Tissue engineering of cartilage using an injectable and adhesive chitosan-based cell-delivery vehicle.

OBJECTIVE: Adult articular cartilage shows a limited intrinsic repair response to traumatic injury. To regenerate damaged cartilage, cell-assisted repair is thus viewed as a promising therapy, despite being limited by the lack of a suitable technique to deliver and retain chondrogenic cells at the defect site. DESIGN: We have developed a cytocompatible chitosan solution that is space-filling, gels within minutes, and adheres to cartilage and bone in situ. This unique combination of properties suggested significant potential for its use as an arthroscopically injectable vehicle for cell-assisted cartilage repair. The primary goal of this study was to assess the ability of this polymer system, when loaded with primary articular chondrocytes, to support cartilage formation in vitro and in vivo. The chitosan gel was cultured in vitro, with and without chondrocytes, as well as injected subcutaneously in nude mice to form subcutaneous dorsal implants. In vitro and in vivo constructs were collectively analyzed histologically, for chondrocyte mRNA and protein expression, for biochemical levels of glycosaminoglycan, collagen, and DNA, and for mechanical properties. RESULTS: Resulting tissue constructs revealed histochemical, biochemical and mechanical properties comparable to those observed in vitro for primary chondrocytes cultured in 2% agarose. Moreover, the gel was retained after injection into a surgically prepared, rabbit full-thickness chondral defect after 1 day in vivo, and in rabbit osteochondral defects, up to 1 week. CONCLUSIONS: The in situ-gelling chitosan solution described here can support in vitro and in vivo accumulation of cartilage matrix by primary chondrocytes, while persisting in osteochondral defects at least 1 week in vivo.

Detection and analysis of cartilage degeneration by spatially resolved streaming potentials.

Cartilage molecular changes in osteoarthritis are most commonly related to the degradation and loss of proteoglycan and collagen fibrils of the extracellular matrix, which directly influence tissue stiffness and compression-generated streaming potentials. In this study, we evaluated the potential of a new technique, spatially resolved mapping of streaming potentials, to non-destructively indicate cartilage health or degeneration. Matched pairs of bovine cartilage/bone explant disks were cultured for 11 days in a serum free medium with and without interleukin-lalpha (IL-1alpha). The electromechanical properties (static stiffness, dynamic stiffness and streaming potentials) of cartilage disks were measured during unconfined compression using a mechanical tester coupled with a linear array of eight 50 microm diameter platinum-iridium microelectrodes. After 11 days of culture, the proteoglycan content of IL-1alpha treated disks was significantly reduced and the denatured and cleaved collagen content was increased compared to control disks. These biochemical alterations were concomitant with the reductions in the amplitudes of the static stiffness, the dynamic stiffness and the streaming potential profile as well as changes in the shape of the streaming potential profile. We found that spatial mapping of streaming potentials presents several advantages for the development of a clinical instrument to evaluate the degeneration of articular cartilage.

Streaming potentials maps are spatially resolved indicators of amplitude, frequency and ionic strength dependant responses of articular cartilage to load.

Streaming potential distributions were measured on the surface of articular cartilage in uniaxial unconfined compression using a linear array of microelectrodes. Potential profiles were obtained for sinusoidal and ramp/stress-relaxation displacements and exhibited dependencies on radial position, sinusoidal amplitude and frequency, time during stress relaxation, and on ionic strength. The measurements agreed with trends predicted by biphasic and related models. In particular, the absolute potential amplitude was maximal at the disk center, as was the predicted fluid pressure and the potential gradient (the electric field) was seen to be maximal at the disk periphery, as was the predicted fluid velocity. We also observed a similarity between non-linear behavior of streaming potential amplitude and load amplitude with respect to sinusoidal displacement amplitude. Taken together, these results support many of the phenomena concerning relative fluid-solid movement and fluid pressurization predicted by biphasic and related models, and they indicate the general utility of spatially resolved measurements of streaming potentials for the investigation of electromechanical phenomena in tissues. For example, these streaming potential maps could be used to non-destructively diagnose cartilage extracellular matrix composition and function, as well as to quantify spatially and temporally varying physical signals in cartilage that can induce cellular and extracellular biological responses to load.

