Linda A. Brouwer

Attachment of glycosaminoglycans to collagenous matrices modulates the tissue response in rats

Biocompatibility and tissue regenerating capacity are essential characteristics in the design of collagenous biomaterials for tissue engineering. Attachment of glycosaminoglycans (GAGs) to collagen may add to these characteristics by creating an appropriate micro-environment. In this study, porous type I collagen matrices were crosslinked using 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide, in the presence and absence of chondroitin sulfate and heparan sulfate. The tissue response to these matrices was evaluated after subcutaneous implantation in rats. Biocompatibility of the matrices was established by the induction of a transitional inflammatory response, and the generation of new host tissue. Non-crosslinked collagen was gradually resorbed and replaced by collagenous connective tissue. By contrast, crosslinked matrices, with and without GAGs. retained their scaffold integrity during implantation, and supported the interstitial deposition and organization of extracellular matrix. In addition, crosslinking decreased tissue reactions at late time intervals. No calcification in any of the implants was observed. The presence of GAGs preserved porous lamellar matrix structures. Heparan sulfate in particular promoted angiogenesis at weeks 2 and 4, predominantly at the matrix periphery. The almost complete absence of macrophages and giant cells associated with collagen-GAG matrices, after 10 weeks implantation, indicated a reduced foreign body reaction. It is concluded that attachment of GAGs to collagen matrices modulates the tissue response. The potential of these biocompatible scaffolds for tissue engineering is increased by preserving porous matrix integrity. promoting angiogenesis and reducing foreign body reactions.

In vivo biocompatibility of dextran-based hydrogels.

Dextran-based hydrogels were obtained by polymerization of aqueous solutions of methacrylated dextran (dex-MA) or lactate-hydroxyethyl methacrylate-derivatized dextran (dex-lactate-HEMA). Both nondegradable dex-MA and degradable dex-lactate-HEMA disk-shaped hydrogels, varying in initial water content and degree of substitution (DS, the number of methacrylate groups per 100 glucose units), were implanted subcutaneously in rats. The tissue reaction was evaluated over a period of 6 weeks. The initial foreign-body reaction to the dex-MA hydrogels was characterized by infiltration of granulocytes and macrophages and the formation of fibrin, and exudate, as well as new blood vessels. This reaction depended on the initial water content as well as on the DS of the hydrogel and decreased within 10 days. The mildest tissue response was observed for the gel with the highest water content and intermediate DS. At day 21 all dex-MA hydrogels were surrounded by a fibrous capsule and no toxic effects on the surrounding tissue were found. No signs of degradation were observed. The initial foreign-body reaction to the degradable dex-lactate-HEMA hydrogels was less severe compared with the dex-MA gels. In general, the size of the dex-lactate-HEMA hydrogels increased progressively with time and finally the gels completely dissolved. Degradation of the dex-lactate-HEMA hydrogels was associated with infiltration of macrophages and the formation of giant cells, both of which phagocytosed pieces of the hydrogel. A good correlation between the in vitro and the in vivo degradation time was found. This suggests that extra-cellular degradation is not caused by enzymes but depends only on hydrolysis of the ester and/or carbonate bonds present in the crosslinks of the hydrogels. After 21 days, the degradable hydrogels, as such, could not be retrieved, but accumulation of macrophages and giant cells was observed, both of which contained particles of the gels intracellularly. As for the dex-MA hydrogels, no toxic effects on the surrounding tissue were found. The results presented in this study demonstrate that dextran-based hydrogels can be considered as biocompatible materials, making these hydrogels attractive systems for drug delivery purposes.

In vitro and in vivo evaluation of gelatin-chondroitin sulphate hydrogels for controlled release of antibacterial proteins.

Chemically cross-linked gelatin-chondroitin sulphate (ChS) hydrogels, impregnated in Dacron, were evaluated as drug delivery systems for antibacterial proteins. The gelatin-chondroitin sulphate gels, plain or impregnated in Dacron, were cross-linked with a water-soluble carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The release of lysozyme and recombinant thrombocidin (rTC-1), an antibacterial protein derived from human blood platelets, from the gelatin-ChS gels in Dacron in phosphate-buffered saline at 37 degrees C was determined, and compared to the release from gelatin gels in Dacron and plain gelatin-ChS gels. The incorporation of chondroitin sulphate into gelatin gels, caused a marked increase in lysozyme loading capacity, and a slower release rate. The relative release profiles for rTC-1 and lysozyme were equal for cross-linked gelatin as well as for cross-linked gelatin-ChS gels. Furthermore, rTC-1 showed no loss of antibacterial activity after 1 week of release. The lysozyme concentration profiles in the samples and in the surrounding medium as a function of time were calculated using mathematical solutions for Ficks second law of diffusion for a semi-infinite composite medium, which is a schematic representation of a slab in a surrounding medium. The biocompatibility and degradation of the Dacron matrices impregnated with gelatin-ChS gels was studied after implantation in subcutaneous pockets in rats. Chemically cross-linked gelatin-Ch5 gels showed a mild tissue reaction, and almost complete degradation within 18 weeks of implantation.

