Biomedical subjects

Achim Göpferich

Publications and source records attributed to Achim Göpferich.

10 recordsLinked to original sources

PubMed2003

The use of poly(ethylene glycol)-block-poly(lactic acid) derived copolymers for the rapid creation of biomimetic surfaces.

For many tissue engineering applications biomimetic or bioactive polymers would allow for a more precise control of cell behavior in growing tissues than has so far been possible. For this application recently developed amine reactive diblock copolymers (N-succinimidyl tartrate monoamine poly(ethylene glycol)-block-poly(D,L-lactic acid) [ST-NH-PEGxPLAy]) were investigated concerning their reactivity in binding model substances. Their ability to covalently immobilize proteins on their surfaces was examined using polymer films with amine reactive surfaces. Furthermore, thiol reactive polymers were obtained by attaching N-succinimidyl 3-maleinimido propionate, a thiol reactive linker to monoamine poly(ethylene glycol)-block-poly(D,L-lactic acid) [H2N-PEGxPLAy]. This allowed the immobilization of proteins carrying free thiol groups. The amine and thiol reactive polymers were characterized by 1H-NMR spectroscopy and gel permeation chromatography (GPC). Investigation of glass transitions temperatures using modulated differential scanning calorimetry proved suitability for the fabrication of polymeric scaffolds for tissue engineering applications. The functionality of the polymers was demonstrated by investigating their ability to bind model amines, like the fluorescent dye EDANS. Moreover, insulin and somatostatin were covalently attached to the active linker groups via amine and thiol groups. The polymers will permit covalently attaching different bioactive molecules, such as growth and differentiation factors, with fast and gentle procedures securing their biological activity.

Amino Acid Sequence↗

PubMed2003

Towards biomimetic scaffolds: anhydrous scaffold fabrication from biodegradable amine-reactive diblock copolymers.

The development of biomimetic materials and their processing into three-dimensional cell carrying scaffolds is one promising tissue engineering strategy to improve cell adhesion, growth and differentiation on polymeric constructs developing mature and viable tissue. This study was concerned with the fabrication of scaffolds made from amine-reactive diblock copolymers, N-succinimidyl tartrate monoamine poly(ethylene glycol)-block-poly(D,L-lactic acid), which are able to suppress unspecific protein adsorption and to covalently bind proteins or peptides. An appropriate technique for their processing had to be both anhydrous, to avoid hydrolysis of the active ester, and suitable for the generation of interconnected porous structures. Attempts to fabricate scaffolds utilizing hard paraffin microparticles as hexane-extractable porogens failed. Consequently, a technique was developed involving lipid microparticles, which served as biocompatible porogens on which the scaffold forming polymer was precipitated in the porogen extraction media (n-hexane). Porogen melting during the extraction and polymer precipitation step led to an interconnected network of pores. Suitable lipid mixtures and their melting points, extraction conditions (temperature and time) and a low-toxic polymer solvent system were determined for their use in processing diblock copolymers of different molecular weights (22 and 42 kDa) into highly porous off-the-shelf cell carriers ready for easy surface modification towards biomimetic scaffolds. Insulin was employed to demonstrate the principal of instant protein coupling to a prefabricated scaffold.

Biocompatible Materials↗

PubMed2003

Acylation of peptides by lactic acid solutions.

To simplify the search for effective mechanisms to suppress peptide acylation inside drug delivery devices made of poly(D,L-lactic acid) (PLA) and poly(lactic-co-glycolic acid), we were looking for a suitable model system that would allow screening of strategies for peptide stabilization. With their low pH and the presence of lactic acid oligomers, diluted lactic acid solutions promised to be a suitable test system that mimics the microclimate in degrading PLA devices. We created solutions of 1-50% (w/w) lactic acid by dilution of concentrated lactic acid. Using high performance liquid chromatography (HPLC) and high performance liquid chromatography coupled with mass spectrometry (HPLC-MS) analysis, oligomer hydrolysis was monitored during the equilibration process of the diluted solutions. Their final oligomer content was determined by titration and by calculations based on HPLC data. HPLC-MS analysis of human atrial natriuretic peptide (ANP) stability in different lactic acid solutions at 37 degrees C for 4 weeks demonstrated that ANP underwent acylation even in diluted solutions containing only 0.05% (w/w) lactic acid oligomers. Purity analysis of lactic acid solutions allowed us to compare the conditions in the solution test-system to the microclimate that prevails inside degrading PLA microspheres.

Acylation↗

PubMed2002

Innovative frontal sinus stent acting as a local drug-releasing system.