Increased density of glucagon receptors in liver from endurance-trained rats.

The binding properties of glucagon receptors were determined in plasma membranes isolated from liver of untrained (n = 6) and swimming endurance-trained Sprague-Dawley male rats (n = 7; 3 h/day, 5 days/wk, for 8 wk). Plasma membranes were purified from liver by aqueous two-phase affinity partitioning, and saturation kinetics were obtained by incubation of plasma membranes (10 microg of proteins/150 microl) with (125)I-labeled glucagon at concentrations ranging from 0.15 to 3.0 nM for 30 min at 30 degrees C. Saturating curve analysis indicated no difference in the affinity of glucagon receptors (0.57 +/- 0.06 and 0.77 +/- 0.09 nM in untrained and trained groups, respectively) but a significant higher glucagon receptor density in liver from untrained vs. trained rats (3.09 +/- 0.12 vs. 4.28 +/- 0.19 pmol/mg proteins). These results suggest that the reported increase in liver glucagon sensitivity in endurance-trained subjects (Drouin R, Lavoie C, Bourque J, Ducros F, Poisson D, and Chiasson J-L. Am J Physiol Endocrinol Metab 274: E23-E28, 1998) could be partly due to an increased glucagon receptor density in response to training.

The recanalized umbilical vein in portal hypertension: a myth.

The demonstration of a vessel in the falciform ligament, traditionally presumed to be a reopened umbilical vein, is an important sonographic sign of portal hypertension. This vessel was sought in 200 umbilicoportographies (all portal hypertensive) and in 41 autopsy-dissected falciform ligaments (34 normal and seven cirrhotic). The normal falciform ligament contained one to three tiny collapsed paraumbilical veins. In cirrhotics, the number and caliber of paraumbilical veins increased. A reopened umbilical vein was never found. The authors conclude that the umbilical vein does not recanalize in portal hypertension. The vessel involved is actually an enlarged paraumbilical vein.

Idiopathic portal hypertension.

Idiopathic portal hypertension is reported in five cases including one case of chronic arsenical intake and one case of chronic industrial vinyl chloride exposure. In all five cases the patients presented with gastrointestinal bleeding as the chief complaint. Physical examination was within normal limits except for splenomegaly in all. Results of liver function tests were normal, except for the relative clearance of sulfobromophtalein. A surgical liver biopsy specimen was obtained in all cases and showed moderate degrees of portal fibrosis, but no cirrhosis. Combined umbilicoportal, hepatic vein and superior mesenteric artery catheterization was performed in all cases. Hepatoportographies showed distortion of the intrahepatic portal venous system and cut-off of small portal venules. Porto-hepatic gradients ranged from 14.0 to 20.5 mm Hg. The portal hypertension was both sinusoidal and presinusoidal in nature but mainly presinusoidal. Hepatic extraction of indocyanine green and of albumin microaggregates was normal, thereby suggesting normal functional portal blood supply to the liver. The patients with arsenical or vinyl chloride exposure could not be differentiated from the other three patients with idiopathic portal hypertension. These results suggest that idiopathic portal hypertension may be related to domestic or industrial exposure to other hepatotoxins.

Noncirrhotic presinusoidal portal hypertension associated with chronic arsenical intoxication.

A 39-year-old male with bleeding esophageal varices due to portal hypertension was observed. The patient had taken an arsenical preparation during a period of 12 yr because of psoriasis and subsequently developed keratotic changes of the palms and soles of his feet and an epithelioma of the scrotum. Physical examination was unremarkable except for splenomegaly and skin lesions. Liver function tests were normal; a needle biopsy of the liver (right lobe) showed nonspecific changes. Combined hepatic and umbilicoportal catheterization revealed, on splenography and portography, huge esophageal varices and patent portal vein; dilation, distortion, and cut-off of many intrahepatic portal branches were found. A marked gradient existed between the free portal venous pressure (25 mm Hg) and the wedged hepatic venous pressure (9.5 mm Hg). Hepatic blood flow, portal PO2, cardiac output, cardiac index, and blOOD volume were within normal range. Arteriographies did not reveal arteriovenous shunts in the splanchnic or splenic vessels. A splenorenal shunt were performed and a wedged biopsy of the liver (left lobe) revealed nonspecific changes. Three years later the patient had not experienced any episode of hemorrhage or hepatic encephalopathy but developed an epithelioma of the tongue. No known cause could be incriminated in the pathogenesis of the portal hypertension. However, there was unequivocal chronic arsenic intoxication. Toxic hepatitis, cirrhosis, noncirrhotic portal hypertension, and hemangiosarcoma of the liver have been reported with the intake of arsenicals. Thus, it is suggested that in this patient, presinusoidal portal hypertension was secondary to chronic arsenical intake associated with marked intrahepatic vascular changes seen on portography.