A comparative biocompatibility study of microspheres based on crosslinked dextran or poly(lactic‐co‐glycolic)acid after subcutaneous injection in rats

Microspheres based on methacrylated dextran (dex-MA), dextran derivatized with lactate-hydroxyethyl methacrylate (dex-lactate-HEMA) or derivatized with HEMA (dex-HEMA) were prepared. The microspheres were injected subcutaneously in rats and the effect of the particle size and network characteristics [initial water content and degree of methacrylate substitution (DS)] on the tissue reaction was investigated for 6 weeks. As a control, poly(lactic-co-glycolic)acid (PLGA) microspheres with varying sizes (unsized, smaller than 10 microm, smaller and larger than 20 microm) were injected as well. A mild tissue reaction to the PLGA microspheres was observed, characterized by infiltration of macrophages (MØs) and some granulocytes. Six weeks postinjection, the PLGA microspheres were still present. However, their size was decreased indicating degradation and many spheres had been phagocytosed. The tissue reaction was hardly affected by size differences, except for particles smaller than 10 microm, which induced an extensive tissue reaction. The initial tissue reaction to nondegradable dex-MA microspheres was stronger than towards the PLGA microspheres, but at day 10 the tissue reactions were comparable for both groups. Six weeks postinjection, the dex-MA microspheres were completely phagocytosed, and no signs of degradation were observed. The size and initial water content of dex-MA microspheres hardly affected the tissue response, although less granulocytes were observed for microspheres with higher DS. Slowly degrading dextran microspheres composed of dex-(lactate(1)-)HEMA induced a tissue reaction comparable to the PLGA microspheres. However, degradation of the dex-(lactate(1,3)-)HEMA microspheres was associated with an increased number of MØs and giant cells, both phagocytosing the microspheres and their degradation products. Similar to PLGA, no adverse reactions were observed for the nondegradable dex-MA and degradable dextran microspheres. This study shows that both nondegradable and degradable dextran-based microspheres are well tolerated after subcutaneous injection in rats, which make them interesting candidates as controlled drug delivery systems.

Endothelial-to-mesenchymal transition contributes to fibro-proliferative vascular disease and is modulated by fluid shear stress.

AIMS Neointimal hyperplasia is a common feature of fibro-proliferative vascular disease and characterizes initial stages of atherosclerosis. Neointimal lesions mainly comprise smooth muscle-like cells. The presence of these lesions is related to local differences in shear stress. Neointimal cells may arise through migration and proliferation of smooth muscle cells from the media. However, a role for the endothelium as a source of smooth muscle-like cells has largely been disregarded. Here, we investigated the role of endothelial-to-mesenchymal transition (EndMT) in neointimal hyperplasia and atherogenesis, and studied its modulation by shear stress. METHODS AND RESULTS In human atherosclerotic plaques and porcine aortic tissues, myo-endothelial cells were identified, suggestive for EndMT. Flow disturbance by thoracic-aortic constriction in mice similarly showed the presence of myo-endothelial cells specifically in regions exposed to disturbed flow. While uniform laminar shear stress (LSS) was found to inhibit EndMT, endothelial cells exposed to disturbed flow underwent EndMT, in vitro and in vivo, and showed atherogenic differentiation. Gain- and loss-of-function studies using a constitutive active mutant of MEK5 and short hairpins targeting ERK5 established a pivotal role for ERK5 signalling in the inhibition of EndMT. CONCLUSION Together, these data suggest that EndMT contributes to neointimal hyperplasia and induces atherogenic differentiation of endothelial cells. Importantly, we uncovered that EndMT is modulated by shear stress in an ERK5-dependent manner. These findings provide new insights in the role of adverse endothelial plasticity in vascular disease and identify a novel atheroprotective mechanism of uniform LSS, namely inhibition of EndMT.

