Calcification of subcutaneously implanted type I collagen sponges. Effects of formaldehyde and glutaraldehyde pretreatments.

Although collagen-containing implants are widely used in various surgical applications, there has been relatively little attention paid to the possibility that this type of biomaterial may undergo pathologic calcification which could compromise its function. The present study reports for the first time the calcification of a series of implants of purified collagen sponges prepared with graded degrees of aldehyde-induced cross-linkages (assessed by shrinkage-temperature, wetting time, and collagenase digestibility). Type I collagen sponges were pretreated with either glutaraldehyde (0.1% to 2.0% aqueous solution, for 5-180 minutes) or formaldehyde (as vapors for 15 minutes to 15 hours), and implanted subcutaneously for 21 days in weanling rats. Although specimens not pretreated with either aldehyde reagent and the formaldehyde sponges pretreated for 15 minutes were resorbed without evidence of calcification, all other aldehyde-pretreated implants mineralized. The degree of calcification did not correlate with extent of cross-linking. Formaldehyde-pretreated implants calcified more extensively (Ca2+ = 87.8 +/- 2.8 micrograms/mg, mean +/- standard error of the mean; n = 58) than did glutaraldehyde-pretreated implants (Ca2+ = 40.9 +/- 1.4 micrograms/mg; n = 52). It is concluded that both glutaraldehyde- and formaldehyde-pretreated Type I collagen sponges calcify after subdermal implantation in young rats. Although aldehyde pretreatment of Type I collagen sponge implants is a prerequisite for their eventual mineralization, the threshold level of aldehyde-induced cross-linking required to potentiate their maximal pathologic calcification is low.     

Longterm histopathologic follow-up of bronchial arteries after therapeutic embolization with polyvinyl alcohol (Ivalon) in patients with cystic fibrosis

We used light microscopy to examine, at autopsy, bronchial arteries in three patients with cystic fibrosis who died, respectively, 10, 16, and 28 months after bronchial artery embolization with barium sulfate-impregnated polyvinyl alcohol (PVA) to control hemoptysis. PVA was not identified beyond the midsegmental bronchus in any patient. Persistent focal fibrovascular occlusion was noted in two patients, and recanalized and/or partially obstructed vessels were associated with PVA in all. The histologic reaction to PVA included fibrosis, mild chronic inflammation, localized foreign body reaction, and, in two patients, focal calcification of PVA spicules. Within the inflammatory milieu were numerous macrophages containing BaSO4. Extensive vascular mural destruction and fibrosis associated with PVA were also observed. Both PVA and BaSO4 were also frequently present in the perivascular connective tissue. These findings indicate that, although longterm occlusion persists after therapeutic arterial embolization with PVA, focal recanalization also occurs. The extent of vascular mural injury following PVA embolization in humans has been previously underestimated by animal experiments. Finally, perivascular deposition of PVA represents a common reaction to diverse foreign body emboli in both systemic and pulmonary arteries.     

Ultrastructure of the giant cell infiltrate of subcutaneously implanted bone particles in rats and mice

The giant cells of soft tissues and those of mineralized tissues (osteoclasts) have distinctly different cell surface receptors and ultrastructural characteristics. Recently, the removal of dead bone particles in a subcutaneous environment has been described as a prototype of bone resorption, and a major issue is whether the giant cells that surround these ectopic bone implants and the processes involved in the disruption of bone surfaces are the same as those in the skeleton. We have compared the cytology and ultrastructure of giant cells recruited to subcutaneously implanted isogeneic bone particles with similar features of osteoclasts in metaphyseal bone of young normal rats and mice. Giant cells on surfaces of bone particles 2, 3, and 4 weeks after implantation were multinucleated, had a homogeneous, nonvacuolated cytoplasm, and had a bone surface interface unremarkable by light microscopy. In a few cells randomly distributed, small cytoplasmic vacuoles were present and large vacuoles were noted next to the bone surface at high magnification. By transmission electron microscopy, folded membrane configurations forming extensive interdigitations with adjacent cells were prominent features on most surfaces of giant cells. In instances where these interdigitations abutted bone surfaces, configuration resembling a ruffled border were noted, but these regions were always part of two different cells when examined at lower magnification or in serial sections. Breakdown of bone particles appeared to be by phagocytosis of small pieces and subsequent intracellular digestion in electron-dense cytoplasmic vacuoles. Osteoclasts from these same young animals were smaller with fewer nuclei, had cytoplasmic vacuoles concentrated next to bone surfaces, and had characteristic ruffled borders and clear zones. These results confirm those of others that native osteoclasts and multinucleated giant cells on dead bone particles are distinctly different with respect to both ultrastructure and mechanism of disruption of bone surfaces.     

