Marc Hendriks

Successive epoxy and carbodiimide cross-linking of dermal sheep collagen

Cross-linking of dermal sheep collagen (N-DSC, T(S) = 46 degrees C, number of amine groups = 31 (n/1000)) with 1,4-butanediol diglycidyl ether (BDDGE) at pH 9.0 resulted in a material (BD90) with a high T(S)(69 degrees C), a decreased number of amine groups of 15 (n/1000) and a high resistance towards collagenase and pronase degradation. Reaction of DSC with BDDGE at pH 4.5 yielded a material (BD45) with a T(S) of 64 degrees C, hardly any reduction in amine groups and a lower stability towards enzymatic degradation as compared to BD90. The tensile strength of BD45 (9.2 MPa) was substantially improved as compared to N-DSC (2.4 MPa), whereas the elongation at break was reduced from 210 to 140%. BD90 had a tensile strength of 2.6 MPa and an elongation at break of only 93%. To improve the resistance to enzymes and to retain the favorable tensile properties, BD45 was post-treated with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) in the presence of N-hydroxysuccinimide (NHS) to give BD45EN. Additional cross-linking via the formation of amide bonds took place as indicated by the T(S) of 81 degrees C and the residual number of amine groups of 19 (n/1000). BD45EN was stable during exposure to both collagenase and pronase solutions. The tensile properties (tensile strength 7.2 MPa, elongation at break 100%) were comparable to those of BD45 and glutaraldehyde treated controls (G-DSC). Acylation of the residual amine groups of BD45 with acetic acid N-hydroxysuccinimide ester (HAc-NHS) yielded BD45HAc with a large reduction in amine groups to 10 (n/1000) and a small reduction in T(S) to 62 degrees C. The stability towards enzymatic degradation was reduced, but the tensile properties were comparable to BD45.

α-Amino Acid Containing Degradable Polymers as Functional Biomaterials: Rational Design, Synthetic Pathway, and Biomedical Applications

Currently, biomedical engineering is rapidly expanding, especially in the areas of drug delivery, gene transfer, tissue engineering, and regenerative medicine. A prerequisite for further development is the design and synthesis of novel multifunctional biomaterials that are biocompatible and biologically active, are biodegradable with a controlled degradation rate, and have tunable mechanical properties. In the past decades, different types of α-amino acid-containing degradable polymers have been actively developed with the aim to obtain biomimicking functional biomaterials. The use of α-amino acids as building units for degradable polymers may offer several advantages: (i) imparting chemical functionality, such as hydroxyl, amine, carboxyl, and thiol groups, which not only results in improved hydrophilicity and possible interactions with proteins and genes, but also facilitates further modification with bioactive molecules (e.g., drugs or biological cues); (ii) possibly improving materials biological properties, including cell-materials interactions (e.g., cell adhesion, migration) and degradability; (iii) enhancing thermal and mechanical properties; and (iv) providing metabolizable building units/blocks. In this paper, recent developments in the field of α-amino acid-containing degradable polymers are reviewed. First, synthetic approaches to prepare α-amino acid-containing degradable polymers will be discussed. Subsequently, the biomedical applications of these polymers in areas such as drug delivery, gene delivery and tissue engineering will be reviewed. Finally, the future perspectives of α-amino acid-containing degradable polymers will be evaluated.

Crosslinking and modification of dermal sheep collagen using 1,4-butanediol diglycidyl ether

