A hyaluronan-based nerve guide: in vitro cytotoxicity, subcutaneous tissue reactions, and degradation in the rat.

We investigated possible cytotoxic effects, biocompatibility, and degradation of a hyaluronan-based conduit for peripheral nerve repair. We subjected the conduits to an in vitro fibroblast cytotoxicity test and concluded that the conduits were not cytotoxic. Subsequently, we implanted the conduits subcutaneously in rats, in order to investigate tissue reactions and biodegradation. Initially, a fibrin matrix was formed around the material, while the surroundings were relatively quiet. Macrophages (MØ) migrated to the conduits and formed giant cells next to the material after 5 days. The maximum presence of MØ was found after 3-6 weeks. The appearance of MHC class II cells showed a similar pattern. Highest numbers of giants reached a maximum after 6-12 weeks. Angiogenesis was started in the surroundings of the hyaluronan-based conduit within a few days. Massive ingrowth of blood vessels into the biomaterial was found after 6 weeks as well as cellular ingrowth into the lumen of the tube. At that time the tubular structure of the conduit was lost and loose biomaterial fibers were observed. The results show that a hyaluronan-based conduit is not cytotoxic and shows good biocompatibility. Such a conduit may be suitable as a guide in peripheral nerve repair.

In vivo behavior of poly(1,3-trimethylene carbonate) and copolymers of 1,3-trimethylene carbonate with D,L-lactide or epsilon-caprolactone: Degradation and tissue response.

The degradation and the tissue response evoked by poly(1,3-trimethylene carbonate) [poly(TMC)] and copolymers of TMC with either 52 mol % D,L-lactide (DLLA) or 89 mol % epsilon-caprolactone (CL) were evaluated in vivo by subcutaneous implantation of polymer films in rats for periods up to one year. Poly(TMC) specimens were extensively degraded after 3 weeks and, as confirmed by histology, totally resorbed in less than a year. A fast linear decrease in thickness and mass without a change in molecular weight was observed. Initially an acute sterile inflammatory tissue reaction, caused by the implantation procedure, was observed, followed by a mild macrophage-mediated foreign body reaction that lasted during the resorption period of the polymer. It is concluded that in vivo, poly(TMC) is degraded via surface erosion involving cellular-mediated processes. The degradation of the copolymers was slower than that of poly(TMC), taking place via autocatalyzed bulk hydrolysis, preferentially of ester bonds. The TMC-DLLA copolymer degraded 20 times faster than the TMC-CL one. In both cases, the tissue reaction upon implantation resembled a sterile inflammatory reaction followed by a foreign body reaction that led to the polymer encapsulation. Significant mass loss was only observed for the TMC-DLLA copolymer, which underwent 96% mass loss in 1 year. When extensive mass loss started, a mild-to-moderate secondary foreign body reaction, related to clearance of the polymer fragments, was triggered. The results presented in this study demonstrate that poly(TMC) and both TMC copolymers are biodegradable and biocompatible materials, making these polymers attractive for the preparation of short- and long-term degradable devices for soft tissue engineering.

Cardiac tissue engineering: characteristics of in unison contracting two- and three-dimensional neonatal rat ventricle cell (co)-cultures.

