In vivo performance of equine and bovine tendon non cross-linked and EDC 1 cross-linked and bovine corium EDC 1 cross-linked minirods were analyzed macroscopically and microscopically. In macroscopic studies, implants could be observed over 28d. The implants could be differentiated well from the surrounding tissue, because minirods were darker and denser than the
surrounding tissue. Minirods were swollen, but their cylindrical shape was retained (see Figure 4-62).
Figure 4-62 Sections of equine non cross-linked minirod 7d (cross-section), 14d (lateral section) and 28d (cross-section) post implantation (from left to right)
To evaluate the biocompatibility of a material, in general two histological investigations are performed (Anderson; 1994). On the one hand the tissue response is evaluated with respect to degeneration of necrotic changes, inflammation, foreign body reactions and fibrosis. On the other hand, the cell types are characterized in more detail (Anderson; 1994). Histological evaluations of the minirods were more difficult than macroscopic examinations, because the collagen extrudates contain collagen type I with traces of collagen type III. Since these collagen types are also present in tissue, differentiation between implants and surrounding tissue was challenging, especially because pig dermal collagen appears denser than human collagen structures (Burke et al.; 1983). Minirods were identified by denser, more stained areas with atypical, non-native collagen bundle structures (Burke et al.; 1985). Additionally, especially at the later time points, cell accumulations gave hints where the minirods had been implanted.
After surgical implantation acute and chronic inflammation reactions occur dependent on the implanted material, implantation site and the size, shape and motion of the device (Anderson et al.; 1981; Anderson; 1994). Usually antigenic reactions are detected during the first 72h post implantation (Soo et al.; 1993).
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The first response to an injury is the migration of leukocytes, predominantly neutrophiles, and the exudation of fluids and plasma proteins within minutes to days. This is followed by a chronic inflammation characterized by an increase in macrophages, monocytes and lymphocytes and proliferation of blood vessels and connective tissue. If normal wound healing takes place, the healing response is initiated by proliferation of macrophages and monocytes followed by appearance of fibroblasts and vascular endothelial cells and formation of granulation tissue. Subsequently, the foreign body reaction follows in which macrophages, fibroblasts and foreign body giant cells take part. This may lead finally to fibrosis or the formation of a fibrous capsule when particles with a size greater than 10µm were implanted (Medlicott et al.; 2004). If biomaterials are less biocompatible, body response ends at the stage of chronic inflammation and no further healing process is initiated (Anderson; 1994).
Almost no inflammation could be detected by interferon 1β staining for all investigated insoluble collagen materials between day 5 and 56. Only superficial, slight inflammatory areas were observed, resulting from the administration of the solid, but rapidly swollen matrix (Hirasawa et al.; 1997) and the motion of the implant (Anderson et al.; 1981). Inflammatory reactions could be detected by TGFβ staining and cell accumulations, most likely leucocytes.
Figure 4-63 Equine non cross-linked (a) and EDC 1 cross-linked (b) minirod 3d post implantation (TGFβ staining, solid arrows indicate boundaries of implant, dashed arrows: capsule)
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TGFβ staining was performed between day 3 and 28. Positive TGFβ stained areas were detected near the implant boundaries up to day 14 (see Figure 4-63). This observation corresponded to the interferon 1β staining studies. Differentiation between mono-, lympho- and granulocytes was difficult, because no cell specific staining was performed. However, it was assumed that the observed cells were mainly mono- or lymphocytes due to the short life time of granulocytes. The immunogenic response was slightly more pronounced for non cross-linked materials which could be due to less washing steps during raw material preparation (Park et al.; 2002) and a faster collagen degradation. Since gelatin and gelatin peptides are chemotactic for neutrophils, an acute inflammatory response can be elevated (Weadock et al.; 1984). No further increase in cell number was observed for EDC 1 cross-linked matrices, indicating that this cross-linking ratio was suitable for in vivo applications (van Wachem et al.; 1994a). In general, equal performance was observed for materials with and without PCA-BSA, indicating that the in vivo performance of minirods was mainly dependent on the matrix material and not on the incorporated labeled model compound.