Crosslinking of dermal sheep collagen (DSC) was accomplished using 1, 4-butanediol diglycidyl ether (BDDGE). At pH values > 8.0, epoxide groups of BDDGE will react with amine groups of collagen. The effects of BDDGE concentration, pH, time, and temperature were studied. Utilization of a 4-wt % BDDGE instead of 1-wt % resulted in a faster reaction. Whereas similar values of shrinkage temperature were obtained, fewer primary amine groups had reacted at a lower BDDGE concentration, which implies that the crosslinking reaction had a higher efficacy. An increase in pH from 8.5 to 10.5 resulted in a faster reaction but reduced crosslink efficacy. Furthermore, an increase in reaction temperature accelerated the reaction without changing the crosslink efficacy. Crosslinking under acidic conditions (pH < 6.0) evoked a reaction between epoxide groups and carboxylic acid groups of collagen. Additional studies showed that no oligomeric crosslinks could be formed. However, hydrolysis of the epoxide groups played a role in the crosslink mechanism especially under acidic reaction conditions. The macroscopic properties of these materials were dependent on the crosslinking method. Whereas a flexible and soft tissue was found if crosslinking was performed at pH < 6.0, a stiff sponge was obtained under alkaline conditions. Reaction of DSC with a monofunctional compound (glycidyl isopropyl ether) led to comparable trends in reaction rate and in similar macroscopical differences in materials as observed with BDDGE.

Covalently-Bound Heparin Makes Collagen Thromboresistant

Objective—Blood compatibility of artificial surfaces depends on their immunogenic and thrombogenic properties. Collagen’s weak antigenicity makes it an attractive candidate for stent coatings or fabrication of vascular grafts. However, the thrombogenic nature of collagen limits its application. We examined whether heparinization can make collagen more thromboresistant. Methods and Results—Collagen was heparinized by crosslinking collagen with extensively periodate oxidized heparin and/or by covalently bonding of mildly periodate oxidized heparin. Both ways of heparinization have no effect on platelet adhesion and could not abolish induction of platelet procoagulant activity. However, thrombin generation was completely prevented under static and flow conditions. The functionality of immobilized heparin was confirmed by specific uptake of antithrombin, 13.5±4.7 pmol/cm2 and 1.95±0.21 pmol/cm2 for mildly and heavily periodated heparin, respectively. Conclusions—These results indicate that immobilization of heparin on collagen, even as a crosslinker, is a very effective way to prevent surface thrombus formation. These data encourage the application of heparinized collagen as stent-graft material in animal and eventually human studies.

Optimization of diamine bridges in glutaraldehyde treated bioprosthetic aortic wall tissue.

OBJECTIVE Bioprosthetic calcification can be significantly mitigated by both increased concentrations of glutaraldehyde (GA) and the introduction of diamine (DA) bridges. The purpose of the present study was to evaluate whether an optimal effect of DA-enhanced fixation can be achieved by titration of dialdehyde and diamine concentrations. METHODS Porcine aortic roots were fixed at 0.05% GA (under-fixation) or 0.2% GA and 0.7% GA (commercial fixation). An interim step of DA treatment (L-Lysine; 0, 25, 50 or 100 mM; 37 degrees C; 2 days) was followed by completion of the GA fixation (37 degrees C; 5 days). Aortic wall coupons (12 mm) were punched out and implanted subcutaneously into seven-week old Long-Evans rats for 60 days. Calcium content was assessed by atomic absorption spectroscopy and histology. RESULTS Increasing the L-Lysine concentrations beyond 25 mM was essential to achieve the anti-calcific effect of DA-enhanced fixation. This effect was proportional to the GA concentrations applied. Compared to non-enhanced GA fixation (0 mM DA), calcification increased by 17.4% (p = 0.2114) in 0.05% fixed tissue but decreased by 32.0% (p < 0.0001) and 45.1% (p < 0.0002) in 0.2% and 0.7% GA, respectively, when the DA concentration was 100 mM. Histologically the extent, but not the pattern of calcification, was affected. CONCLUSION The calcium mitigating effect of diamine-treatment as an interim step of glutaraldehyde fixation is proportional to the GA concentration applied. Within commercial 0.7% GA fixation 100 mM DA has the potential to practically halve aortic wall calcification.