Combined hepatic vein, umbilicoportal vein, and superior mesenteric artery catheterization in portal hypertension: estimation of the portal fraction of total hepatic blood flow in cirrhotic patients.

Hemodynamic data were obtained in 13 cirrhotic patients with severe portal hypertension, undergoing combined hepatic vein, umbilicoportal vein, and superior mesenteric artery catheterization. The relative clearance of indocyanine green, the portohepatic gradient (difference between the free portal venous pressure and the free hepatic venous pressure), and the estimated hepatic blood flow were measured. The portal fraction (PF) of total hepatic blood flow was calculated in all patients using indicator dilution curves obtained from the portal bifurcation, a right hepatic vein, and when possible a left hepatic vein (six cases) after injection of (51)Cr-labeled red blood cells ((51)Cr RBC) into the superior mesenteric artery. Flows were overestimated because of loss of indicator through spontaneous portosystemic shunts; however, the ratio between hepatic and portal indicator dilution curves can be used to calculate the portal fraction of total hepatic blood flow since no extrahepatic shunts existed after the bifurcation of the portal vein (as shown on portography). In 10 patients, 15 series of curves were calculable and the PF varied between 30.1 and 100% (mean ± SE: 71.1 ± 6.2%). In the three other patients, only delayed activity from recirculation was detected from portal and hepatic vein samples and PF was 0%; in these three cases, portography and arteriography revealed spontaneous portacaval shunting with reverse and/or stagnant circulation in the portal vein. In the 13 patients, no correlation existed between PF and the relative clearance of indocyanine green or the portohepatic gradient, parameters generally used as indices of severity in cirrhosis. In 10 patients, no correlation was found between PF and the estimated hepatic blood flow.These data indicate that (51)Cr RBC dilution curves can be used for the estimation of the portal fraction of total hepatic blood flow in conscious cirrhotic patients before portacaval shunts. Using this methodology, it could be assessed whether any critical level of portal fraction exists above which poor clinical results occur after portacaval shunting. This measurement could eventually be helpful in determining the appropriate surgical procedure to be applied in individual cases.

Bleeding from esophageal varices in cirrhosis of the liver. Hemodynamic and radiological criteria for the selection of potential bleeders through hepatic and umbilicoportal catheterization studies.

Combined hepatic and umbilicoportal catheterization was performed in 38 compensated cirrhotics. Portohepatography with opacification of the coronary vein was obtained in all cases. The free portal venous pressure (FPVP) and the wedged (WHVP) and free (FHVP) hepatic venous pressures were recorded. The portohepatic gradient (FPVP-FHVP) was used as an index of portal hypertension. The coronary vein was separately re-evaluated for varices and graded as 1+ to 4+.Eighteen patients had varices graded as 3+ or 4+ (Group A) and all had a portohepatic gradient of 10 mm. Hg or more. The other 20 cirrhotics (Group B) had varices graded as 1+ or 2+ and 15 had a portohepatic gradient of less than 10 mm. Hg. The difference between gradients of Group A and Group B was highly significant.Of the 38 cirrhotics studied, eight had bled from varices and all are included in Group A. There is no significant difference between the gradients of both bleeders and non-bleeders of Group A.There is a significant correlation between the presence of large varices with a portohepatic gradient of 10 mm. Hg or more and a high risk of variceal bleeding. The radiological and hemodynamic data obtained by combined hepatic and umbilicoportal catheterization in cirrhosis of the liver can be of significant help in the selection of potential bleeders.