Biologic determinants of dystrophic calcification and osteocalcin deposition in glutaraldehyde-preserved porcine aortic valve leaflets implanted subcutaneously in rats.

Bioprosthetic cardiac valve calcification is a frequent complication after long-term valve replacement. In this study the authors sought to examine the biologic determinants of this type of dystrophic calcification using subcutaneous implants of glutaraldehyde-preserved porcine aortic valve leaflets (GPVs) in rats. GPVs and clinical valvular bioprostheses were prepared identically. Retrieved implants were examined for calcification and the deposition of osteocalcin (OC), a vitamin K-dependent, bone-derived protein, that is found in other dystrophic and ectopic calcifications. GPVs implanted in 3-week-old rats calcified progressively (GPV Ca2+, 122.9 +/- 6.0 micrograms/mg) after 21 days, with mineral deposition occurring in a morphologic pattern comparable to that noted in clinical retrievals. Calcified GPVs accumulated osteocalcin (OC, 183.4 +/- 19.4 ng/mg); Nonpreserved porcine aortic leaflet implants did not calcify (Ca2+ + 5.6 +/- 1.0 micrograms/mg). Millipore diffusion chamber (0.45-mu pore size enclosed GPV implants accumulated calcium and adsorbed osteocalcin despite the absence of attached host cells. GPVs implanted for 21 days in 8-month-old rats calcified less (GPV Ca2+, 22.4 +/- 5.0 micrograms/mg) than did GPVs implanted in 3-week-old rats (see above). High-dose warfarin therapy (80 mg/kg) did not alter GPV calcification (GPV Ca2+, 39.6 +/- 2.9 micrograms/mg) in 72-hour subcutaneous implants in 3-week-old male rats, compared with control rats (GPV Ca2+, 40.8 +/- 4.8 micrograms/mg).     

A morphological assessment of macrophages attaching to subcutaneously implanted coverslips in dogs with chronic lymphoedema.

In an experimental model of chronic lymphoedema in the dog leg it has been shown that the presence of lymphoedema was associated with reduced numbers of macrophages found on subcutaneously implanted coverslips. Total numbers of mononuclear cells were not, however, significantly affected. When expressed as a percentage of the total mononuclear cells attached, the lymphoedematous limbs always had only half those of the corresponding normal limbs. Similarly, the percentage of macrophages with two or more pseudopods on the coverslips in the lymphoedematous limbs was only 1/3 of that for the same morphological criteria in normal limbs. However, when the macrophages with vacuoles were expressed as a percentage of the total macrophages, there was always a higher percentage in the lymphoedematous limbs compared with the corresponding normal ones.     

Calcification of poly(2-hydroxyethyl methacrylate) hydrogel sponges implanted in the rabbit cornea: A 3-month study

Poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogels have been used in the past as ocular implants. In a recent development, PHEMA sponges have shown suitable properties as materials for the peripheral component of an artificial cornea (keratoprosthesis). However, the propensity of PHEMA to calcify could threaten the long-term stability of the implanted devices. In an attempt to improve the understanding of the calcification mechanism, the dynamics, extent, and nature of calcified deposits within PHEMA sponges implanted in the cornea were investigated in this study, and the possible correlation between necrosis of cells and calcification was critically examined. Samples of a PHEMA sponge were implanted in rabbit corneas and explanted at predetermined time points (2, 4, and 12 weeks). The samples were examined by microscopy (light, transmission, scanning) and energy dispersive analysis of X-rays. Histological assessment and semiquantitative analysis of the amount of calcium deposited was performed using image analysis. An in vitro experiment was also performed by incubating sponge samples for 2 weeks in a solution of calcium and phosphate ions at a ratio similar to that in hydroxyapatite, in the absence of cells. Calcification was not seen in the 2- and 4-week explants, however, small deposits were detected in two of the 12-week explants, both within and on the sponge's constituent polymer particles. The deposit volumes represented 0.094% and 0.21%, respectively, of the total sponge volumes. Calcium deposits were present in large amounts both within the constituent polymer particles and on the surface of the sponges incubated in the abiotic calcifying solution. Cooperative mechanisms are suggested for the calcification of PHEMA sponges in vivo. The initial event may occur at a molecular level, when plasma proteins are adsorbed onto the polymer surface and bound through chelation to the calcium ions present in the medium. After their natural degradation, these structures may act as nucleation sites for calcium phosphate crystallization. Concurrently, the calcium ions can diffuse into the hydrogel particles and then the spontaneous precipitation of calcium phosphate may be caused by supersaturation due to the lower content of water in polymer, an effect which is likely predominant in vitro. The second event is the recruitment of phagocytic cells to clear calcium debris. Degeneration of these cells may then form nucleation sites for secondary calcification.     

