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.

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.

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.

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.

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.

Inhibition of the tissue reaction to a biodegradable biomaterial by monoclonal antibodies to IFN-gamma.

Biomaterials are increasingly used for clinical applications. However, loss of function may occur owing to tissue reactions, which are mainly caused by a variety of inflammatory reactions. Recently, we demonstrated that macrophages (MO) and T cells play key roles in these reactions. Since immunological studies showed that the T cell-derived cytokine interferon-gamma (IFN-gamma) activates MO, the aim of this study was to investigate the possibility of modulating tissue reactions to biodegradable biomaterials by inactivating IFN-gamma. Dermal sheep collagen (DSC) was used as a test biomaterial. DSC impregnated with anti-IFN-gamma or phosphate-buffered saline (control) was implanted in rats. The results showed that cellular ingrowth and formation and function of giant cells were strongly delayed by anti-IFN-gamma. Also, MHC class II expression was strongly inhibited. In the treated DSC, some huge giant cells were formed at the interface but association with the DSC bundles did not occur. Finally, in both the control and treated DSC, T cells and NK cells were rarely detected. This study demonstrates that IFN-gamma plays an important role in the inflammatory reaction to biomaterials. This reaction can be modulated by anti-IFN-gamma, which warrants further studies of anti-IFN-gamma for clinical application to prevent unwanted tissue reactions to biomaterials.

Modulation of the tissue reaction to biomaterials. II. The function of T cells in the inflammatory reaction to crosslinked collagen implanted in T-cell-deficient rats.

Unwanted tissue reactions are often observed resulting in events such as early resorption of the biomaterial, loosening of the implant, or a chronic (immunologic) response. From immunologic studies it is known that inflammatory reactions can be modulated by use of (anti)-growth factors or anti-inflammatory drugs. Before this can be employed with respect to biomaterials, the role of individual factors (humoral and cellular) has to be studied. In this part of the investigation, the role of T cells was studied by use of T-cell-deficient (nude) rats and control (AO) rats. Hexamethylenediisocyanate-crosslinked dermal sheep collagen (HDSC) was selected as the test material. The results showed that T cells or T cell-related factors played a prominent role in the attraction of macrophages and the formation of giant cells, their antigen presentation, and their phagocytotic capacity. As a consequence, degradation of HDSC was strongly delayed. This study also showed that infiltration of fibroblasts and creation of stromal areas in HDSC was restricted to areas subjected to degradation. However, in time, absence of T cells resulted in increased formation and maturation of autologous rat collagen. Results obtained suggest that the inflammatory reaction to biomaterials might be modulated by controlling T-cell activation.

Clonal dysregulation of the antibody response to tetanus-toxoid after bone marrow transplantation.

After bone marrow transplantation (BMT), a prolonged dysregulation of humoral immunity can be observed. In the present study, we investigated whether this is reflected in an abnormal production of specific antibodies (Ab) to the T-cell-dependent recall antigen tetanus-toxoid (TT). The study group consisted of children receiving transplants of an unmodified allogeneic graft and of adults receiving either a T-cell-depleted allogeneic or an unmodified autologous BM graft. Findings were compared with those in healthy controls. In pediatric graft recipients, who were routinely revaccinated early after BMT, the Ab response was quantitatively superior to that in adult graft recipients who did not receive early revaccination. In the majority of graft recipients, the time period after vaccination required to reach the peak level of antibodies was prolonged and the number of responding TT-specific B-cell clones was markedly decreased in comparison with controls. In controls, a low frequency of dominant B-cell clones may produce low quantities of homogeneous Ab components (H-Ab) against a heterogeneous background. However, in BM graft recipients, "overshooting" of Ab production by separate B-cell clones was observed, resulting in the development of H-Ab at a relatively high concentration. These abnormalities were present up to 10 years after BMT, irrespective of either the age of the recipient, the modulation of the graft, or the vaccination schedule used. It is hypothesized that the dysregulated Ab production is the consequence of activation of a restricted number of resting memory B cells, present in germinal centers, repopulating gradually after BMT. Our data show that routine revaccination early after BMT improves the humoral immune response. However, because of a clonally dysregulated Ab production, long-lasting qualitative defects may be present even after normalization of Ab titers.