The kinetics of 1,4-butanediol diglycidyl ether crosslinking of dermal sheep collagen

Dermal sheep collagen was crosslinked with 1,4-butanediol diglycidyl ether (BDDGE) or modified with glycidyl isopropyl ether (PGE). The reduction in amine groups as a function of time was followed to study the overall reaction kinetics of collagen with either BDDGE or PGE. Linearization of the experimental data resulted in a reaction order of 2 with respect to the amine groups in the PGE masking reaction, whereas a reaction order of 2.5 was obtained in the BDDGE crosslinking reaction. The reaction orders were independent of the pH in the range of 8.5-10.5 and the reagent concentration (1-4 wt %). The reaction order with respect to epoxide groups was equal to 1 for both reagents. As expected, the reaction rate was favored by a higher reagent concentration and a higher solution pH. Because the BDDGE crosslinking reaction occurs via two distinct reaction steps, the content of pendant epoxide groups in the collagen matrix was determined by treating the collagen with either O-phosphoryl ethanolamine or lysine methyl ester. The increase in either phosphor or primary amine groups was related to the content of pendant groups. Crosslinking at pH 9.0 resulted in a low reaction rate but in a high crosslink efficacy, especially after prolonged reaction times. A maximum concentration of pendant epoxide groups was detected after 50 h. Reaction at pH 10.0 was faster, but a lower crosslinking efficacy was obtained. At pH 10.0, the ratio between pendant epoxide groups and crosslinks was almost equal to 1 during the course of the crosslinking reaction.

Enzymatically and Reductively Degradable α-Amino Acid-Based Poly(ester amide)s: Synthesis, Cell Compatibility, and Intracellular Anticancer Drug Delivery

A novel and versatile family of enzymatically and reductively degradable α-amino acid-based poly(ester amide)s (SS-PEAs) were developed from solution polycondensation of disulfide-containing di-p-toluenesulfonic acid salts of bis-l-phenylalanine diesters (SS-Phe-2TsOH) with di-p-nitrophenyl adipate (NA) in N,N-dimethylformamide (DMF). SS-PEAs with Mn ranging from 16.6 to 23.6 kg/mol were obtained, depending on NA/SS-Phe-2TsOH molar ratios. The chemical structures of SS-PEAs were confirmed by (1)H NMR and FTIR spectra. Thermal analyses showed that the obtained SS-PEAs were amorphous with a glass transition temperature (Tg) in the range of 35.2-39.5 °C. The in vitro degradation studies of SS-PEA films revealed that SS-PEAs underwent surface erosion in the presence of 0.1 mg/mL α-chymotrypsin and bulk degradation under a reductive environment containing 10 mM dithiothreitol (DTT). The preliminary cell culture studies displayed that SS-PEA films could well support adhesion and proliferation of L929 fibroblast cells, indicating that SS-PEAs have excellent cell compatibility. The nanoparticles prepared from SS-PEA with PVA as a surfactant had an average size of 167 nm in phosphate buffer (PB, 10 mM, pH 7.4). SS-PEA nanoparticles while stable under physiological environment undergo rapid disintegration under an enzymatic or reductive condition. The in vitro drug release studies showed that DOX release was accelerated in the presence of 0.1 mg/mL α-chymotrypsin or 10 mM DTT. Confocal microscopy observation displayed that SS-PEA nanoparticles effectively transported DOX into both drug-sensitive and -resistant MCF-7 cells. MTT assays revealed that DOX-loaded SS-PEA nanoparticles had a high antitumor activity approaching that of free DOX in drug-sensitive MCF-7 cells, while more than 10 times higher than free DOX in drug-resistant MCF-7/ADR cells. These enzymatically and reductively degradable α-amino acid-based poly(ester amide)s have provided an appealing platform for biomedical technology in particular controlled drug delivery applications.

(Electron) microscopic observations on tissue integration of collagen-immobilized polyurethane.

