Sandra L. Johnson

Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells

We have derived from normal human, mouse, and rat postnatal bone marrow primitive, multipotent adult progenitor cells (MAPCs) that can differentiate into most mesodermal cells and neuroectodermal cells in vitro and into all embryonic lineages in vivo. Here, we show that MAPCs can also differentiate into hepatocyte-like cells in vitro. Human, mouse, and rat MAPCs, cultured on Matrigel with FGF-4 and HGF, differentiated into epithelioid cells that expressed hepatocyte nuclear factor-3beta (HNF-3beta), GATA4, cytokeratin 19 (CK19), transthyretin, and alpha-fetoprotein by day 7, and expressed CK18, HNF-4, and HNF-1alpha on days 14-28. Virtually all human, as well as a majority of rodent cells stained positive for albumin and CK18 on day 21; 5% (rodent) to 25% (human) cells were binucleated by day 21. These cells also acquired functional characteristics of hepatocytes: they secreted urea and albumin, had phenobarbital-inducible cytochrome p450, could take up LDL, and stored glycogen. MAPCs, which can be expanded in vitro and maintained in an undifferentiated state for more than 100 population doublings, can thus differentiate into cells with morphological, phenotypic, and functional characteristics of hepatocytes. MAPCs may therefore be an ideal cell for in vivo therapies for liver disorders or for use in bioartificial liver devices.

Functional tissue-engineered valves from cell-remodeled fibrin with commissural alignment of cell-produced collagen.

Heart valve replacements composed of living tissue that can adapt, repair, and grow with a patient would provide a more clinically beneficial option than current inert replacements. Bioartificial valves were produced by entrapping human dermal fibroblasts within a fibrin gel. Using a mold design that presents appropriate mechanical constraints to the cell-induced fibrin gel compaction, gross fiber alignment (commissure-to-commissure alignment in the leaflets and circumferential alignment in the root) and the basic geometry of a native aortic valve were obtained. After static incubation on the mold in complete medium supplemented with transforming growth factor beta 1, insulin, and ascorbate, collagen fibers produced by the entrapped cells were found to coalign with the fibrin based on histological analyses. The resultant tensile mechanical properties were anisotropic. Ultimate tensile strength and tensile modulus of the leaflets in the commissural direction were 0.53 and 2.34 MPa, respectively. The constructs were capable of withstanding backpressure commensurate with porcine aortic valves in regurgitation tests (330 mmHg) and opened and closed under physiological pressure swings of 10 and 20 mmHg, respectively. These data support proof of principle of using cell-remodeled fibrin gel to produce tissue-engineered valve replacements.

Fibronectin and Its Fragments Increase With Degeneration in the Human Intervertebral Disc

Study Design. This laboratory-based experiment correlates fibronectin content of intervertebral disc with a morphologic grade of degeneration. Objectives. To correlate the fibronectin content of the anulus fibrosus and nucleus pulposus with a gross morphologic grade of disc degeneration, and to determine the molecular size of the extractable fibronectin. Summary of Background Data. Intervertebral disc degeneration increases with age and can lead to low back pain. Fibronectin helps to organize the extracellular matrix and provides environmental cues by interaction with cell surface integrins. In other tissues, its synthesis is elevated in response to injury. Fibronectin fragments can stimulate cells to produce metalloproteases and cytokines and inhibit matrix synthesis. Methods. In this study, 17 anuli fibrosis and 18 nuclei pulposus from 11 spines were graded by Thompson’s gross morphologic scale. Fibronectin was sequentially extracted with 4 mol/L guanidine hydrochloride and trypsin, and then quantitated by enzyme-linked immunoassay. The size of extractable fibronectin was determined by Western blot analyses. Results. The fibronectin content of the disc increased with grade and was significantly elevated between Grades 3 and 4. The percentage of extractable fibronectin varied widely, but it was more extractable from the nucleus. In both the nucleus and anulus, 30% to 40% of the extractable fibronectin existed as fragments. Many of the fragments contained functional heparin or collagen-binding sites. Conclusions. Fibronectin is elevated in degenerated discs and frequently present as fragments. Elevated levels of fibronectin suggest that disc cells are responding to the altered environment. Fibronectin fragments resulting from normal or enhanced proteolytic activity could be a mechanism that induces the cell to degrade the matrix further.