Patients with heart failure have, in spite of improved palliative therapies, bad prognosis. Cardiac tissue engineering by use of a temporary bioscaffold and cardiomyocytes may help to find answers for future treatments in heart failure. For that purpose two neonatal rat heart ventricular cell fractions were obtained after a gradient cell separation. Time related characteristics of Fractions I and II were established in two-dimensional (2-D) and three-dimensional (3-D) cell cultures. The 3-D cardiac constructs were obtained by use of a bovine type I collagen matrix after culturing either under static conditions or in the HARV bioreactor. With the 2-D cultures contracting cells were present after 1 day, and reached confluency from day 5 on and this was maintained up to 135 days. In Fraction-I some non-contracting cells were always noticed between the (in time in unison) contracting cells. Transmission electron microscopy (TEM) revealed that these mainly concerned fibroblasts. Differences in the expression of alpha-SM-1 actin and troponin-T were observed between the two fractions. In both fractions endothelial cells and macrophages were only sporadically observed. All through the 3-D matrix pendant-like single cell and clustered cell contractions were present after 1-2 days, resulting in time in unison contracting of cells with the collagen matrices. The whole event was faster with Fraction-I and was observed up to 3 weeks. At this time point clusters of troponin-T positive cells were found scattered through the collagen matrices. Additionally, TEM revealed healthy layers of connected cardiomyocytes with intercalated discs, in this case on and in between the collagen fibres. These findings provide evidence that in unison contracting structurally organized cell-matrix cardiac constructs can be engineered by use of co-cultures (neonatal cardiomyocytes and fibroblasts) and collagen matrices, which is very promising for the repair of larger scar areas of the myocardium.

Loading of collagen-heparan sulfate matrices with bFGF promotes angiogenesis and tissue generation in rats.

The loading of biocompatible matrices with growth factors offers the opportunity to induce specific cell behavior. The attachment of heparan sulfate (HS) to these matrices may promote the binding, modulation, and sustained release of signaling molecules. In this study, basic fibroblast growth factor (bFGF) was bound to crosslinked collagenous matrices with and without covalently attached HS. The tissue response to these matrices was evaluated after subcutaneous implantation in rats. Attachment of HS to collagen matrices increased the bFGF binding capacity threefold and resulted in a more gradual and sustained release of the growth factor in vitro. bFGF primarily was located at the matrix margins. In vivo, the presence of HS without bFGF resulted in a transient vascularization, predominantly at the matrix periphery. Angiogenesis was further enhanced by combining HS with bFGF. In contrast to collagen-HS and collagen/bFGF matrices, collagen-HS/bFGF matrices remained highly vascularized throughout the matrix during the 10-week implantation period. In addition, these latter matrices revealed an intense and prolonged tissue response and considerably promoted the generation of new tissue. Foreign body reactions were only observed sporadically at this time interval. It is concluded that bFGF loading of collagen-HS matrices has additional value for those tissue-engineering applications that require enhanced angiogenesis and generation of new tissue.

(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.

Animal study on surface-modified defibrillator systems: Indications for enhanced infection resistance.

One of the most important problems with ICD systems is infection. The aim of this study was an in vivo evaluation of the efficacy of defibrillator systems in terms of infection resistance. The polyurethane leads were coupled with heparin and loaded with the antibiotic gentamicin, while the PGs were modified to release gentamicin. Group I was comprised of 10 pigs implanted with either a standard or a modified system for 2 weeks; group II was implanted during 4 weeks. The lead was inserted into the heart wall via the jugular vein. The other end was subcutaneously tunneled to the armpit where the PG was positioned. A cocktail of Staphylococcus aureus and epidermidis was injected at the site of the PG. Evaluation was performed macroscopically, by taking bacterial swabs during explantation and by microscopic processing. The results showed that 3 out of 5 modified defibrillator-systems in group I and 1-2 out of 5 in group II were judged as noninfected, whereas all standard systems were infected. Infection rates of the remaining modified defibrillators showed variances, as found with the standards, from slight to moderate to high, to even high/severe in group II (1x standard and 1x modified). With the modified systems, this may be related to production of humoral factors by an intensified early tissue reaction, as indicated by a swelling at day 6 at the site of the PG. When infected, whether or not modified, usually only Staphylococcus aureus was present. Spreading of infection seemed to occur by inoculation via blood, for example, based on the observation that group II in general showed an increase in infected fibrotic overgrowth in the heart, while infectious problems were low in the jugular vein. It is concluded that the modification at short term shows enhanced infection resistance. An increased infection rate already at 4 weeks, however, indicates that the modification may not hold in the long run. Special attention is needed concerning the more intense early tissue reaction.

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 biocompatibility of carbodiimide-crosslinked collagen matrices: Effects of crosslink density, heparin immobilization, and bFGF loading.

