Novel polyurethanes with interconnected porous structure induce in vivo tissue remodeling and accompanied vascularization.

Tissue engineering and regenerative medicine have furnished a vast range of modalities to treat either damaged tissue or loss of soft tissue or its function. In most approaches, a temporary porous scaffold is required to support tissue regeneration. The scaffold should be designed such that the turnover synchronizes with tissue remodeling and regeneration at the implant site. Segmented polyester urethanes (PUs) used in this study were based on epsilon-caprolactone (CL) and co-monomers D,L-lactide (D,L-L) and gamma-butyrolactone (BL), and 1,4-butanediisocyanate (BDI). In vitro, the PUs were nontoxic and haemocompatible. To test in vivo biocompatibility, the PUs were further processed into porous structures and subcutaneously implanted in rats for a period up to 21 days. Tissue remodeling and scaffold turnover was associated with a mild tissue response. The tissue response was characterized by extensive vascularization through the interconnected pores, with low numbers of macrophages on the edges and stroma formation inside the pores of the implants. The tissue ingrowth appeared to be related to the extent of microphase separation of the PUs and foam morphology. By day 21, all of the PU implants were highly vascularized, confirming the pores were interconnected. Degradation of P(CL/D,L-L)-PU was observed at this time, whereas the other two PU types remained intact. The robust method reported here of manufacturing and processing, good mechanical properties, and in vivo tissue response of the porous P(CL/D,L-L)-PU and PBCL-PU makes them excellent candidates as biomaterials with an application for soft tissue remodeling, for example, for cardiovascular regeneration.

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.

In vitro biocompatibility of biodegradable dextran-based hydrogels tested with human fibroblasts.

The cytotoxicity of dextran T40, methacrylated dextran (dex-MA) and hydroxyethyl-methacrylated dextran (dex-HEMA), dextran-based hydrogel discs and microspheres, and their degradation products, was studied by measuring the cell proliferation inhibition index (CPII) on human fibroblasts in vitro. In addition, during the 72 h incubation period light-microscopic observations were performed daily. After 24 h of incubation with dextran and dex-HEMA polymers, the cells showed elongated or spider-like forms, some lipid droplets and intracellular granula, indicative of pinocytosis and internalization of the polymers. During the next two days, the fibroblasts' appearance did not change. Methacrylic acid (MAA), formed by hydrolysis of dex-HEMA, did not influence the cell morphology. Dex-HEMA polymer solutions with a low and high degree of substitution (DS) at 100 mg/ml caused a CPII of 30-40% after 72 h. This is less than 10% growth inhibition per cell cycle and statistically not different from the CPII induced by 100 mg/ml dextran T40. Growth inhibition induced by MAA was also low. The various dex-MA hydrogel discs caused similar low growth inhibition. Interestingly, hydrogel microspheres of dex-MA and dex-(lactate-)HEMA caused a CPII of only 0-20% after 72 h. The results presented in this study demonstrate that methacrylate-derivatized dextran hydrogels show good biocompatibility in vitro making these degradable biomaterials promising systems for drug delivery purposes.

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.

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.

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 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.

TGF-beta and bFGF affect the differentiation of proliferating porcine fibroblasts into myofibroblasts in vitro.

Fibroblasts and myofibroblasts are involved in the foreign body reaction to biomaterials, especially in capsule formation. However, contraction or detachment of the capsule can lead to complications. Biocompatibility of biomaterials may be improved by the application of proteins regulating the differentiation or activation of (myo)fibroblasts. Myofibroblasts, differentiating from fibroblasts can be identified by the expression of alpha-smooth muscle actin (alpha-SM actin). We investigated the influence of proliferation and quiescence on the differentiation of porcine dermal cells and whether transforming growth factor-beta (TGF-beta) and basic fibroblast growth factor (bFGF) are involved in the differentiation of proliferating cells. Porcine cells were used because pigs increasingly function as in vivo models while little is known of the characteristics of their cells. Serum-free cultured, quiescent fibroblasts differentiated into myofibroblasts, while proliferating fibroblasts cultured in the presence of serum containing TGF-beta, formed alpha-SM actin-negative cell clusters. After reaching confluency, these clusters started to expressing alpha-SM actin. Moreover, these proliferating cells produced TGF-beta from day 4 onwards while bFGF did not. Differentiation into myofibroblasts was inhibited by bFGF and to an even greater extent by antibodies to TGF-beta. Further, two theories concerning the role of the myofibroblast in tissue contraction in view of two biomaterial application will be discussed.