Juergen Groll

Patterned melt electrospun substrates for tissue engineering

Tissue engineering scaffolds can be built with patterning techniques that allow discrete placement of structures. In this study, electrospun fibres are collected in focused spots; the patterning and drawing of a cell adhesive scaffold is shown. Blends of biodegradable poly(ethylene glycol)-block-poly(epsilon-caprolactone) (PEG-b-PCL) and PCL were melt electrospun onto glass collectors, and the optimal electrospinning parameters determined. The quality of the fibre was largely influenced by the flow rate of the melt to the spinneret; however, this can be adjusted with the voltage. A collection distance between 3 cm and 5 cm was optimal, and at 10 cm the fibres became unfocused in their deposition although the diameter remained similar (0.96 +/- 0.19 microm). Aligned lines of electrospun fibres 200-400 microm in width could be applied onto the slide with an x-y stage, continuously and discretely. Lines of electrospun fibres could be applied on top of one another and were very uniform in diameter. Fibroblasts adhered primarily in the fibre region, due to the poor cell adhesion to the PEG substrate. Improvements in depositing hydrophilic electrospun fibres that wet and adhere to in vitro substrates and the use of stage automation for the writing interface could provide scaffold-building devices suitable for tissue engineering applications.

Structure and properties of urea-crosslinked star poly[(ethylene oxide)-ran-(propylene oxide)] hydrogels.

Isocyanate-terminated six armed star shaped macromers with a statistical copolymer backbone consisting of 80% EO and 20% PO have previously demonstrated excellent protein and cell repellence as nano-layered surfaces. In this study, various macromers are mixed with water and provide a spectrum of materials that range from particles to uniform hydrogels. Due to hydrophobic end groups, 3 kDa molecular weight macromers result in micro and nano-particles, while 18 kDa macromers completely dissolve and consequently uniform, transparent, high water content hydrogels are formed. Oriented channels may be induced into these hydrogels through the controlled freezing of water in the preformed hydrogel.

Bone tissue engineering in osteoporosis

Osteoporosis is a polygenetic, environmentally modifiable disease, which precipitates into fragility fractures of vertebrae, hip and radius and also confers a high risk of fractures in accidents and trauma. Aging and the genetic molecular background of osteoporosis cause delayed healing and impair regeneration. The worldwide burden of disease is huge and steadily increasing while the average life expectancy is also on the rise. The clinical need for bone regeneration applications, systemic or in situ guided bone regeneration and bone tissue engineering, will increase and become a challenge for health care systems. Apart from in situ guided tissue regeneration classical ex vivo tissue engineering of bone has not yet reached the level of routine clinical application although a wealth of scaffolds and growth factors has been developed. Engineering of complex bone constructs in vitro requires scaffolds, growth and differentiation factors, precursor cells for angiogenesis and osteogenesis and suitable bioreactors in various combinations. The development of applications for ex vivo tissue engineering of bone faces technical challenges concerning rapid vascularization for the survival of constructs in vivo. Recent new ideas and developments in the fields of bone biology, materials science and bioreactor technology will enable us to develop standard operating procedures for ex vivo tissue engineering of bone in the near future. Once prototyped such applications will rapidly be tailored for compromised conditions like vitamin D and sex hormone deficiencies, cellular deficits and high production of regeneration inhibitors, as they are prevalent in osteoporosis and in higher age.

Strength reliability and in vitro degradation of three-dimensional powder printed strontium-substituted magnesium phosphate scaffolds.

