Tissue engineering: selecting the optimal fixative for immunohistochemistry.

BACKGROUND: In the immunohistochemical analysis of tissue-engineered structures, aggressive treatments for fixation and antigen retrieval can impair the quality of specimen staining and visualization. HYPOTHESIS: We hypothesized that the adequate choice of fixative and antigen-retrieval method might improve the quality of immunohistochemical staining. METHODS: Tissue-engineered vascular grafts were fixed using formalin, Carnoy's, or HOPE(®) fixative. Antigen retrieval was performed where necessary and samples from each group were stained using hematoxylin and eosin to assess overall tissue preservation. For a set of proteins relevant to cardiovascular tissue development, immunohistochemical staining was applied to formalin-, Carnoy's-, and HOPE-fixed specimens to allow a comparative analysis. RESULTS: In tissue-engineered constructs, antigen retrieval methods necessary after formalin fixation led to significant destruction of the overall tissue structure. Carnoy's fixation resulted in good overall tissue preservation and adequate results for immunohistochemical staining of alpha-smooth muscle actin (α-SMA), vimentin, type I collagen, elastin, and laminin. HOPE fixative led to a loosened tissue structure and a swollen appearance but showed adequate results for staining against type III collagen and elastin. Formalin fixation without antigen retrieval led to inadequate visualization of α-SMA, vimentin, type I- and type III collagen, and laminin. CONCLUSION: Based on the present study, we recommend that Carnoy's fixative is employed for the preservation of tissue-engineered constructs to allow immunohistochemical analysis of type I- and type III collagen, elastin, laminin, α-SMA, and vimentin. However, it is clear that the technique requires optimization based on the particular tissue engineering application.

The BioStent: novel concept for a viable stent structure.

OBJECTIVES: Percutaneous stenting of occluded peripheral vessels is a well-established technique in clinical practice. Unfortunately, the patency rates of small-caliber vessels after stenting remain unsatisfactory. The aim of the BioStent concept is to overcome in-stent restenosis by excluding the diseased vessel segment entirely from the blood stream, in addition to providing an intact endothelial cell layer. DESIGN: The concept combines the principles of vascular tissue engineering with a self-expanding stent: casting of the stent within a cellularized fibrin gel structure, followed by bioreactor conditioning, allows complete integration of the stent within engineered tissue. MATERIALS AND METHODS: Small-caliber BioStents (Ø=6 mm; n=4) were produced by casting a nitinol stent within a thin fibrin/vascular smooth muscle cell (vSMC) mixture, followed by luminal endothelial cell seeding, and conditioning of the BioStent within a bioreactor system. The potential remodeling of the fibrin component into tissue was analyzed using routine histological methods. Scanning electron microscopy was used to assess the luminal endothelial cell coverage following the conditioning phase and crimping of the stent. RESULTS: The BioStent was shown to be noncytotoxic, with no significant effect on cell proliferation. Gross and microscopic analysis revealed complete integration of the nitinol component within a viable tissue structure. Hematoxylin and eosin staining revealed a homogenous distribution of vSMCs throughout the thickness of the BioStent, while a smooth, confluent luminal endothelial cell lining was evident and not significantly affected by the crimping/release process. CONCLUSIONS: The BioStent concept is a platform technology offering a novel opportunity to generate a viable, self-expanding stent structure with a functional endothelial cell lining. This platform technology can be transferred to different applications depending on the luminal cell lining required.