Contemporary endonasal sinus surgery has given rise to distinct extended procedures focusing on the frontal sinus. However, surgical results sometimes are flawed, with reactive scarring leading to a relapse of insufficiency of drainage and ventilation. Topical application of medicines may offer help, but the hidden operative field around the frontal sinus is not reached by the usual nasal drugs. The effectiveness of an intraoperative insertion of stents is still a subject of debate in the literature. In previous studies we have seen some positive results. Based on this fact we looked for additional modalities to boost the effectiveness of fronto-nasal stents. We present a new device acting both as a stent and also as a local drug-releasing system. The combination of two therapeutic modalities may improve our treatment results in endonasal frontal sinus surgery. The pharmaceutical basics of our device are presented, and the first clinical data are shown. The first clinical trial was completely successful. Modification of the polymer and also of the released pharmaceutical agents may provide future improvements and may allow additional applications of the system in other areas of our surgical specialty.

Anti-Inflammatory Agents↗

PubMed2002

Determination of oxygen gradients in engineered tissue using a fluorescent sensor.

Nutrient and oxygen supply of cells are crucial to tissue engineering in general. If a sufficient supply cannot be maintained, the development of the tissue will slow down or even fail completely. Previous studies on oxygen supply have focused on measurement of oxygen partial pressures (pO(2)) in culture media or described the use of invasive techniques with spatially limited resolution. The experimental setup described here allows for continuous, noninvasive, high-resolution pO(2) measurements over the cross-section of cultivated tissues. Applying a recently developed technique for time-resolved pO(2) sensing using optical sensor foils, containing luminescent O(2)-sensitive indicator dyes, we were able to monitor and analyze gradients in the oxygen supply in a tissue over a 3-week culture period. Cylindrical tissue samples were immobilized on top of the sensors. By measuring the luminescence decay time, two-dimensional pO(2) distributions across the tissue section in contact with the foil surface were determined. We applied this technique to cartilage explants and to tissue-engineered cartilage. For both tissue types, changes were detected in monotonously decreasing gradients of pO(2) from the surface with high pO(2) to minimum pO(2) values in the center of the samples. Nearly anoxic conditions were observed in tissue constructs ( approximately 0 Torr) but not in excised cartilage discs ( approximately 20 Torr) after 1 day. Furthermore, the oxygen supply seemed to strongly depend on cell density and cell function. Additionally, histological analysis revealed a maximum depth of approximately 1.3 mm of regular cartilage development in constructs grown under the applied culture conditions. Correlating analytical and histological analysis with the oxygen distributions, we found that pO(2) values below 11 Torr might impair proper tissue development in the center. The results illustrate that the method developed is an ideal one to precisely assess the oxygen demand of cartilage cultures.

Animals↗

PubMed2002

The effect of poly(ethylene glycol)-poly(D,L-lactic acid) diblock copolymers on peptide acylation.

The combination of poly(ethylene glycol) (PEG) with a biodegradable poly(ester), such as poly(D,L-lactic acid) (PLA), is an approach that has been successfully used for the stabilization of proteins and peptides in several biodegradable delivery devices. The acylation of peptides inside degrading PLA microspheres has been described only recently as another instability mechanism related to the accumulation of polymer degradation products inside eroding PLA. We investigated whether the block copolymerization of PLA with PEG reduces peptide acylation inside degrading microspheres. Diblock copolymers consisting of poly(D,L-lactic acid) covalently bound to poly(ethylene glycol)-monomethyl ether (Me.PEG-PLA) were used for these investigations. Human atrial natriuretic peptide (ANP) was incorporated into microspheres manufactured from Me.PEG5-PLA45, a diblock copolymer with an overall PEG content of 10%. Peptide integrity inside the microspheres was monitored by HPLC-MS analysis during 4 weeks of microsphere degradation in isotonic phosphate buffer (pH 7.4) at 37 degrees C. Inside the degrading Me.PEG5-PLA45 microspheres, acylation products as well as an oxidation product of ANP were formed. The results demonstrate that the combination of PEG with PLA does not necessarily display a favorable effect concerning peptide acylation inside degrading polymer microspheres. However, they also suggested that the acylation reaction is mainly driven by the formation and accumulation of polymer degradation products inside the degrading microspheres.

Acylation↗

PubMed2002

Why degradable polymers undergo surface erosion or bulk erosion.