UNLABELLED Strontium ions (Sr(2+)) are known to prevent osteoporosis and also encourage bone formation. Such twin requirements have motivated researchers to develop Sr-substituted biomaterials for orthopaedic applications. The present study demonstrates a new concept of developing Sr-substituted Mg3(PO4)2 - based biodegradable scaffolds. In particular, this work reports the fabrication, mechanical properties with an emphasis on strength reliability as well as in vitro degradation of highly biodegradable strontium-incorporated magnesium phosphate cements. These implantable scaffolds were fabricated using three-dimensional powder printing, followed by high temperature sintering and/or chemical conversion, a technique adaptable to develop patient-specific implants. A moderate combination of strength properties of 36.7MPa (compression), 24.2MPa (bending) and 10.7MPa (tension) were measured. A reasonably modest Weibull modulus of up to 8.8 was recorded after uniaxial compression or diametral tensile tests on 3D printed scaffolds. A comparison among scaffolds with varying compositions or among sintered or chemically hardened scaffolds reveals that the strength reliability is not compromised in Sr-substituted scaffolds compared to baseline Mg3(PO4)2. The micro-computed tomography analysis reveals the presence of highly interconnected porous architecture in three-dimension with lognormal pore size distribution having median in the range of 17.74-26.29μm for the investigated scaffolds. The results of extensive in vitro ion release study revealed passive degradation with a reduced Mg(2+) release and slow but sustained release of Sr(2+) from strontium-substituted magnesium phosphate scaffolds. Taken together, the present study unequivocally illustrates that the newly designed Sr-substituted magnesium phosphate scaffolds with good strength reliability could be used for biomedical applications requiring consistent Sr(2+)- release, while the scaffold degrades in physiological medium. STATEMENT OF SIGNIFICANCE The study investigates the additive manufacturing of scaffolds based on different strontium-substituted magnesium phosphate bone cements by means of three-dimensional powder printing technique (3DPP). Magnesium phosphates were chosen due to their higher biodegradability compared to calcium phosphates, which is due to both a higher solubility as well as the absence of phase changes (to low soluble hydroxyapatite) in vivo. Since strontium ions are known to promote bone formation by stimulating osteoblast growth, we aimed to establish such a highly degradable magnesium phosphate ceramic with an enhanced bioactivity for new bone ingrowth. After post-processing, mechanical strengths of up to 36.7MPa (compression), 24.2MPa (bending) and 10.7MPa (tension) could be achieved. Simultaneously, the failure reliability of those bioceramic implant materials, measured by Weibull modulus calculations, were in the range of 4.3-8.8. Passive dissolution studies in vitro proved an ion release of Mg(2+) and PO4(3-) as well as Sr(2+), which is fundamental for in vivo degradation and a bone growth promoting effect. In our opinion, this work broadens the range of bioceramic bone replacement materials suitable for additive manufacturing processing. The high biodegradability of MPC ceramics together with the anticipated promoting effect on osseointegration opens up the way for a patient-specific treatment with the prospect of a fast and complete healing of bone fractures.

Reinforcement Strategies for Load-Bearing Calcium Phosphate Biocements

Calcium phosphate biocements based on calcium phosphate chemistry are well-established biomaterials for the repair of non-load bearing bone defects due to the brittle nature and low flexural strength of such cements. This article features reinforcement strategies of biocements based on various intrinsic or extrinsic material modifications to improve their strength and toughness. Altering particle size distribution in conjunction with using liquefiers reduces the amount of cement liquid necessary for cement paste preparation. This in turn decreases cement porosity and increases the mechanical performance, but does not change the brittle nature of the cements. The use of fibers may lead to a reinforcement of the matrix with a toughness increase of up to two orders of magnitude, but restricts at the same time cement injection for minimal invasive application techniques. A novel promising approach is the concept of dual-setting cements, in which a second hydrogel phase is simultaneously formed during setting, leading to more ductile cement–hydrogel composites with largely unaffected application properties.

Tailored hyaluronic acid hydrogels through hydrophilic prepolymer cross-linkers

Despite its enormous biological importance, a lack of mechanical stability and fast degradability often hampers more intense application of hyaluronic acid (HA) for biomedical applications. We present a method to prepare hyaluronic acid based hydrogels with tailored stability, degradability and porosity by cross-linking with functional 18 kDa six arm star shaped poly(ethylene oxide-stat-propylene oxide) (sP(EO-stat-PO)) prepoylmers in aqueous solutions. In the case of NCO groups at the distal ends of the arms, cross-linking occurs through reaction with OH groups of the HA resulting in urethane bridges between star molecules and HA. In parallel, slow hydrolysis of isocyanates to amines that subsequently rapidly aminolyze other isocyanate groups and lead to urea bridges between prepolymers occurs as a side reaction. Overall, this process leads to cross-linking within 5 to 15 min depending on the molar ratios of NCO-sP(EO-stat-PO) to HA. A chemically orthogonal way of cross-linking can be achieved with acrylate functional prepolymers and thiolated HA where cross-linking exclusively results from Michael-type addition of the thiol to the acrylate groups. This process also results in gelation times of 5 to 15 min. The two mechanisms are compared and the resulting gels are analyzed regarding chemical composition, equilibrium water content, degree of sol-content, porosity, mechanical stability and resistance towards enzymatic degradation by hyaluronidase.