Uptake of polyvinyl alcohol by macrophages in the glomerular mesangium of rats. Histologic and functional studies

Rats received daily subcutaneous injections of the synthetic polysaccharide polyvinyl alcohol (PVA) for 1-28 days. The amount of PVA localized in the glomerular mesangium increased progressively during this time. By 28 days, all glomeruli showed extensive intracellular mesangial sequestrations of PVA, causing marked widening of mesangial areas, while the peripheral capillary loops were unaltered. Overall glomerular hypercellularity was mild to moderate, occurring mainly in areas of PVA deposition. Follow-up studies after 6, 12, 26, and 40 weeks revealed partial reduction of glomerular PVA masses. The PVA deposits were frequently associated with nonspecific esterase (NSE)-positive cells. The number of NSE-positive cells per glomerular tuft section increased from 0.1 in controls, to 2.1, 4.0, and 4.5 after 3, 14, and 28 days of PVA treatment, respectively. Similarly, glomerular counts for Ia-antigen-bearing cells rose from 2.1 in controls to 4.8 on Day 3 and showed further increases at later time periods with confluent staining of clusters of Ia-positive cells. In glomeruli, Ia-bearing cells were mainly noted in PVA-positive mesangial areas. These results indicate that PVA is taken up in the glomerulus primarily by cells that are NSE- and Ia-antigen-positive, suggesting that these cells are activated blood-borne monocyte-macrophages that sequester this polysaccharide. Clearance studies revealed that the glomerular filtration rate and effective renal plasma flow remained normal after 4 weeks of PVA injections. PVA-treated rats showed only mild elevations of urinary protein excretion. These findings indicate that confinement of marked structural and cellular alterations to the mesangium, even including the presence of infiltrating monocyte-macrophages, is compatible with absent or minimal dysfunction of the glomerular ultrafilter.     

Apoptosis mediates the decrease in cellularity during the transition between granulation tissue and scar.

Granulation tissue formation and contraction is an important step of second intention wound healing. Granulation tissue develops from the connective tissue surrounding the damaged or missing area and its cellular components are mainly small vessel and inflammatory cells as well as fibroblasts and myofibroblasts. As the wound closes and evolves into a scar, there is an important decrease in cellularity; in particular myofibroblasts disappear. The question arises as to which process is responsible for this cellular loss. During a previous investigation on the expression of alpha-smooth muscle actin in myofibroblasts (Darby I, Skalli O, Gabbiani G, Lab Invest, 1990, 63:21-29), we have observed that in late phases of wound healing, many myofibroblasts show changes compatible with apoptosis and suggested that this type of cell death could be responsible for the disappearance of myofibroblasts. We have now tested this hypothesis by means of morphometry at the electron microscopic level and by in situ end labeling of fragmented DNA. Our results indicate that the number of myofibroblastic and vascular cells undergoing apoptosis increases as the wound closes and support the assumption that this is the mechanism of granulation tissue evolution into a scar. The regulation of apoptotic phenomena during wound healing may be important in scar establishment and development of pathological scarring.     

Ontogeny of ethanol intake in alcohol preferring (P) and alcohol nonpreferring (NP) rats

There is a scarcity of research on ethanol affinity in alcohol‐preferring (P) rats before weaning and it is unknown if neonate P rats exhibit ethanol intake preferences comparable to those observed in adult P rats. This study examined ethanol intake in P and alcohol‐nonpreferring (NP) rats 3 hr after birth (Experiment 1, surrogate nipple test), at postnatal days (PD) 8, 12, and 18 (Experiment 2, consumption from the floor procedure) and at adolescence (Experiment 3, two‐bottle choice test at PD32). The high‐preference genotype was readily expressed 3 hr after birth. P neonates drank twice as much ethanol as their NP counterparts. This heightened ethanol preference transiently reversed at P8, reemerged as weaning approached (P18) and was fully expressed during adolescence. These results help to clarify the ontogeny of genetic predisposition for ethanol. Genetic predisposition for higher ethanol intake in P than in NP rats seems to be present immediately following birth. © 2010 Wiley Periodicals, Inc. Dev Psychobiol 53:234–245, 2011.     

Analysis of the leukotriene D4 receptor in the granulation tissue of allergic inflammation in rats.

