to bone loss, including Paget's bone disease, osteoporosis, fibrous dysplasia and bone metastases. In this work, we have synthesized and characterized bioinspired nAp and evaluated their functionalization with alendronate. In vitro tests will be used to evaluate the efficacy of the functionalized compound to impede the formation of osteoclasts and to show that alendronate-functionalized nAp can significantly reduce osteoclasteogenesis. Finally, alendronate-functionalized nAp (FnAp) has been deposited on titanium implants via the electrospray deposition technique in order to develop inorganic-organic coatings for bone implants with improved functionality.

Abstract: Periodontitis is one of the most common inflammatory diseases, which can lead to early tooth loss. The conventional treatment of periodontitis is to arrest the disease progression. Most reconstructive procedures involve application of bone substitutes, barrier membranes or a combination of both into the bony defects. Calcium phosphate cements (CPCs) are the predominant type of bone substitute material used for reasons of injectability and hence perfect filling potential for bone defects. Recently, injectable apatitic CPCs demonstrated to be more rapidly degradable when combined with poly (lactic-co-glycolic) acid (PLGA) microspheres. Further, PLGA microspheres can be used as a delivery vehicle for growth factors. In this study, the performance of injectable CPCs as a bone substitute material for alveolar bone defects created in Beagle dogs was evaluated. Four CPC-formulations were generated by incorporating hollow or dense PLGA microspheres, either or not loaded with the growth factors (platelet derived growth factor (PDGF) and insulin-like growth factor (IGF). Implantation period was 8 weeks. Bone formation was based on histological and histomorphometrical evaluation. The results demonstrated that filling alveolar bone defects with CPC-dense PLGA revealed significant more bone formation compared to CPC-hollow PLGA either or not loaded with IGF and PDGF. In summary, we conclude that injectable CPC-dense PLGA composites proved to be the most suitable material for a potential use as off the shelf material due to its good biocompatibility, enhanced degradability and subsequent bone formation.

Abstract: Calcium phosphate cements (CPCs) can be a suitable scaffold material for bone tissue engineering because of their osteoconductivity and perfect fit with the surrounding tissue when injected in situ. However, the main disadvantage of hydroxyapatite (HA) forming CPC is its slow degradation rate, which hinders complete bone regeneration. A new approach is to use hydraulic apatite cement with mainly α/β-tricalciumphosphate (TCP) instead of α-TCP. After hydrolysis the α/β-TCP transforms in a partially non-absorbable HA and a completely resorbable β-TCP phase. Therefore, α-TCP material was thermally treated at several temperatures and times resulting in different α/β-TCP ratios. In this experiment, we developed and evaluated injectable biphasic calcium phosphate cements (BCPC) in vitro. Biphasic α/β-TCP powder was produced by heating α-TCP ranging from 1000-11250°C. Setting time and compressive strength of the CPCs were analyzed after soaking in PBS for 6 weeks. Results demonstrated that the phase composition can be controlled by the sintering temperature. Heat treatment of α-TCP, resulted in 100%, 75% and 25% of α-to β-TCP transformation, respectively. Incorporation of these sintered BCP powder into the cement formulation increased the setting time of the CPC paste. Compressive strength decreased with increasing β-TCP content. In this study, biphasic CPCs were produced and characterized in vitro. This injectable biphasic CPC presented comparable properties to an apatitic CPC.

Abstract: The aim of this study was to evaluate potential effects of DNA-coatings on calcium phosphate (CaP) nucleation from simulated body fluids (SBF) and subsequently the effects of DNA-coatings and SBF-immersed DNA coatings on the behavior of osteoblast-like cells. DNAcoatings demonstrated to enhance the nucleation and deposition of CaP from SBF compared to titanium controls. The behavior of osteoblast-like cells was affected on SBF-immersed DNAcoatings, showing an increased deposition of the extracellular matrix protein osteocalcin compared to titanium controls. These results indicate bone-bonding capacity of DNA-coatings, which needs to be confirmed using future animal experiments.

Abstract: Protein thin films were prepared by electrostatic spray deposition (ESD) from aqueous solutions of the enzyme alkaline phosphatase (ALP). ALP thin films with preserved functional properties were successfully deposited using the ESD technique. The biological activity of the deposited ALP films was investigated in vitro by immersing the ESD-coated titanium substrates in both simulated body fluid (SBF) and cell-culture medium. Mineral deposition occurred on substrates immersed in both SBF and medium. Various analytical techniques (SEM,XRD,FTIR,EDS) showed that the calcium phosphate layers deposited in SBF and medium differ in both crystallinity and morphology. The results demonstrated that ESD is a successful method for the deposition of biomedical coatings with enhanced functionality.