The foreign body reactions to collagen-immobilized polyurethane (PU-CI) films during subcutaneous implantation in rats were characterized. The underlying concept is that collagen-immobilization will improve the tissue integration. Since the method of collagen-immobilization involves the covalent coupling of collagen to an acrylic acid (AA) based surface graft, both non-modified PU and PU-AA were used as controls. Bare PU has a flat surface, whereas both PU-AA and PU-CI displayed a slightly roughened surface. Implantation showed that PU-CI induced early after implantation a far more intense foreign body reaction than PU and PU-AA. This reaction consisted of increased presence of fibrin, granulocytes and macrophages. Roughening of the surface as with PU-AA induced only a small increase in fibrin formation and cellular migration. At day 5 the reaction to PU-CI had slowed down; giant cell formation now slowly started but was decreased compared to PU and PU-AA. At day 10 capsules around each type of material looked similar, but in contrast to PU. PU-CI films could no longer be dissected from their capsules. Only at week 3 this also occurred with PU, at which time point again similar capsules with the three materials were observed. At week 6, of the three materials PU-CI showed the thinnest capsule with most immediate adherence of connective tissue. These results show that collagen-immobilization of PU increased the early tissue reaction and therefore the tissue integration. The thin capsule observed at 6 weeks may be beneficial in e.g. infectious circumstances, when easy access for immune reactions is needed. This, and the long-term performance of PU-CI will be a matter of future investigations.

In vivo behavior of epoxy-crosslinked porcine heart valve cusps and walls

Calcification limits the long-term durability of xenograft glutaraldehyde-crosslinked heart valves. In this study, epoxy-crosslinked porcine aortic valve tissue was evaluated after subcutaneous implantation in weanling rats. Non-crosslinked valves and valves crosslinked with glutaraldehyde or carbodiimide functioned as control. Epoxy-crosslinked valves had somewhat lower shrinkage temperatures than the crosslinked controls, and within the series also some macroscopic and microscopic differences were obvious. After 8 weeks implantation, cusps from non-crosslinked valves were not retrieved. The matching walls were more degraded than the epoxy- and control-crosslinked walls. This was observed from the higher cellular ingrowth with fibroblasts, macrophages, and giant cells. Furthermore, non-crosslinked walls showed highest numbers of lymphocytes, which were most obvious in the capsules. Epoxy- and control-crosslinked cusps and walls induced lower reactions. Calcification, measured by von Kossa-staining and by Ca-analysis, was always observed. Crosslinked cusps calcified more than walls. Of all wall samples, the non-crosslinked walls showed the highest calcification. It is concluded that epoxy-crosslinked valve tissue induced a foreign body and calcification reaction similar to the two crosslinked controls. Therefore, epoxy-crosslinking does not represent a solution for the calcification problem of heart valve bioprostheses.

Tissue reactions to bacteria-inoculated rat lead samples. II. Effect of local gentamicin release through surface-modified polyurethane tubing

A surface modification technique was developed to achieve controlled release of gentamicin from implanted polyurethane (PU) rat lead samples. PU tubing first was provided with an acrylic acid/acrylamide copolymer surface graft and then loaded with gentamicin. This surface modification technique resulted in release of gentamicin base (GB) and was applied either to the inner luminal surface only (PU-GB-1x) or to both the inner and outer surfaces (PU-GB-2x). First we investigated whether the early tissue response was harmfully compromised when surface-modified rat lead samples were implanted without any infectious challenge. Additionally, the efficacy of this type of local gentamicin therapy was investigated by establishing its effect on tissue response and its ability to prevent lead-related infections after inoculation with Staphylococcus aureus. It was demonstrated that the applied surface modification(s) did not induce adverse effects although an increase in the infiltration of granulocytes and macrophages and an increase in the formation of wound fluid and fibrin were observed. This effect was stronger with PU-GB-2x than with PU-GB-1x. With bacterial inoculation the applied surface modification successfully suppressed the infectious challenge, PU-GB-2x more effectively than PU-GB-1x. PU-GB-2x also was more effective when compared to the gentamicin-delivery methods discussed in the first part of this two-part study, i.e., release through a vicinal gentamicin-containing collagen sponge and preoperative gentamicin solution-dipping of rat lead samples.