Collagen matrices, crosslinked using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (E) and N-hydroxysuccinimide (N), were previously developed as a substrate for endothelial cell seeding of small-diameter vascular grafts. In the present study, the biocompatibility of various EN-crosslinked collagen matrices was evaluated following subcutaneous implantation in rats for periods up to 10 weeks. The effects of the crosslink density, referred to as the number of free primary amino groups per 1,000 amino acid residues (EN10, EN14, EN18, or EN22), the amount of heparin immobilized to EN14, and the effect of preloading heparinized EN14 with basic fibroblast growth factor (bFGF) on the induced tissue reaction were studied. EN-crosslinked collagen was biocompatible at both early and late time intervals, and matrices with high crosslink densities (i.e., EN14, EN10) especially demonstrated a significantly decreased antigenic response when compared to noncrosslinked collagen. Furthermore, increased crosslinking resulted in a decreased degradation rate. Immobilization of heparin onto EN14 resulted in a similar to EN14 (thus without heparin) or somewhat reduced tissue reaction, but fibrin formation and vascularization were increased with increasing quantities of immobilized heparin. Matrices preloaded with bFGF also demonstrated good biocompatibility, especially in combination with higher amounts of immobilized heparin. The latter matrices [EN14 with high heparin and bFGF, thus EN14-H (0.4)F and EN14-H(1.0)F] demonstrated significantly increased vascularization for periods up to 3 weeks. Neither heparin immobilization nor bFGF preloading induced an increased antigenic response. It is concluded that the results of this study justify further evaluation of bFGF preloaded, heparin immobilized EN14 collagen, as a matrix for endothelial cell seeding in experimental animals.

Tissue reactions to epoxy-crosslinked porcine heart valves post-treated with detergents or a dicarboxylic acid.

Calcification limits the long-term durability of xenograft glutaraldehyde (GA)-crosslinked heart valves. Previously, a study in rats showed that epoxy-crosslinked heart valves reduced lymphocyte reactions to the same extent as the GA-crosslinked control and induced a similar foreign-body response and calcification reaction. The present study was aimed at reducing the occurrence of calcification of epoxy-crosslinked tissue. Two modifications were carried out and their influence on cellular reactions and the extent of calcification after 8 weeks' implantation in weanling rats was evaluated. First, epoxy-crosslinked valves were post-treated with two detergents to remove cellular elements, phospholipids and small soluble proteins, known to act as nucleation sites for calcification. The second approach was to study the effect of the impaired balance between negatively and positively charged amino acids by an additional crosslinking step with a dicarboxylic acid. The detergent treatment resulted in a washed-out appearance of especially the cusp tissue. With the dicarboxylic acid, both the cusps and the walls had a limited washed-out appearance. The wall also demonstrated some detachment of the subendothelium. After implantation, both detergent and dicarboxylic acid post-treatment histologically resulted in reduced calcification at the edges of cusps and walls. However, total amounts of calcification, measured by atomic emission spectroscopy, were not significantly reduced. Data concerning the presence of lymphocytes varied slightly, but were in the same range as the GA-crosslinked control, i.e., clearly reduced compared with a noncrosslinked control. It is concluded that both the double detergent and the dicarboxylic acid post-treatment of epoxy-crosslinked heart valve tissue do not represent a sound alternative in the fabrication of heart valve bioprostheses.

Enzyme and cytokine effects on the impaired onset of the murine foreign-body reaction to dermal sheep collagen.