A theoretical model was developed that allows to predict the erosion mechanism of water insoluble biodegradable polymer matrices. The model shows that all degradable polymers can undergo surface erosion or bulk erosion. Which way a polymer matrix erodes after all depends on the diffusivity of water inside the matrix, the degradation rate of the polymer's functional groups and the matrix dimensions. From these parameters the model allows to calculate for an individual polymer matrix a dimensionless 'erosion number' epsilon. The value of epsilon indicates the mode of erosion. Based on epsilon, a critical device dimension Lcritical can be calculated. If a matrix is larger than Lcritical it will undergo surface erosion, if not it will be bulk eroding. Lcritical values for polymers were estimated based on literature data. Polyanhydrides were found to be surface eroding down to a size of approximately Lcritical = 10(-4) m while poly(alpha-hydroxy esters) matrices need to be larger than Lcritical = 10(-1) m to lose their bulk erosion properties. To support our theoretical findings it was shown experimentally that poly(alpha-hydroxy ester) matrices, which are considered classical bulk eroding materials, can also undergo surface erosion.

Biocompatible Materials↗

PubMed2002

Peptide acylation by poly(alpha-hydroxy esters).

PURPOSE: Poly(lactic acid) (PLA) and poly(lactic-co-glycolic acid) (PLGA) microspheres were investigated concerning the possible acylation of incorporated peptides. METHODS: Atrial natriuretic peptide (ANP) and salmon calcitonin (sCT) were encapsulated into PLA and PLGA microspheres. Peptide integrity was monitored by HPLC-MS analysis during microsphere degradation for four weeks. sCT fragmentation with endoproteinase Glu-C was used for identifying modified amino acids. Peptide stability in lactic acid solutions was investigated to elucidate possible mechanisms for preventing peptide acylation. RESULTS: Both peptides were acylated by lactic and glycolic acid units inside degrading microspheres in a time-dependent manner. After 21 days, 60% ANP and 7% sCT inside PLA microspheres were acylated. Fragmentation of sCT with endoproteinase Glu-C revealed that besides the N-terminal amine group, lysine, tyrosine or serine are further possible targets to acylation. Stability studies of the peptides in lactic acid solutions suggest that oligomers are the major acylation source and that lower oligomer concentration and higher pH substantially decreased the reaction velocity. CONCLUSIONS: The use of PLA and PLGA for drug delivery needs substantially more circumspection. As, according to FDA standards. the potential hazards of peptide acylation products need to be assessed, our findings may have significant implications for products already on the market. Techniques to minimize the acylation reaction are suggested.

Acylation↗

PubMed2002

Effects of hedgehog proteins on tissue engineering of cartilage in vitro.

The effects of three derivatives of the N-terminal signaling domain of hedgehog proteins on cartilage engineered in vitro were investigated, with specific focus on the ability to increase tissue growth rate and concentrations of major extracellular matrix components, that is, glycosaminoglycans (GAG) and collagen, and on the effects on morphological appearance of the tissue. Bovine articular chondrocytes were cultured on biodegradable polyglycolic acid (PGA) scaffolds with or without the addition of dipalmitoylated sonic hedgehog (dp-shh), dipalmitoylated indian hedgehog (dp-ihh), or sonic hedgehog dimer (shh-dimer) to medium with either 1% or 10% fetal bovine serum (FBS). All three hedgehog proteins dose-dependently increased construct weights (by up to 1.95-fold, dp-shh at 1,000 ng/mL) and the fraction of GAG over 4 weeks (by up to 2.7-fold, dp-shh at 1,000 ng/mL), as compared to control constructs. Dp-shh and dp-ihh elicited similar responses; a 10-fold higher concentration of nonacylated shh-dimer was necessary to reach comparable results. Positive hedgehog effects were more pronounced in medium containing 1% FBS than in medium containing 10% FBS; however, at either FBS concentration, cartilaginous tissues grown in the presence of hedgehog proteins appeared morphologically more mature. Hedgehog derivatives thus appear as promising candidates to improve the development and composition of engineered cartilage.

Animals↗

PubMedDate not supplied

Amine-reactive biodegradable diblock copolymers.

A new class of diblock copolymers was synthesized from biodegradable poly(lactic acid) and poly(ethylene glycol)minus signmonoamine. These polymers were activated by covalently attaching linkers such as disuccinimidyl tartrate or disuccinimidyl succinate to the hydrophilic polymer chain. The polymers were characterized by (1)H NMR spectroscopy, (13)C NMR spectroscopy and gel permeation chromatography (GPC). These investigations indicated that the polymers were obtained with the correct composition, in high purities, and the expected molecular weight. By using dyes containing primary amine groups such as 5-aminoeosin as model substrates, it was possible to show that the polymers are able to bind such compounds covalently. The diblock copolymers were developed to suppress unspecific protein adsorption and allow the binding of bioactive molecules by instant surface modification. The polymers are intended to be used for tissue engineering applications where surface immobilized cell adhesion peptides or growth factors are needed to control cell behavior.

Amines↗