Ultrathin Coatings with Change in Reactivity over Time Enable Functional In Vitro Networks Of Insect Neurons

Its just not cricket! A novel coating system that enables covalent attachment of biomolecules in a nonfouling environment without use of additional chemical crosslinkers is presented. Concanavalin A is patterned on the coatings to direct cell adhesion and growth of neurons from the cricket Gryllus bimaculatus and generate functional, patterned in vitro insect neuronal networks for the first time.

Nanostructured Ordering of Fluorescent Markers and Single Proteins on Substrates

Highly ordered hexagonal nanopatterns of gold clusters on glass substrates were used as anchoring points for the specifc attachment of fluorescence dyes and proteins labeled with fluorescence dyes. Thiol‐ or disulfide‐containing linker molecules were used for the binding to the gold dots. In order to ensure specific binding on the gold dots only, the surface area in between the dots was protected against unspecific adsorption. For the attachment of polar low‐molecular‐weight fluorescence dyes, an octadecyltrichlorosilane self‐assembled monolayer was prepared on the surface in between the gold dots, whereas a layer prepared from star‐shaped poly(ethylene oxide‐stat‐propylene oxide) prepolymers was used to prevent unspecific adsorption of proteins between the gold dots. Fluorescence microscopy proved the specific binding of the dyes as well as of the proteins. Scanning force microscopy studies show that each gold dot is only capable of binding one protein at a time.

Additive Manufacturing of a Photo-Cross-Linkable Polymer via Direct Melt Electrospinning Writing for Producing High Strength Structures

Melt electrospinning writing (MEW) is an emerging additive manufacturing technique that enables the design and fabrication of micrometer-thin fibrous scaffolds made of biocompatible and biodegradable polymers. By using a computer-aided deposition process, a unique control over pore size and interconnectivity of the resulting scaffolds is achieved, features highly interesting for tissue engineering applications. However, MEW has been mainly used to process low melting point thermoplastics such as poly(ε-caprolactone). Since this polymer exhibits creep and a reduction in modulus upon hydration, we manufactured scaffolds of poly(L-lactide-co-ε-caprolactone-co-acryloyl carbonate) (poly(LLA-ε-CL-AC)), a photo-cross-linkable and biodegradable polymer, for the first time. We show that the stiffness of the scaffolds increases significantly (up to ∼10-fold) after cross-linking by UV irradiation at room temperature, compared with un-cross-linked microfiber scaffolds. The preservation of stiffness and high average fiber modulus (370 ± 166 MPa) within the cross-linked hydrated scaffolds upon repetitive loading (10% strain at 1 Hz up to 200,000 cycles) suggests that the prepared scaffolds may be of potential interest for soft connective tissue engineering applications. Moreover, the approach can be readily adapted through manipulation of polymer properties and scaffold geometry to prepare structures with mechanical properties suitable for other tissue engineering applications.

Controlled intramyocardial release of engineered chemokines by biodegradable hydrogels as a treatment approach of myocardial infarction.

Myocardial infarction (MI) induces a complex inflammatory immune response, followed by the remodelling of the heart muscle and scar formation. The rapid regeneration of the blood vessel network system by the attraction of hematopoietic stem cells is beneficial for heart function. Despite the important role of chemokines in these processes, their use in clinical practice has so far been limited by their limited availability over a long time‐span in vivo. Here, a method is presented to increase physiological availability of chemokines at the site of injury over a defined time‐span and simultaneously control their release using biodegradable hydrogels. Two different biodegradable hydrogels were implemented, a fast degradable hydrogel (FDH) for delivering Met‐CCL5 over 24 hrs and a slow degradable hydrogel (SDH) for a gradual release of protease‐resistant CXCL12 (S4V) over 4 weeks. We demonstrate that the time‐controlled release using Met‐CCL5‐FDH and CXCL12 (S4V)‐SDH suppressed initial neutrophil infiltration, promoted neovascularization and reduced apoptosis in the infarcted myocardium. Thus, we were able to significantly preserve the cardiac function after MI. This study demonstrates that time‐controlled, biopolymer‐mediated delivery of chemokines represents a novel and feasible strategy to support the endogenous reparatory mechanisms after MI and may compliment cell‐based therapies.