Subcutaneous implantation of biodegradable hexamethylenediisocyanate crosslinked dermal sheep collagen (HDSC) elicited little foreign-body reaction in mice in contrast to rats. If the factor(s) resulting in this minor foreign-body reaction are better understood, this knowledge can be used to modulate unwanted foreign-body reactions. Therefore, we investigated whether the phagocytic potential of murine macrophages and giant cells could be enhanced. Disks of HDSC were predegraded with collagenase or impregnated with tumor necrosis factor-alpha (TNF-alpha) or interferon-gamma (IFN-gamma) before implantation in 129 SVEV mice. Explantation was performed on days 7, 14, 21, and 28 and the disks were evaluated at the (immuno) light and transmission electron-microscopic levels. More giant cells were present in the predegraded disks. Cells were associated with the HDSC bundles, and the onset of phagocytosis started on day 28, in contrast to the controls and the disks impregnated with the cytokines. Expression of MHC class II was minimal in all groups. The matrix metalloproteinases MMP-2 and MMP-9 were expressed in all groups although on day 28 MMP-9 expression was higher in the predegraded disks. Thus, predegradation only slightly enhanced the onset of the foreign-body reaction to HDSC in mice, and impregnation with cytokines not at all. This suggests that lack of proteolytic enzymes or TNF-alpha or IFN-gamma is not the cause of the impaired onset of the foreign-body reaction.

The foreign body reaction to a biodegradable biomaterial differs between rats and mice.

Before a biomaterial can be applied in the clinic, biocompatibility must be tested in in vivo models, by monitoring the foreign body reaction. In this study, we compared the foreign body reaction (FBR) to the biodegradable biomaterial hexamethylenediisocyanate crosslinked dermal sheep collagen (HDSC) between several strains of rats and mice. HDSC disks were implanted subcutaneously on the backs of AO, BN, F344, LEW, and PVG rats and on the backs of 129 SVEV, BALB/c, and C57BL/6 mice. Materials were explanted after 7, 14, 21, and 28 days and processed for (immuno) light and transmission electron microscopic evaluation. In all rat strains, giant cell formation and phagocytosis of HDSC bundles were comparable. In addition, in the PVG rat, many plasma cells infiltrated the HDSC disks. Only a few T cells were present in AO and PVG rats, whereas, in F344 and LEW rats, the presence of T cells was more pronounced. BN rats showed an intermediate T-cell infiltration. In mice, the FBR to HDSC was comparable between the different strains. Compared with rats, giant cell formation was limited, whereas stroma formation was more abundant. Phagocytosis of HDSC bundles rarely occurred in mice, whereas calcification was observed more often. It is concluded that the FBR to HDSC clearly differs between rats and mice. This has consequences for assessment studies on biocompatibility and also on fundamental biomaterial research.

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.

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 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.

In vivo compatibility and degradation of crosslinked gelatin gels incorporated in knitted Dacron.

Gelatin gels were applied to porous Dacron meshes with the aim of using these gels for local drug delivery. In this article, the biocompatibility and degradation of gelatin gels with different crosslink densities applied in Dacron were studied in vivo by subcutaneous implantation in rats. Dacron discs were treated with carbon dioxide gas plasma to improve hydrophilicity, and subsequently impregnated with gelatin type B. The gelatin samples were crosslinked to different extents using various amounts of water-soluble carbodiimide (EDC) and N-hydroxysuccinimide (NHS). After 6 h, 2, 5, and 10 days, and 3, 6, and 10 weeks of postimplantation, the tissue reactions and biodegradation were studied by light microscopy. The early reaction of macrophages and polymorphonuclear cells to crosslinked gelatin was similar to or milder than Dacron. Giant cell formation was predominantly aimed at Dacron fibers and was markedly reduced in the presence of a crosslinked gelatin coating. At week 10 of implantation, the crosslinked gelatin gels were still present in the Dacron matrix. The gelatin degradation was less for samples with the highest crosslink density. The gelatin gel with the lowest crosslink density showed clear cellular ingrowth, starting after 6 weeks of implantation. The intermediate and high crosslinked gelatin gels showed little or no ingrowth. In these gels, giant cells were involved in the phagocytosis of gelatin parts at week 10. Application of carbodiimide crosslinked gelatin gels in Dacron is suitable for medical applications because of the good biocompatibility of the gels and the possibility of adapting the degradation rate of gelatin to a specific application.

In vivo and in vitro release of lysozyme from cross-linked gelatin hydrogels: a model system for the delivery of antibacterial proteins from prosthetic heart valves.