Linkage Analysis between the Major Histocompatibility System and Insulin-dependent Diabetes in Families with Patients in Two Consecutive Generations

We have histocompatibility (HLA) genotyped 28 families with insulin-dependent diabetics in two or more consecutive generations, usually parent and child. This strategy of ascertainment was used to maximize the likelihood of obtaining a homogeneous type of disease within a family, and an autosomal dominant mode of inheritance. 76 diabetics and 169 nondiabetics were studied in these families. The frequencies of the antigens Dw3 and Dw4, and the genotype Dw3/Dw4 among the diabetics are 59, 68, and 30%, respectively, as compared with 15, 12, and 2% in normal controls, and 43, 41, and 10% in the nondiabetic relatives of the diabetics. Dw2 is present in only one diabetic (4%), as compared with 18% in normal controls and 17% in nondiabetic relatives.HLA haplotype concordance was analyzed for sib pairs in relation to the haplotype shared by the affected parent/child pair, and for the diabetic sib pairs within each sibship. The results failed to reveal deviations in the expected HLA haplotype assortment. Assuming an autosomal dominant mode and several penetrance levels, linkage analysis between the HLA and diabetes was performed. The total lod score is 0.37 for a recombination fraction of 0.29 at 50% penetrance. Although the linkage and concordance analysis results are inconclusive, they seem to be different from those reported by us for families with normal parents and two or more diabetic sibs. Because ascertainment biases may have influenced these results in an unquantifiable manner, it is not certain whether the two types of families are genetically different. However, the marked difference in the lod scores for the 50% penetrant autosomal recessive model between the two types of families is compatible with a genetic dissimilarity between them. The high frequency of the Dw3 and Dw4 antigens, the Dw3/Dw4 genotype, and the decreased frequency of Dw2, however, indicate the existence of two or more important diabetic genetic factors associated with the D region of the HLA in these families.

Collagen architecture and failure processes in bovine patellar cartilage

Cartilage fails by fibrillation and wearing away. This study was designed to identify the microscopic failure processes in the collagen network of bovine cartilage using scanning electron microscopy. Cartilage samples from fibrillated cartilage from the bovine patella were removed from the bone, fixed, digested to remove proteoglycans, freeze‐fractured, and processed for SEM. The architecture of the collagen network in the normal cartilage was first defined, and then the failure processes were identified by examining sites of fibrillation and at crack tips. The bovine patellar cartilage was organised with a superficial layer composed of 3–5 lamina, attached to a sub‐superficial tissue by angled bridging fibrils. Collagen in the sub‐superficial tissue was organised in lamina oriented in the radial direction up to the transition zone. Failure of the system occurred by cracks forming in superficial layer and lamina, creating flaps of lamina that rolled up into the larger ‘fronds’. Larger cracks not following the laminar planes occurred in the transition, mid, and deep zones. Failure at the crack tips in the sub‐superficial tissue appeared to be by peeling of collagen fibrils, as opposed to breaking of collagen fibrils, suggesting a ‘glue’ bonding the collagen fibrils in a parallel fashion. Cracks propagated by breaking these bonds. This bond could be a site of disease action, since weakening of the bond would accelerate crack propagation.

Ruthenium-catalyzed photo cross-linking of fibrin-based engineered tissue

Most cross-linking methods utilize chemistry or physical processes that are detrimental to cells and tissue development. Those that are not as harmful often do not provide a level of strength that ultimately meets the required application. The purpose of this work was to investigate the use of a ruthenium-sodium persulfate cross-linking system to form dityrosine in fibrin-based engineered tissue. By utilizing the tyrosine residues inherent to fibrin and cell-deposited proteins, at least 3-fold mechanical strength increases and 10-fold stiffness increases were achieved after cross-linking. This strengthening and stiffening effect was found to increase with culture duration prior to cross-linking such that physiologically relevant properties were obtained. Fibrin was not required for this effect as demonstrated by testing with collagen-based engineered tissue. Cross-linked tissues were implanted subcutaneously and shown to have minimal inflammation after 30 days, similar to non-cross-linked controls. Overall, the method employed is rapid, non-toxic, minimally inflammatory, and is capable of increasing strength and stiffness of engineered tissues to physiological levels.

