Abstract
Abstract A cell extraction process was employed to remove the cellular components from bovine pericardia, leaving a framework of largely insoluble collagen, elastin, and tightly bound glycosaminoglycans (GAGs). The acellular tissues then were fixed with a naturally occurring crosslinking agent (genipin) as a tissue-engineering extracellular matrix (ECM). Effects of the ECM porous structure, GAGs content and its degradation rate as well as incorporation with a novel angiogenic agent (ginsenoside Rg1) on the tissue regeneration patterns in the acellular ECM were investigated subcutaneously in a rat model. Applications of the acellular ECM as a patch in repairing defects in the pulmonary artery in a canine model were evaluated. In the first study, a cell extraction process was used to remove cellular components from bovine pericardia. Varying pore sizes and porosities of the acellular tissues were then created using acetic acid and collagenase. These tissue samples were fixed with genipin. The study was to investigate the ultrastructures of these acellular tissues and their biochemical and mechanical properties. Additionally, the effect of porous structures of acellular tissues on their in vivo angiogenesis was investigated subcutaneously in a rat model. After cell extraction, electron microscopy indicated that all cellular constituents were removed from the tissue. The acellular tissues formed distinct patterns in pore size, porosity following treatment with acetic acid and collagenase. Biochemical analyses confirmed that these acellular tissues with distinct porous structures consisted primarily of insoluble collagen, elastin, and tightly bound glycosaminoglycans. The thermal stability, and mechanical properties of the bovine pericardial tissue remained unaltered after cell extraction. The porous structures of the implanted samples seem to determine whether successful microvessel-ingrowth takes place. The acetic-acid-treated and collagenase-treated tissues, due to their high pore size and porosity, showed a large number of microvessels infiltrating into the interstices of the implanted samples. In contrast, a low density of microvessels was observed infiltrating into the acellular tissue and penetration of microvessels into the cellular tissue was never encountered. In the second study, the acellular tissues then were fixed with genipin at various known concentrations to obtain varying degrees of crosslinking. It was shown in the in vitro degradation study that after fixing with genipin, the resistance against enzymatic degradation of the acellular tissue increased significantly with increasing its crosslinking degree. In the in vivo subcutaneous study, it was found that cells (inflammatory cells, fibroblasts, endothelial cells, and red blood cells) were able to infiltrate into acellular tissues. Generally, the depth of cell infiltration into the acellular tissue decreased with increasing its crosslinking degree. Infiltration of inflammatory cells was accompanied by degradation of the acellular tissue. Due to early degradation, no tissue regeneration was observed within fresh (without crosslinking) and the 30%-degree-crosslinking acellular tissues. This is because the scaffolds provided by these two samples were already completed degraded before the infiltrated cells began to secrete their own extracellular matrix. In contrast, tissue regeneration (fibroblasts, neo-collagen fibrils, and neo-capillaries) was observed for the 60%- and 95%-degree-crosslinking acellular tissues by the histological examination, immunohistological staining, transmission electron microscopy, and denaturation temperature measurement. The 95%-degree-crosslinking acellular tissue was more resistant against enzymatic degradation than its 60%-degree-crosslinking counterpart. Consequently, tissue regeneration was limited in the outer layer of the 95%-degree-crosslinking acellular tissue throughout the entire course of the study (1-year postoperatively), while tissue regeneration was observed within the entire sample for the 60%-degree-crosslinking acellular tissue. In conclusion, the crosslinking degree determines the degradation rate of the acellular tissue and its tissue regeneration pattern. In the third study, the tissue regeneration patterns in acellular bovine pericardia fixed with glutaraldehyde or genipin as a biological patch to repair a defect in the pulmonary trunk in a canine model. The implanted samples were retrieved at distinct durations postoperatively. The structural remodeling of retrieved samples was then examined. It was found that the degree of inflammatory reaction observed for the genipin-fixed acellular patch was significantly less than its glutaraldehyde-fixed counterpart. At 1-month postoperatively, intimal thickening was found on the inner surfaces of both studied groups. The intimal thickening observed on the glutaraldehyde-fixed acellular patch was significantly thicker than its genipin-fixed counterpart. An intact layer of endothelial cells was found on the intimal thickening of the genipin-fixed acellular patch, whereas endothelial cells did not universally and totally cover the entire surface of the glutaraldehyde-fixed acellular patch. Additionally, fibroblasts with neocollagen fibrils and myofibroblasts were observed in the acellular patches for both studied groups, an indication of tissue regeneration. This phenomenon was more prominent for the genipin-fixed acellular patch than its glutaraldehyde-fixed counterpart. At 6-month postoperatively, foci of chondroid and/or bony metaplasia were found in each retrieved sample for both studied groups. The observed adverse response of chondroid metaplasia may be attributed to a compliance mismatch at the implanted site of the canine pulmonary trunk following implantation or a lack of angiogenesis in the regenerated tissue observed at 1-month postoperatively. Bony metaplasia may then develop as in other chondroid tissues. It was reported that ischemia is a usual cause of metaplasia. In the fourth study, effects of ginsenoside Rg1 (Rg1), a natural compound isolated from Panax ginseng, on angiogenesis and tissue regeneration in a genipin-fixed acellular tissue (ECM) in vivo were investigated. Basic fibroblast growth factor (bFGF) was used as a control. The results obtained at 1-week postoperatively showed that the extent of angiogenesis in the ECM was significantly enhanced by bFGF or Rg1. At 1-month postoperatively, vascularzied neo-connective tissues were found to fill the pores within the ECMs loaded with bFGF or Rg1. There was a significant increase in the neo-capillary density from 1 week to 1 month for the ECM loaded with Rg1, while that observed in the ECM loaded with bFGF stayed approximately the same because of the limitations of protein stability. These results suggested that Rg1 may be a new class of angiogenic agent and may be loaded in the ECM for accelerating tissue regeneration. In the fifth study, the distinct varying GAGs content of genipin-fixed acellular tissue (ECM) loaded with bFGF, and its release from the ECM was analyzed. The tissue response to the ECM loaded with bFGF was evaluated by H&E after subcutaneous implantaton in rats. It was found that attachment of GAGs to ECM increased the bFGF binding capacity double and resulted in a more gradual and sustained release of bFGF in vitro. The in vivo results obtained at 1-week postoperatively showed that the extent of angiogenesis in the attachment of GAGs to ECM was significantly enhanced by bFGF.