Leukotriene (LT) D4 receptor in the granulation tissue formed in the air pouch-type allergic inflammation model in rats was analyzed. Membrane preparation of the granulation tissue obtained 3-9 days after the antigen challenge has specific binding sites of [3H]LTD4. Scatchard analysis showed that the affinity (Kd) and the density (Bmax) were not changed among the granulation tissue obtained 3-9 days after the antigen challenge. The Kd value in the granulation tissue (0.90 +/- 0.12 nM) was close to that in the rat lung (1.00 +/- 0.24 nM) and the guinea pig lung (0.86 nM). On the other hand, Bmax (62 +/- 8 fmol/mg protein) in the granulation tissue was higher than that in the rat lung (21 +/- 4 fmol/mg protein) but was far less than that in the guinea pig lung (405 fmol/mg protein). LTC4 and LTE4 inhibited the binding of [3H]LTD4 to the membrane preparation of the granulation tissue in a concentration-dependent manner. IC50 of LTC4 and LTE4 were 1 x 10(-7) and 2 x 10(-7) M, respectively. A guanine nucleotide, guanyl-5'-yl-imido-diphosphate (GppNHp), reduced [3H]LTD4 binding to the membrane preparation of the granulation tissue suggesting that LTD4 receptors in the granulation tissue are associated with G proteins. These results indicate that LTD4 binding sites in the granulation tissue are high affinity receptors for LTD4. A possible role of LTD4 in the recurrence of allergic inflammation in the chronic phase is discussed.     

Biocompatibility and biodegradation of different hyaluronan derivatives (Hyaff) implanted in rats

Hyaluronan (HL), a naturally occurring glycosaminoglycan, has been chemically modified through the esterification of its carboxylic groups with different types of alcohol. The physico-chemical properties of these new biopolymers allow the preparation of many biomaterials which may be used in several medical applications. In the present study both the biocompatibility and biodegradation of some water-insoluble HL esters have been evaluated, either as raw material or as manufactured devices after subcutaneous and intraperitoneal implantation in male rats. The inflammatory response and the degree of resorption for each tested material are reported. The relationships between the degree of esterification and the type of alcohol used with the above parameters are also investigated.     

Design and manufacture of a polyvinyl alcohol (PVA) cryogel tri-leaflet heart valve prosthesis

Although current artificial heart valves are life sustaining medical devices, improvements are still necessary to address deficiencies. Bioprosthetic valves have a compromised fatigue life, while mechanical valves have better durability but are prone to thromboembolic complications. A novel, one-piece, tricuspid valve, consisting of leaflets, stent and sewing ring, made entirely from the hydrogel, polyvinyl alcohol cryogel (PVA-C), has been developed and demonstrated. This valve has three thin leaflets attached to a cylindrical stent. In order to approximate the complex shape of the surface of the natural heart valve leaflets, two different geometries have been proposed: revolution about an axis of a hyperboloid shape and revolution about an axis of an arc subtending (joining) two straight lines. The parameters of both geometries were examined based on a compromise between avoiding sharp curvature of leaflets and minimization of the central opening of the valve when closed. The revolution of an arc subtending two straight lines was selected as the preferred geometry since it has the benefit of a smaller central opening when the value of the maximum curvature for the leaflets is the same for each valve geometry. A cavity mold has been designed and constructed to form the PVA-C heart valve. The three leaflets were formed and integrated into the stent and sewing ring in a single process. Prototype heart valves were manufactured in the mold from a solution of PVA and water, by controlled freezing and thawing cycles.     

Collagen development in granulation tissue as compared with collagen of skin and aorta from injured and non-injured rats.

Granulation tissue in rats was produced by subcutaneous implantation of viscose cellulose sponges. Granulomas, aortae, and skin samples were taken 4, 8, 14, 22, 33, and 42 days after the sponge implantation and compared with age-matched non-operated rats. 14C-proline was given 4 hours before death to animals killed on day 0, 14, and 42. The 14C-OH-proline activity in salt insoluble collagen was higher in granulation tissue and aorta than in skin. This indicates a faster formation, or an increased stability of the intermolecular cross-links in granulation tissue and aorta, than in skin. The percentage of free OH-proline was than in skin, reflecting a relatively increased collagen degradation in granulation tissue. An increased collagen degradation may also, in part, explain a registered higher alpha/beta ratio in collagen from granulation tissue than from skin, as well as the increase in alpha/beta ratio in the older granulomas. The sponge implantation did not affect the collagen of aorta and skin, but caused a decrease in the dry weight of aorta and skin, and an increase in the number of granulocytes in the blood.     