Severe Hypoglycemia During Maximized Insulin Treatment of Diabetes in a Randomized Clinical Trial

Our experience with hypoglycemia during a randomized clinical trial testing the hypothesis of a causal relationship between hyperglycemia and diabetic complications is described. Patients under maximized control experienced hypoglycemia much more often than patients under standard control. There were five episodes of serious hypoglycemia, none of them on pumps, in the former group, but none in the latter. Lack of compliance (or overcompliance) with certain aspects of intensive treatment increases the danger of grave hypoglycemia and should be part of exclusion criteria when considering maximized Control.

Interfibrillar Collagen Bonding Exists in Matrix Produced by Chondrocytes in Culture: Evidence by Electron Microscopy

Interfibrillar bonding of collagen fibrils in tissue grown from rabbit chondrocytes in culture was examined by a variety of electron microscopy techniques. Interfibrillar bonding is expected to increase tissue strength and may be a desirable feature in engineered cartilage and other soft tissues. The apparent bonding evident by scanning electron microscopy, using standard chemical fixation processing, is suspected to be artifact due to drying. The goal of this article was to establish the existence of interfibrillar bonding, apart from any processing artifacts. Specimens prepared by transmission electron microscopy, scanning electron microscopy (SEM) after notching and fixing under load, and cryo-SEM all showed evidence of bonding, supporting the existence of bonding in the unprocessed tissue. Exclusion from the bond space of gold particles labeled to decorin further supported the existence of natural bonds. Artifactual bonding may still be occurring with some of the methods used, but interfibrillar bonds exist in natural tissue. The bond distance was estimated to be 7-14 nm. Demonstration of the existence of these bonds supports further study of their mechanism and effect on tissue properties.

Functional Tissue-Engineered Valves from Cell-Remodeled Fibrin with Commissural Alignment of Cell-Produced Collagen

A piezoelectric component having resonance characteristics adjusted to a high degree of accuracy is provided. A piezoelectric substrate is provided with a resonating part. A deposit is added onto a surface of the resonating part and is provided with a plurality of indented portions at its surface enclosed by outer edges. The deposit is constituted of a resin which may contain a carbon filler. The indented portions are formed through laser machining.

A completely biological “off-the-shelf” arteriovenous graft that recellularizes in baboons

A decellularized tissue tube grown from human skin cells and fibrin becomes repopulated by recipient cells when tested in baboons as a vascular graft for hemodialysis access. Growing grafts for hemodialysis Patients undergoing hemodialysis for renal failure often receive an arteriovenous fistula, a connection between a vein and an artery. These surgical connections fail or cannot be attempted in some patients with compromised vasculature, who instead require vein grafts. As an alternative to autologous or synthetic grafts, Syedain et al. used a tissue engineering approach to generate vascular grafts from sacrificial fibrin scaffolds and human fibroblasts. Decellularized grafts were implanted into baboons and tested as hemodialysis access points. Over the course of 6 months, the grafts were recellularized with host cells and maintained sufficient burst pressure without evidence of immune rejection. Pending additional testing, these grafts represent an additional surgical option for hemodialysis access. Prosthetic arteriovenous grafts (AVGs) conventionally used for hemodialysis are associated with inferior primary patency rates and increased risk of infection compared with autogenous vein grafts. We tissue-engineered an AVG grown from neonatal human dermal fibroblasts entrapped in bovine fibrin gel that is then decellularized. This graft is both “off-the-shelf” (nonliving) and completely biological. Grafts that are 6 mm in diameter and about 15 cm in length were evaluated in a baboon model of hemodialysis access in an axillary-cephalic or axillary-brachial upper arm AVG construction procedure. Daily antiplatelet therapy was given. Grafts underwent both ultrasound assessment and cannulation at 1, 2, 3, and 6 months and were then explanted for analysis. Excluding grafts with cephalic vein outflow that rapidly clotted during development of the model, 3- and 6-month primary patency rates were 83% (5 of 6) and 60% (3 of 5), respectively. At explant, patent grafts were found to be extensively recellularized (including smoothelin-positive smooth muscle cells with a developing endothelium on the luminal surface). We observed no calcifications, loss of burst strength, or outflow stenosis, which are common failure modes of other graft materials. There was no overt immune response. We thus demonstrate the efficacy of an off-the-shelf AVG that is both acellular and completely biological.