Absence of muscle regeneration after implantation of a collagen matrix seeded with myoblasts

Collagens are widely used as biomaterials for e.g. soft tissue reconstruction. The present study was aimed at reconstruction of abdominal wall muscle using processed dermal sheep collagen (DSC) and myoblast seeding. Myoblasts were harvested from foetal quadriceps muscle of an inbred rat strain, cultured, seeded as non-differentiated cells into DSC-discs and incubated in vitro for 2 h. The discs were implanted in the abdominal wall defects in adult rats. Non-seeded discs functioned as control. Implantation periods till week 6 were chosen. At day 1 and 2 after implantation infiltration of granulocytes and macrophages was clearly more intense in the seeded discs than in the controls. Relatively large numbers of mast cells infiltrated from the side of the adhering omentum. In central areas of the discs, seeded cells were easily recognized till day 5, since non-seeded control discs did not contain such cells. Ingrowth of host cells and tissue at the margins proceeded faster with the seeded discs. Lymphocyte accumulations were observed in the 3 week seeded specimen. At week 3 and week 6, in the seeded discs muscle tissue was not present, in contrast to very large giant-like cells. It is concluded that the chosen method of myoblast seeding did not result in the regeneration of muscle during this observation period. Unfavorable circumstances such as humoral factors, direct cellular interactions (phagocytosis), indirect cellular interactions (cytokines), or initial absence of vascularization, may play a role. Further studies are required.

A novel and simple method for endotracheal intubation of mice.

Endotracheal intubation in mice is necessary for experiments involving intratracheal instillation of various substances, repeated pulmonary function assessments and mechanical ventilation. Previously described methods for endotracheal intubation in mice require the use of injection anaesthesia to immobilize the animal during the intubation procedure or the use of a volatile anaesthetic prior to intubation for immobilization. With these methods, the control of anaesthetic depth during the intubation procedure is absent. We describe a method for simple and rapid intratracheal intubation in mice for mechanical ventilation, using a self-built plastic support to facilitate the intubation procedure. General anaesthesia is maintained by means of inhalation through a non-rebreathing circuit connected to the plastic support. This set-up gives the operator control of anaesthetic depth and sufficient time to perform the intubation procedure. A purpose-made laryngoscopic blade is used to facilitate the intubation tube entering the trachea. The blade of the purpose-made laryngoscope is constructed as a retraction guide and is curved for easy handling. Under direct vision, the epiglottis is gently lifted by the laryngoscopic blade while the intubation tube is pushed into the trachea. Following this novel intubation technique, we were able to mechanically ventilate mice for at least 2 h without severely disturbing blood gases. Histological evaluation of the lungs and microscopic evaluation of the trachea and larynx showed no signs of trauma related to the intubation technique or mechanical ventilation.

Repetitive subcutaneous implantation of different types of (biodegradable) biomaterials alters the foreign body reaction

In the present study two biodegradable materials (cross-linked collagens) and two non-biodegradable materials (polyurethane and silicone) were applied in a repetitive subcutaneous implantation model in rats. In contrast to the first challenge, the second challenge with the same type of material, but at a different subcutaneous site of the same animal, induced an increase of macrophages and giant cells inside the biodegradable materials. Additionally, only after the second challenge clusters and accumulations of plasma cells were present in the surrounding tissue of each type of material. In the same areas an increase of MHC II expression was measured by immunocytochemistry. Differences in the numbers of macrophages and T cells were not observed around the explants. Undifferentiated B cells or NK cells were not present at any time point. The results indicate that alterations observed after the second challenge did not depend on biodegradation of the materials. Significance of these findings should be considered in view of increased and repetitive use of the same type of biomaterial (possibly for different application sites) for implantation in patients.

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.

IL-2 loaded dextran microspheres with attractive histocompatibility properties for local IL-2 cancer therapy

Biodegradable dextran microspheres (MS) were developed as a slow-release system for interleukin-2 (IL-2) to apply them for local IL-2 therapy of cancer. We describe the tissue reactions induced by these MS without or with IL-2 in rats. Dextran MS stain bright red-purple with the periodic acid Schiff (PAS), visualising the exact spot of IL-2 release and its relation to the histological reaction pattern. Subcutaneously injected MS always form a well-circumscribed deposit. In the first 2 days there is a PMN inflammation within the MS-deposit, but the surroundings show only a scanty inflammatory reaction. The PMN reaction is replaced by an abundant macrophage reaction in particular in the MS-deposit. At day 21 a fibrous capsule of about 50 mum surrounds the deposit. The effect of IL-2 administered in its free form is mainly vascular, with vascular dilatation, vascular leakage and oedema. It is remarkable that lymphocytes are present in the injection area already at day 2. When IL-2 releasing MS were used, the various reactions induced by IL-2 and MS were amplified leading to local necrosis. We conclude that neither placebo MS nor IL-2 leads to necrosis after subcutaneous injection in rats. In contrast, when IL-2 was released from MS, then massive necrosis was induced. This might be due to increased phagocytosis or changes in the micro-niche due to the release of humoral factors by the infiltrating cells. This is probably fortuitous for local IL-2 therapy of cancer, as massive necrosis of tumour cells can be expected to lead to an increased antitumour reaction.