Figure 4-64 Remaining pocket of a bovine tendon non cross-linked minirod 5d post implantation (HE staining)
At days 3 and 5 post implantation, difficulties in cutting of bovine tendon specimens occurred. The minirods were pressed out during this preparation step, resulting in a cavity with sharp, cell rich boundaries from the implantation procedure (see Figure 4-64). The increase in cell number gave evidence that cells were attracted by the collagen minirods. Both equine and bovine corium EDC 1 cross-linked minirods were attached firmly enough to the surrounding tissue, to remain at their position. At day 3, cells were already attracted by the equine and bovine corium EDC 1 cross-linked minirods. This resulted in formation of a cellular capsule around the implants (see Figure 4-65b), because the additionally cross-links hindered cells to penetrate into the matrix core. Equine non cross-linked extrudates were also attached well to the surrounding tissue. Cells already started to invade the devices and consequently no distinct capsule around the implants could be detected (see Figure 4-65a).
Figure 4-65 Equine non cross-linked (a) and EDC 1 cross-linked (b) minirod 3d post implantation (HE staining, solid arrows indicate boundaries of implant, dashed arrows: capsule)
After 7d, all materials were incorporated well and cells infiltrated the minirods (see Figure 4-66). Since minirods were swollen and fragmented, a mixed immune response could be observed. In the interior of the implant an acute inflammatory reaction, with a high number of presumably polymorphonuclear leukocytes, occurred. The cell number at the implant boundary decreased
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slightly compared to day 3 and a chronic immune reaction with monocytes and lymphocytes took place (Anderson; 1994), which resulted in the formation of a thin capsule around the minirods.
Figure 4-66 Equine non cross-linked (a) and EDC 1 cross-linked (b) minirod 7d post implantation (HE staining, solid arrows indicate boundaries of implant, dashed arrows: capsule)
Still a lower number of cells could be detected in the interior of equine EDC 1 cross-linked minirods compared to equine non cross-linked matrices (see Figure 4-66).
Looking closer at bovine tendon EDC 1 minirods, it was observed that the implant itself exhibited a porous structure which was surrounded by tissue with numerous cells (see Figure 4-67a). In spite of the different structure, incorporation had taken place, as can be seen by collagen I staining (see Figure 4-67b). Extrudate fragments were stronger stained than tissue. Cells, presumable monocytes, infiltrated the minirods and the implants were degraded. An exact boundary could not be detected anymore. The remodeling process could also be observed with this sample. During wound healing processes, collagen type III is the prevalent collagen type (Burke et al.; 1985) and can already be detected between day 2 and 7 after injury. In later wound healing phases, collagen type III is replaced by collagen type I (Sedlarik; 2005).
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Figure 4-67 Bovine tendon EDC 1 minirod 7d post implantation (a) HE staining, (b) collagen I staining, (c) collagen III staining (arrows indicate boundaries of implant; in figure (b): solid arrows: inner implant, dashed arrows: transition, dotted arrows: tissue)
a b c 100µm 100µm 100µm
Collagen III staining showed, that inside the original extrudate boundaries, collagen type III was present and it was concluded that new collagen structures had been built (see Figure 4-67c).
After two weeks, the bovine corium EDC 1 minirods were degraded completely and at the site at which the sample had been implanted only a large cell accumulation could be detected (see Figure 4-68). This indicated that bovine corium EDC 1 extrudates stimulated a host response which caused fast implant degradation and replacement by newly formed collagen. A similar effect was observed by Burke et al. for a bovine dermal type I collagen (Zyderm®) suspension implanted in pigs (Burke et al.; 1983).
Figure 4-68 Bovine corium EDC 1 minirod 14d post implantation (HE staining, arrow indicates cell accumulation)
Bovine and equine tendon non cross-linked minirods were still present after two weeks and showed good incorporation into the surrounding tissue (see Figure 4-69a).
Figure 4-69 Equine non cross-linked (a) and EDC 1 cross-linked (b) minirod 14d post implantation (HE staining, arrows indicate boundaries of implant)
Both tendon EDC 1 cross-linked extrudates were also incorporated well and retained their homogeneous structure even longer than non cross-linked matrices. Only at the boundaries small cavities were observed, whereas still a compact implant core with several invaded cells existed (see Figure 4-69b). After 28d, equine implants, independent of cross-linking, were infiltrated by many cells and extensive remodeling had taken place (see Figure 4-70). However, in contrast to bovine corium EDC 1 cross-linked extrudates after 14d (see Figure 4-68) shapes of minirods could still be detected.
