Elke Vorndran

Angiogenesis in Calcium Phosphate Scaffolds by Inorganic Copper Ion Release

Angiogenesis in a tissue-engineered device may be induced by incorporating growth factors (e.g., vascular endothelial growth factor [VEGF]), genetically modified cells, and=or vascular cells. It represents an important process during the formation and repair of tissue and is essential for nourishment and supply of reparative and immunological cells. Inorganic angiogenic factors, such as copper ions, are therefore of interest in the fields of regenerative medicine and tissue engineering due to their low cost, higher stability, and potentially greater safety compared with recombinant proteins or genetic engineering approaches. The purpose of this study was to compare tissue responses to 3D printed macroporous bioceramic scaffolds implanted in mice that had been loaded with either VEGF or copper sulfate. These factors were spatially localized at the end of a single macropore some 7 mm from the surface of the scaffold. Controls without angiogenic factors exhibited only poor tissue growth within the blocks; in contrast, low doses of copper sulfate led to the formation of microvessels oriented along the macropore axis. Further, wound tissue ingrowth was particularly sensitive to the quantity of copper sulfate and was enhanced at specific concentrations or in combination with VEGF. The potential to accelerate and guide angiogenesis and wound healing by copper ion release without the expense of inductive protein(s) is highly attractive in the area of tissue-engineered bone and offers significant future potential in the field of regenerative biomaterials.

Fabrication of computationally designed scaffolds by low temperature 3D printing

The development of artificial bone substitutes that mimic the properties of bone and simultaneously promote the desired tissue regeneration is a current issue in bone tissue engineering research. An approach to create scaffolds with such characteristics is based on the combination of novel design and additive manufacturing processes. The objective of this work is to characterize the microstructural and the mechanical properties of scaffolds developed by coupling both topology optimization and a low temperature 3D printing process. The scaffold design was obtained using a topology optimization approach to maximize the permeability with constraints on the mechanical properties. This procedure was studied to be suitable for the fabrication of a cage prototype for tibial tuberosity advancement application, which is one of the most recent and promising techniques to treat cruciate ligament rupture in dogs. The microstructural and mechanical properties of the scaffolds manufactured by reacting α/β-tricalcium phosphate with diluted phosphoric acid were then assessed experimentally and the scaffolds strength reliability was determined. The results demonstrate that the low temperature 3D printing process is a reliable option to create synthetic scaffolds with tailored properties, and when coupled with topology optimization design it can be a powerful tool for the fabrication of patient-specific bone implants.

Modeling vancomycin release kinetics from microporous calcium phosphate ceramics comparing static and dynamic immersion conditions.

The release kinetics of vancomycin from calcium phosphate dihydrate (brushite) matrices and polymer/brushite composites were compared using different fluid replacement regimes, a regular replacement (static conditions) and a continuous flow technique (dynamic conditions). The use of a constantly refreshed flowing resulted in a faster drug release due to a constantly high diffusion gradient between drug loaded matrix and the eluting medium. Drug release was modeled using the Weibull, Peppas and Higuchi equations. The results showed that drug liberation was diffusion controlled for the ceramics matrices, whereas ceramics/polymer composites led to a mixed diffusion and degradation controlled release mechanism. The continuous flow technique was for these materials responsible for a faster release due to an accelerated polymer degradation rate compared with the regular fluid replacement technique.

Ready-to-use injectable calcium phosphate bone cement paste as drug carrier

Current developments in calcium phosphate cement (CPC) technology concern the use of ready-to-use injectable cement pastes by dispersing the cement powder in a water-miscible solvent, such that, after injection into the physiological environment, setting of cements occurs by diffusion of water into the cement paste. It has also been demonstrated recently that the combination of a water-immiscible carrier liquid combined with suitable surfactants facilitates a discontinuous liquid exchange in CPC, enabling the cement setting reaction to take place. This paper reports on the use of these novel cement paste formulations as a controlled release system of antibiotics (gentamicin, vancomycin). Cement pastes were applied either as a one-component material, in which the solid drugs were physically dispersed, or as a two-component system, where the drugs were dissolved in an aqueous phase that was homogeneously mixed with the cement paste using a static mixing device during injection. Drug release profiles of both antibiotics from pre-mixed one- and two-component cements were characterized by an initial burst release of ∼7-28%, followed by a typical square root of time release kinetic for vancomycin. Gentamicin release rates also decreased during the first days of the release study, but after ∼1 week, the release rates were more or less constant over a period of several weeks. This anomalous release kinetic was attributed to participation of the sulfate counter ion in the cement setting reaction altering the drug solubility. The drug-loaded cement pastes showed high antimicrobial potency against Staphylococcus aureus in an agar diffusion test regime, while other cement properties such as mechanical performance or phase composition after setting were only marginally affected.