Prosthetic valve endocarditis may be reduced by the local delivery of antibacterial proteins from the Dacron sewing ring of a prosthetic heart valve. Dacron discs were treated with a carbon dioxide gas plasma to improve the hydrophilicity and thereby enabling homogeneous impregnation with gelatin type B. The gelatin samples were cross-linked to different degrees using various amounts of water-soluble carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Lysozyme, a model protein for antibacterial proteins, was loaded into (non)-cross-linked gelatin gels incorporated in Dacron, or adsorbed onto non-treated and gas plasma-treated Dacron. The in vivo lysozyme release was measured after subcutaneous implantation of lysozyme-loaded samples in rats. The lysozyme content of the samples, and the lysozyme level of the surrounding tissue were determined at different explantation times (ranging from 6 h up to 1 week). For cross-linked gelatin gels, the lysozyme tissue level was elevated up to 2 days after implantation. In vitro release was measured using agarose medium or phosphate buffer. Lysozyme release in buffer solution under sink conditions was in good agreement with the in vivo lysozyme release profiles, and therefore considered a good model to describe in vivo release characteristics. The release was modelled with a solution of Fick's second law of diffusion using the appropriate boundary conditions. In this way the lysozyme concentration in the gel and the surrounding tissue as a function of time and distance was obtained. The presence of cross-linked gelatin in Dacron did lead to an increased uptake of lysozyme and a delayed release during 30 h after implantation, whereas a burst release took place from Dacron, gas plasma-treated Dacron, or Dacron containing non-cross-linked gelatin.

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.

Foreign-body reaction to dermal sheep collagen in interferon-gamma-receptor knock-out mice.

This study was performed to gain more insight into the role of interferon-gamma (IFN-gamma), a potent macrophage activator, in the foreign-body reaction to hexamethylenediisocyanate-crosslinked dermal sheep collagen (HDSC). Because the results of earlier studies aimed at modulating the foreign-body reaction in AO rats by local or systemic treatment with anti-IFN-gamma were not completely unambiguous, we extended our investigations to IFN-gamma-receptor knock-out (KO) mice. Several parameters (i.e., macrophages, giant cells, T-cells, B-cells, granulocytes, expression of MHC class II, stroma formation, and degradation and calcification of the biomaterial) were compared between wild-type (WT) and KO mice. Remarkably, the foreign-body reaction was very similar in WT and KO mice. In both, giant cells were formed, but in contrast to previous results in AO rats, phagocytosis of HDSC bundles occurred hardly at all up to 9 weeks, and MHC class II expression was minimal. Stroma formation and vascularization were high and calcification occurred. T-cells comprised less than 1%; a few plasma cells were present in the KO mice at later time points. Granulocytes, mainly eosinophils, were present at all explantation time points. Because of the similar results in WT and KO mice, we question whether IFN-gamma plays a role at all in the foreign-body reaction in mice.

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.

Systemic anti-IFN-gamma treatment and role of macrophage subsets in the foreign body reaction to dermal sheep collagen in rats.

The application of a biomaterial induces a foreign body reaction. By controlling this reaction, biocompatibility could be improved. We previously demonstrated that impregnation of a biodegradable biomaterial with antibodies against interferon-gamma (IFN-gamma) inhibits the foreign body reaction. In this study we investigate whether systemic administration of the antibody can induce similar reactions. Several parameters are compared between control and anti-IFN-gamma-treated rats: cellular ingrowth; degradation of the biomaterial; ingrowth of macrophage (MO) subsets, T cells, B cells, NK cells, and granulocytes; and expression of the major histocompatibility complex class II (MHC class II) molecule on antigen presenting cells. Treatment with anti-IFN-gamma results in increased cellular ingrowth and biomaterial degradation and a decreased expression of MHC class II. Overall, systemic treatment with anti-IFN-gamma is insufficient to modulate the foreign body reaction. This suggests an alternative mechanism for MO activation besides IFN-gamma. The role of T cells and MO subsets in the foreign body reaction is discussed.