Abstract: Composites of gelatin microspheres and injectable calcium phosphate cement were prepared to increase cement resorption and improve tissue ingrowth. To further enhance these properties, osteoinductive growth factors can be introduced into the microspheres. In this study, the in vitro release of preset gelatin microsphere/CaP composites was followed for 6 weeks by use of 125I-labelled rhBMP-2, rhTGF-β and rh-bFGF. Results for all gelatin microsphere composites showed a release curve that consisted of a small burst, followed by a sustained release. The magnitude of the sustained release was dependent on the growth factor used, and showed a slight dependency on the loading method and type of gelatin. Furthermore, no differences in release pattern or efficiency were found when growth factor concentration increased.

Abstract: The RF magnetron sputter technique was used to deposit Bioglass (BG) and hydroxyapatite (HA) coatings onto titanium substrates. The aim of this study was evaluated the growth behavior of rat bone marrow cells of various deposited coatings. The EDS measurements demonstrated that the composition BG coating was changed during magnetron sputtering. The rat bone marrow derived osteoblast-like cells showed improved osteogenic response on crystalline magnetron sputtered HA coatings compared BG coatings. Scanning electron microscopical examination showed an extensive mineralization after 16 days of culture, while on the surface of the BG coating only a multilayer without mineralization could be observed.

Abstract: The aim of this study is to explore the effects of quenching and milling processing on setting property of calcium phosphate cement (CPC). For this purpose, non-quenched α-tricalcium phosphate (α-TCP) and quenched α-TCP were synthesized and their corresponding cement systems were prepared. The particle size of α-TCP powder was introduced as a variable. Then, setting properties of these CPC systems were estimated. By a comparison between non-quenched CPC and the quenched one, it is found that milling processing mainly influences the initial setting stage by decreasing reactant particle size whereas the quenching treatment affects the final setting stage by changing α-TCP content, which supports that CPC setting initially depends on the surface area of reactants and subsequently on the diffusion through the hydrated layer formed around the reactants.

Abstract: The aim of this study was to evaluate the osteogenic properties of magnetron sputtered dicalcium pyrophosphate (DCPP) and hydroxylapatite (HA) coatings. Therefore, DCPP and HA coatings were deposited on grit-blasted titanium discs. The substrates were used as-prepared or received an additional heat treatment with changed the amorphous coating structure to a crystalline structure. Subsequently, rat bone marrow stromal cells were cultured for 1-24 days on the various substrates. DNA and alkaline phosphatase activity was determined after 1, 3, 5, 8 and 12 days of incubation. Osteocalcin expression was evaluated after 8, 12, 16 and 24 days of incubation. Scanning electron microscopical analysis of cell morphology and coating characteristics was done after 8 and 16 days of incubation. All assays were done in duplicate and in each assay all specimens were present in fourfold. Results demonstrated that the cells did not proliferate and differentiate on all amorphous coatings. SEM revealed that the amorphous coatings showed significant dissolution. On the crystalline DCPP and HA coatings an increase in DNA and alkaline phosphatase activity was seen starting at day 8 of incubation. Osteocalcin expression on the crystalline coatings started to increase at day 16 of incubation. SEM showed that the growth and differentiation of the cells was associated with extensive collage fiber formation and surface mineralization in the form of globular accretions. Further, statistical testing revealed that proliferation and differentiation of the rat bone marrow stromal cells started significantly earlier on the crystalline HA coatings than on the crystalline DCPP coatings. These results demonstrate that the rat bone marrow stromal cells proliferated and differentiated only on crystalline magnetron sputtered DCPP as well as HA coatings, which warrants the further in vivo analysis of the bone healing supporting properties of these coatings.

Abstract: In situ measurements of electrospray droplet sizes and velocities were performed by Phase Doppler Anemometry during Electrostatic Spray Deposition (ESD) of calcium phosphate (CaP) coatings. Numerous processing parameters were varied (nozzle-to-substrate distance, deposition temperature, nozzle geometry, and composition of the precursor solution), whereafter the morphological characteristics of these ESD-derived CaP coatings were correlated with measured droplet characteristics. Equal droplet sizes and velocities were measured for nozzle-to-substrate distances up to 40 mm and deposition temperatures up to 400 °C, indicating that electrospray droplets did not shrink at all during droplet flight using an involatile solvent butyl carbitol with a high boiling point (Tb = 231 °C). Nevertheless, coatings with considerably different surface morphologies were obtained under these conditions, varying from microporous structures with coalesced pore walls to morphologies revealing isolated rings on top of dense or grainy underlayers. The chemical composition of the precursor solutions and the mixing characteristics of the calcium and phosphate precursor components strongly influenced the initial droplet sizes, precipitation kinetics of the CaP solute, and subsequent coating morphology. Unique, reticular coating morphologies were deposited at a deposition rate of 3.2 µm/hour, which have a graded structure consisting of a dense underlayer, a submicron-porous intermediate layer, and a roughened toplayer revealing droplet-derived features such as isolated rings or coalesced, hollow surface pits.