The effect of Cu(II)-loaded brushite scaffolds on growth and activity of osteoblastic cells

Bone substitute materials such as calcium phosphate cements (CPC) are frequently used as growth factor carriers for the stimulation of osteoblast-formation around an implant. However, biological modification based on delicate protein factors like extracellular matrix proteins or growth factors is subject to a number of shortcomings like the need for storage below room temperature and cost of production. The aim of this study was to investigate ionic modification as an alternative bioinorganic route for implant modification. Although it is known that Cu(II) plays a role in angiogenesis and bone formation, not all involved processes are well understood yet. In this study the in vitro effect of Cu(II) on growth and activity of osteoblastic cells seeded on brushite (CaHPO(4) · 2 H(2) O) scaffolds as well as on glass discs was investigated. The results show that Cu(II) enhances cell activity and proliferation of osteoblastic cells on CPC and furthermore affects the expression of several bone specific proteins such as bone sialo protein or osteocalcin. Therefore, the modification of CPC with Cu(II) may offer a promising alternative to protein based modification to stimulate cellular activity for an improved bone healing.

Application of a 3D printed customized implant for canine cruciate ligament treatment by tibial tuberosity advancement.

Fabrication of customized implants based on patient bone defect characteristics is required for successful clinical application of bone tissue engineering. Recently a new surgical procedure, tibial tuberosity advancement (TTA), has been used to treat cranial cruciate ligament (CrCL) deficient stifle joints in dogs, which involves an osteotomy and the use of substitutes to restore the bone. However, limitations in the use of non-biodegradable implants have been reported. To overcome these limitations, this study presents the development of a bioceramic customized cage to treat a large domestic dog assigned for TTA treatment. A cage was designed using a suitable topology optimization methodology in order to maximize its permeability whilst maintaining the structural integrity, and was manufactured using low temperature 3D printing and implanted in a dog. The cage material and structure was adequately characterized prior to implantation and the in vivo response was carefully monitored regarding the biological response and patient limb function. The manufacturing process resulted in a cage composed of brushite, monetite and tricalcium phosphate, and a highly permeable porous morphology. An overall porosity of 59.2% was achieved by the combination of a microporosity of approximately 40% and a designed interconnected macropore network with pore sizes of 845 μm. The mechanical properties were in the range of the trabecular bone although limitations in the cages reliability and capacity to absorb energy were identified. The dogs limb function was completely restored without patient lameness or any adverse complications and also the local biocompatibility and osteoconductivity were improved. Based on these observations it was possible to conclude that the successful design, fabrication and application of a customized cage for a dog CrCL treatment using a modified TTA technique is a promising method for the future fabrication of patient-specific bone implants, although clinical trials are required.

The effect of porosity on drug release kinetics from vancomycin microsphere/calcium phosphate cement composites

The influence of porosity on release profiles of antibiotics from calcium phosphate composites was investigated to optimize the duration of treatment. We hypothesized, that by the encapsulation of vancomycin-HCl into biodegradable microspheres prior admixing to calcium phosphate bone cement, the influence of porosity of the cement matrix on vancomycin release could be reduced. Encapsulation of vancomycin into a biodegradable poly(lactic co-glycolic acid) copolymer (PLGA) was performed by spray drying; drug-loaded microparticles were added to calcium phosphate cement (CPC) at different powder to liquid ratios (P/L), resulting in different porosities of the cement composites. The effect of differences in P/L ratio on drug release kinetics was compared for both the direct addition of vancomycin-HCl to the cement liquid and for cement composites modified with vancomycin-HCl-loaded microspheres. Scanning electron microscopy (SEM) was used to visualize surface and cross section morphology of the different composites. Brunauer, Emmett, and Teller-plots (BET) was used to determine the specific surface area and pore size distribution of these matrices. It could be clearly shown, that variations in P/L ratio influenced both the porosity of cement and vancomycin release profiles. Antibiotic activity during release study was successfully measured using an agar diffusion assay. However, vancomycin-HCl encapsulation into PLGA polymer microspheres decreased porosity influence of cement on drug release while maintaining antibiotic activity of the embedded substance.