Figure 4-70 Equine non cross-linked (a) and EDC 1 cross-linked (b) minirod 28d post implantation (HE staining, arrows indicate boundaries of implant)
a b 100µm 100µm a b 100µm 100µm
Collagen forms a swollen hydrogel after contact with physiological fluids. The swollen matrix is soft and elastic and minimizes mechanical and frictional irritation of the surrounding tissue (Anderson et al.; 1981). Consequently, a normal wound healing process was observed for collagen minirods. Additionally, protein adsorption can be prevented because of the hydrophilic mobile surface and the low adverse interaction between collagen and the aqueous biological environment (Park et al.; 1996). Taken these facts together, collagen has a good biocompatibility. Based on the histological data, the in vivo persistence of minirods was put in the following chronological order: bovine corium EDC 1 cross-linked minirods were taken up first (after 14d), followed by bovine tendon non cross-linked and EDC 1 cross-linked matrices and equine tendon non cross-linked extrudates. As was expected from DSC data (see 4.1.1.2), swelling (see 4.1.2.1.1) and degradation studies (see 4.3.3), the slowest degradation was observed for equine EDC 1 cross-linked minirods. All materials were degraded in a comparable way. Digestion occurred simultaneously to formation of new collagen structures without abnormally high inflammatory reactions. This was in agreement with the common assumption that only low inflammation takes place after collagen implantation, because of almost similar amino acid sequences of collagen flavors extracted from different species (Gilbert; 1988b). Consistent observations were made for the in vivo digestion of minipellets (Hirasawa et al.; 1997).
4.4.6 Summary
In vitro and in vivo studies were performed with BSA loaded minirods. For drug release studies, BSA was labeled with PCA. During labeling no protein aggregation occurred and BSA was not hydrolyzed in the presence of a crude bacterial collagenase mixture. In vitro, 80% PCA-BSA was released from bovine tendon non cross-linked minirods within 48h in absence of collagenase and after 7h in the presence of 0.1µg/ml enzyme. Bovine corium EDC 1 cross-linked matrices released 80% drug within approximately 80h and 12h, respectively. From bovine tendon EDC 1 cross-linked devices 80% release occurred after 168h without addition of collagenase and after 95h if 0.1µg/ml collagenase was present. The sequence of drug release was consistent for both experimental
setups. Hence, drug release and degradation were dependent on the used collagen material; more cross-links led to lower matrix degradation and decreased PCA-BSA release rates. In vitro drug release of FITC dextran 70 was compared with the in vitro PCA-BSA delivery and good correlation was found. This indicated that FITC dextran 70 is suitable as a model “protein” drug and correlations based on the molecular weight can be made. In vivo PCA-BSA release was slightly more delayed than in vitro, because swelling of the minirods was more restricted and the devices were degraded more slowly in pigs compared to the in vitro setup. 80% PCA-BSA was delivered after 96h from bovine tendon non cross-linked and bovine corium EDC 1 cross-linked extrudates, whereas bovine tendon EDC 1 cross-linked minirods showed 80% release after 120h.
Mechanistic studies, performed in vitro, showed that swelling and diffusion controls the drug release in pure Tris buffer pH 7.5 for minirods, irrespective of cross-linking degree. For bovine tendon non cross-linked matrices this was also the dominating mechanism if collagenase was present. In contrast, EDC 1 cross-linked devices exhibited a release which was also dependent on matrix erosion. In the in vivo studies, similar release mechanisms as in the in vitro investigations with 0.1µg/ml collagenase were detected for all three collagen materials. Since it was possible to investigate different parts of the minirods in the in vivo experiments, it could further be demonstrated that the PCA-BSA release started at the matrix surface before release occured inside the minirods. In vitro - in vivo – comparison demonstrated that minirods behave equally in vitro and in vivo and that the period of release was in the same magnitude of time.
Histological investigations of seven different minirods were performed (bovine tendon non cross-linked and EDC 1 cross-linked, bovine corium EDC 1 cross- linked and equine tendon non cross-linked and EDC 1 cross-linked collagen matrices with incorporated PCA-BSA and drug free equine and bovine tendon non cross-linked collagen minirods). All devices were tolerated well, because no severe foreign body reaction was detected. The time course of the observed host digestion, resembling normal tissue turnover, was dependent on the
collagen material. Bovine corium EDC 1 cross-linked minirods were taken up fastest, followed by the bovine tendon non cross-linked, the bovine tendon EDC 1 cross-linked and the equine non cross-linked matrices. The slowest adsorption was observed for equine EDC 1 cross-linked minirods. This order was consistent with the increase in cross-links from bovine to equine collagen materials. Since this remodeling process is required to avoid a surgical implant removal after drug depletion, prospects for in vivo application were promising.