Porcine small intestine submucosa (SIS) is not an acellular collagenous matrix and contains porcine DNA: possible implications in human implantation.

Porcine small intestinal submucosa (SIS) has been recommended as a cell-free, biocompatible biomaterial for the repair of rotator cuff tendon tear. However, we have observed noninfectious edema and severe pain in patients who have undergone SIS implantation for tendon repair. The aim of this study was to conduct an independent assessment of the safety and efficacy of Restore SIS membrane. The Restore orthobiologic implant was examined by histology and the nested PCR technique using porcine immunoreceptor DAP12 gene to examine if SIS membrane contained porcine cells or DNA, respectively. The material was also implanted into mice and rabbits for the evaluation of biological reaction and inflammatory response. Restore SIS was found to contain multiple layers of porcine cells. Chloroacetate esterase staining showed that some of these cells were mast cells. Nested PCR of the DAP12 gene demonstrated that Restore SIS contained porcine DNA material. Subcutaneous implantation of Restore SIS membrane in mice, and in rabbits for rotator cuff tendon repair, showed that the membrane caused an inflammatory reaction characterized by massive lymphocyte infiltration. In conclusion, Restore SIS is not an acellular collagenous matrix, and contains porcine DNA. Our results contradict the current view that Restore SIS is a cell-free biomaterial, and that no inflammatory response is elicited by its implantation. We suggest that further studies should be conducted to evaluate the clinical safety and efficacy of SIS implant biomaterials.

Molecular characterisation of chondrocytes in autologous chondrocyte implantation.

Autologous chondrocyte implantation (ACI) relies on the use of cultured cells. However, the biosynthetic profile of cultured chondrocytes is shown to be altered during in vitro expansion. The purpose of this study therefore, was to examine the cellular phenotype of chondrocytes cultured for ACI and to determine the apoptotic index of cells implanted into patients. Using electron microscopy, immunohistochemistry, RT-PCR and flow cytometry analyses, we have investigated protein and gene expression of several chondrocyte-specific, or associated markers in cultured cells used for implantation in patients. They included S-100, type I and II collagen, aggrecan, transforming growth factor beta, glucocorticoid receptor alpha and beta and vitamin D3 receptor. We have also examined the apoptotic index of chondrocytes. Our results demonstrated that cultured cells for ACI display the characteristics of chondrocytes. These cells are round in shape, contain numerous small surface processes of cytoplasmic membrane and have an accumulation of glycogen within the cytoplasm. They express S-100, aggrecan TGF-beta, glucocorticoid receptor alpha and vitamin D3 receptor as evidenced by either immunohistochemistry or RT-PCR, however, there is variation in the expression of type I, type II collagen glucocorticoid receptor beta between cases. Chondrocyte used for implantation has relatively low level of apoptosis (<11%). In conclusion, although there was variation in the level of expression of these genetic markers, our data indicate that cultured cells used for ACI were of chondrocytic lineage cells and have low level of apoptotic cells.

Dual phenotypic expression of hepatocytes and bile ductular markers in developing and preneoplastic rat liver.

This study supports the existence of a pluripotent liver stem cell population which has the potential to differentiate into hepatocytes and bile ductular cells. We compared the expression of hepatocyte-specific and bile ductular-specific markers in fetal and preneoplastic rat liver. L-pyruvate kinase (L-PK) and alpha glutathione S-transferase (GST) were used as adult hepatocyte-specific markers, while cytokeratin 19 (CK19) was used as a bile ductular-specific marker. pi GST and M2-pyruvate kinase (M2-PK), which are fetal hepatocyte-specific and expressed at high levels in the oval and duct-like cells, were also used. We characterized fetal liver derived from 13-21 days of gestation (E13-E21). pi GST was detected in the E18 hepatoblasts, which form the intrahepatic bile ducts, while CK19 was detected at E19. Some of these cells express alpha GST and L-PK from E19 to E21. Oval, duct-like and bile ductular cells in rats treated with a choline-deficient diet containing 0.07% ethionine (CDE diet) for up to 8 weeks were characterized by double immunocytochemistry. L-PK and alpha GST are absent from bile ductular cells in the normal adult liver and up to 3 weeks of CDE treatment. After 4-5 weeks on CDE treatment, the majority of bile ductular cells express L-PK, while at 6 weeks some co-express L-PK and alpha GST. There are two populations of oval cells, a major population expressing only the fetal hepatocyte markers, while a minor population expresses the fetal hepatocyte, adult hepatocyte and bile ductular markers. There are at least three different duct-like cell populations which co-express different markers and have characteristics of fetal hepatocytes at sequential stages of differentiation. One population co-expresses pi GST and M2-PK and is similar to fetal hepatocytes derived from E13-E14 fetuses. The second expresses the two fetal markers and L-PK, and this reflects characteristics of E15 hepatocytes. The third expresses pi GST, M2-PK, L-PK and alpha GST which is characteristic of E16-E19 hepatocytes. Upon withdrawal of the CDE diet, autoradiography using tritiated thymidine shows that oval and duct-like cells differentiate into hepatocytes. This study demonstrates that oval and duct-like cells express both hepatocytic and bile ductular markers, and have the capacity to differentiate into hepatocytes, characteristics similar to hepatoblasts in the developing rat liver.

Differentiation of oval cells into duct-like cells in preneoplastic liver of rats placed on a choline-deficient diet supplemented with ethionine.

Feeding male Wistar rats a choline-deficient diet containing 0.07% DL-ethionine (CDE diet) for up to 5 weeks results in the production of two distinct non-parenchymal cell populations, oval and duct-like cells. These cells can undergo replication and display different patterns of expression of glutathione S-transferases (GSTs) and pyruvate kinases (PKs). Oval cells were first detected around the periportal region after 1 week of CDE treatment and infiltrated the parenchyma after 2 weeks. Duct-like structures first appeared as isolated ducts in the parenchymal region at 2 weeks and were easily detected after 2.5 weeks. These duct-like structures differed from the bile ducts which reside in the portal region. Large concentrations of duct-like structures in cyst-like clusters were detected after 5 weeks. Enlargement of these structures from single ducts to clusters of up to 20 ducts was observed over 3-5 weeks of CDE treatment. The number of cells forming a duct increased from 5 to 30 cells. We established a double immunocytochemical staining technique to characterize the oval and duct-like cells for their expression of GSTs and PKs. pi GST and M2-PK, which are fetal hepatocytes isoenzymes, are present in virtually all the oval and duct-like cells. Most of the oval cells are devoid of the adult hepatocytes markers, alpha GST, mu GST and L-PK. There are two sub-populations of duct-like cells, one which expresses only fetal markers and the other which co-expresses the adult and fetal isoenzymes. Hence, oval cells display characteristics of fetal hepatocytes and some duct-like cells appear more mature than oval cells. Using a combination of double immunocytochemical and [3H]thymidine labelling techniques we have established that oval cells differentiate into duct-like cells.