A review of polyvinyl alcohol and its uses in cartilage and orthopedic applications

Polyvinyl alcohol (PVA) is a synthetic polymer derived from polyvinyl acetate through partial or full hydroxylation. PVA is commonly used in medical devices due to its low protein adsorption characteristics, biocompatibility, high water solubility, and chemical resistance. Some of the most common medical uses of PVA are in soft contact lenses, eye drops, embolization particles, tissue adhesion barriers, and as artificial cartilage and meniscus. The purpose of this review is to evaluate the available published information on PVA with respect to its safety as a medical device implant material for cartilage replacement. The review includes historical clinical use of PVA in orthopedics, and in vitro and in vivo biocompatibility studies. Finally, the safety recommendation involving the further development of PVA cryogels for cartilage replacement is addressed.     

Negative affect and voluntary alcohol consumption in Wistar-Kyoto (WKY) and Sprague-Dawley rats.

Based on the assumption that the Wistar-Kyoto (WKY) rat strain represents an animal model for depressive behavior, the purported relationship between depression and alcohol consumption was investigated in three experiments. WKY rats consumed more alcohol than Sprague-Dawley (S-D) rats when offered a choice between a 7% alcohol solution and tap water. Subsequently, the severity of stress-induced stomach ulcers was significantly less in WKY rats that had access to alcohol. In Experiment 2, WKY and S-D rats were assigned to either an alcohol access treatment or to a water-only treatment for 27 days and subsequently observed in the open-field test (OFT) and the elevated plus-maze (EPM). Access to alcohol reduced response latency in the OFT, and increased the percent time in the open arm and the total number of arm entries in the EPM for WKY rats. In Experiment 3, the antidepressant, imipramine, reduced alcohol consumption in both strains and significantly increased percent time in the open arms of the EPM for WKY rats. These studies support the assumption that depression and alcohol consumption may be related.     

Calcification of poly(2-hydroxyethyl methacrylate) hydrogel sponges implanted in the rabbit cornea: a 3-month study

Poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogels have been used in the past as ocular implants. In a recent development, PHEMA sponges have shown suitable properties as materials for the peripheral component of an artificial cornea (keratoprosthesis). However, the propensity of PHEMA to calcify could threaten the long-term stability of the implanted devices. In an attempt to improve the understanding of the calcification mechanism, the dynamics, extent, and nature of calcified deposits within PHEMA sponges implanted in the cornea were investigated in this study, and the possible correlation between necrosis of cells and calcification was critically examined. Samples of a PHEMA sponge were implanted in rabbit corneas and explanted at predetermined time points (2, 4, and 12 weeks). The samples were examined by microscopy (light, transmission, scanning) and energy dispersive analysis of X-rays. Histological assessment and semiquantitative analysis of the amount of calcium deposited was performed using image analysis. An in vitro experiment was also performed by incubating sponge samples for 2 weeks in a solution of calcium and phosphate ions at a ratio similar to that in hydroxyapatite, in the absence of cells. Calcification was not seen in the 2- and 4-week explants, however, small deposits were detected in two of the 12-week explants, both within and on the sponge's constituent polymer particles. The deposit volumes represented 0.094% and 0.21%, respectively, of the total sponge volumes. Calcium deposits were present in large amounts both within the constituent polymer particles and on the surface of the sponges incubated in the abiotic calcifying solution. Cooperative mechanisms are suggested for the calcification of PHEMA sponges in vivo. The initial event may occur at a molecular level, when plasma proteins are adsorbed onto the polymer surface and bound through chelation to the calcium ions present in the medium. After their natural degradation, these structures may act as nucleation sites for calcium phosphate crystallization. Concurrently, the calcium ions can diffuse into the hydrogel particles and then the spontaneous precipitation of calcium phosphate may be caused by supersaturation due to the lower content of water in polymer, an effect which is likely predominant in vitro. The second event is the recruitment of phagocytic cells to clear calcium debris. Degeneration of these cells may then form nucleation sites for secondary calcification.     

Calcification of poly(2-hydroxyethyl methacrylate)-collagen composites implanted in rats

Samples of the polyHEMA-collagen composites with varying collagen content have been implanted into the popliteal region of rats. Three, six and twelve months after the implantation, calcification of the implanted material was determined using a radioactive indicator. At the same time, the implants and surrounding tissue were examined histologically. The degree of calcification of the implants was dependent on the collagen content; it was more pronounced with a higher amount of collagen. The composites with 30% (w/w) or more collagen were biodegraded during the long-term implantation. It is suggested that the composites containing less than 20% (w/w) of fibrillar collagen are used for biomedical applications and that those with a higher collagen content for the in vitro studies.