Mechanical Properties and Drug Release Behavior of Bioactivated PMMA Cements

Septic loosening of cemented implants represents an unresolved long-term problem of total hip endoprostheses. Common treatments of infected prostheses involve the use of temporary antibiotic-loaded PMMA spacer-implants or antibiotic-loaded cements. The latter are either provided by a manufacturer or are obtained by simply mixing specific antibiotic powders according to a microbial sensitivity test with PMMA cement. This study is aimed to investigate the antibiotic release behavior and mechanical properties of novel modified PMMA cements, which were bioactivated by chemical modification of commercial cements with either 0.5% hydroxyethylmethacrylate-phosphate (HEMA-P) or 0.5% hydroxyethylmethacrylate-phosphate + calcium chloride and sodium carbonate as buffer. Tobramycin release experiments from the cements were performed statically by immersion of the drug-loaded samples in PBS buffer following liquid change after different periods of time or during cyclic mechanical loading of the cement samples. Cement modification did not significantly alter the mechanical properties of the cements, but affected the release rate from the matrix. While the unmodified cement released approximately 0.33 mg/cm2 tobramycin after 48 h independent of the testing regime, modification with both HEMA-P and salt buffer increased the antibiotic release to 37–50 mg/cm2 when tested under cyclical mechanical loading.

Comparing physicochemical properties of printed and hand cast biocements designed for ligament replacement

Abstract Abstract In order to combat the low regenerative capabilities of ligaments, full ‘bone to bone’ replacements are required, which will integrate with bone while providing a smooth transition to the replacement soft tissue (tissues surrounding organs in the body, not being bone). This study investigated the use of three-dimensional powder printing technology to form calcium phosphate brackets, previously used for forming bespoke scaffold geometries, to 95±0·1% accuracy of their original computer aided design. The surface and internal structures of the printed samples were characterised both chemically and morphologically and compared with hand moulded cements in the dry state and after 3 days of immersion in phosphate buffered saline. X-ray diffraction, Raman spectroscopy and SEM all showed the presence of brushite in the hand moulded samples and brushite and monetite within the printed samples. Furthermore, the printed structures have a higher level of porosity in the dry state in comparison to the hand moulded samples (36±2·2% compared to 24±0·7%) despite exhibiting a compressive strength of almost double the hand cast material. Although the compressive strength of the printed cements decreases after the 3 day immersion, there was no significant difference between the printed and hand moulded cements under the same conditions. Three-dimensional powder printing technology has enabled the manufacture of bespoke calcium phosphate brackets with properties similar to those reported for hand moulded cements.

Structural changes to resorbable calcium phosphate bioceramic aged in vitro.

This work investigates the effect of mammalian cell culture conditions on 3D printed calcium phosphate scaffolds. The purpose of the studies presented was to characterise the changes in scaffold properties in physiologically relevant conditions. Differences in crystal morphologies were observed between foetal bovine serum-supplemented media and their unsupplemented analogues, but not for supplemented media containing tenocytes. Scaffold porosity was found to increase for all conditions studied, except for tenocyte-seeded scaffolds. The presence of tenocytes on the scaffold surface inhibited any increase in scaffold porosity in the presence of extracellular matrix secreted by the tenocytes. For acellular conditions the presence or absence of sera proteins strongly affected the rate of dissolution and the distribution of pore diameters within the scaffold. Exposure to high sera protein concentrations led to the development of significant numbers of sub-micron pores, which was otherwise not observed. The implication of these results for cell culture research employing calcium phosphate scaffolds is discussed.