Fibrin regulates neutrophil migration in response to interleukin 8, leukotriene B4, tumor necrosis factor, and formyl-methionyl-leucyl-phenylalanine

We have examined the capacity of four different chemoattractants/cytokines to promote directed migration of polymorphonuclear leukocytes (PMN) through three-dimensional gels composed of extracellular matrix proteins. About 20% of PMN migrated through fibrin gels and plasma clots in response to a gradient of interleukin 8 (IL-8) or leukotriene B4 (LTB4). In contrast, < 0.3% of PMN migrated through fibrin gels in response to a gradient of tumor necrosis factor alpha (TNF) or formyl-methionyl-leucyl-phenylalanine (FMLP). All four chemoattractants stimulated PMN to migrate through gels composed of collagen IV or of basement membrane proteins (Matrigel), or through filters to which fibronectin or fibrinogen had been adsorbed. PMN stimulated with TNF or FMLP adhered and formed zones of close apposition to fibrin, as measured by the exclusion of a 10-kD rhodamine-polyethylene glycol probe from the contact zones between PMN and the underlying fibrin gel. By this measure, IL-8- or LTB4-treated PMN adhered loosely to fibrin, since 10 kD rhodamine-polyethylene glycol permeated into the contact zones between these cells and the underlying fibrin gel. PMN stimulated with FMLP and IL-8, or FMLP and LTB4, exhibited very little migration through fibrin gels, and three times as many of these cells excluded 10 kD rhodamine-polyethylene glycol from their zones of contact with fibrin as PMN stimulated with IL-8 or LTB4 alone. These results show that PMN chemotaxis is regulated by both the nature of the chemoattractant and the composition of the extracellular matrix; they suggest that certain combinations of chemoattractants and matrix proteins may limit leukocyte movements and promote their localization in specific tissues in vivo.     

Poly(vinyl alcohol) synthetic polymer foams as scaffolds for cell encapsulation

Poly(vinyl alcohol) (PVA) foams were used as scaffolds in hollow fiber membrane-based cell encapsulation devices. The surrounding permselective membrane serves as an immunoisolation barrier while allowing metabolites and other small molecules to be freely transported. The internal matrix defines the microenvironment for the encapsulated cells. PC12 cell-containing devices represent one possible strategy for safe transplantation of dopamine-secreting cells for the treatment of dopamine-deficient diseases such as Parkinson's disease. PC12 cells--a dopamine-secreting cell line--were encapsulated with PVA foam as a matrix material in the lumen of these hollow fibers. In this work, we demonstrate the presence of the PVA matrix increased the catecholamine secretion efficiency of the cells as compared to devices containing a chitosan matrix. Devices were implanted in vivo into rodent striatum and device output of catecholamines was measured preimplant and post-explant. Evoked stores of dopamine remained constant (preimplant vs explant) for devices encapsulated with the foam matrix and increased with devices encapsulated with chitosan matrix. Cell proliferation within devices was inhibited in the presence of the foam matrix. Cell viability and distribution was significantly improved with the inclusion of the foam matrix in both in vitro and in vivo studies. In comparison to chitosan--a typical matrix material for PC12 cells--addition of a foam-type matrix altered the encapsulated cell microenvironment and resulted in more efficient secretion of catecholamines and improved distribution within the device resulting in smaller necrotic regions and a lower rate of cell proliferation.     

Chondrocyte-seeded hydroxyapatite for repair of large articular cartilage defects. A pilot study in the goat

The aim of this study was to evaluate the potential for restoration of a large cartilage defect in the goat knee with hydroxyapatite (HA) loaded with chondrocytes. Isolated chondrocytes were suspended in fibrin glue, seeded on top of the HA, and then the composite graft was implanted in the defect. After transplantation, cell behaviour, newly synthesised matrix and the HA-glue interface were assessed histologically after 2, 4, 12, 26 and 52 weeks. Special attention was paid to the incorporation process of HA in the subchondral bone and interactions between this biomaterial and the fibrin-glue-chondrocyte suspension. Chondrocytes in the glue proved to survive the transplantation procedure and produced new metachromatically stained matrix two weeks after implantation. The glue-cell suspension had penetrated the superficial porous structure of the HA. Four weeks after surgery, islands of hyaline-like cartilage were observed at the HA-glue interface. A layer of fibrous tissue was formed surrounding the HA graft, resulting in a relatively instable fixation of the HA in the defect. This instability of the graft in the defect, possibly together with early weight bearing, resulted in a gradual loss of the newly formed hyaline cartilage-like repair tissue. Progressive resorption of the HA occurred without any sign of active bone remodelling from the host site. One year after surgery part of the defect which extended down to the cancellous bone had been predominantly restored with newly formed lamellar bone. Only small HA remnants were still present at the bottom of the original defect. Resurfacing of the joint had occurred with fibrocartilaginous repair tissue. The absence of adequate fixation capacity of the HA near the joint space resulted in a relative instability of the graft with progressive resorption. Therefore, HA is not a suitable biomaterial to facilitate the repair of large articular cartilage defects.     

Metabolism of the extracellular matrix formed by intervertebral disc cells cultured in alginate

STUDY DESIGN: Cells from normal rabbit nucleus pulposus (NP) and anulus fibrosus (AF) were cultured in alginate beads for as long as 14 days to allow them to reform a matrix made up of two compartments: the cell-associated matrix (CM) and further removed matrix (FRM). At different time points, the CM and FRM made by each cell population were analyzed using histologic, biochemical, and immunologic assays. OBJECTIVES: To study the metabolism of normal rabbit NP and AF cells in alginate by characterizing the CM and FRM formed by each cell population, and to identify metabolic properties that may shed light on mechanisms at play in disc degeneration. SUMMARY OF BACKGROUND DATA: Little is known about the metabolism of intervertebral disc cells, in part because of the lack of microculture systems appropriate for the study of these cells in vitro. In recent studies from our laboratories, it was suggested that articular chondrocytes cultured in alginate beads remain phenotypically stable and reform a matrix similar to the one they populate in vivo. This culture system appears ideally suited for the study of intervertebral cells available only in limited numbers. METHODS: Rabbit NP and AF cells released from the matrix by sequential enzyme digestion were encapsulated in alginate beads (20,000 cells/bead) and cultured for as long as 14 days. At selected time points, beads were solubilized with calcium chelating agents, and the CM and FRM were isolated. The rate of 35S-sulfate incorporation into proteoglycans, and the contents of various extracellular matrix molecules (total sulfated proteoglycans, antigenic keratan sulfate, hyaluronan, collagen, and pyridinium crosslinks) were measured. RESULTS: Both NP and AF cells remained phenotypically stable in the alginate gel throughout the culture period and reestablished a matrix composed of CM and FRM compartments. The two cell populations exhibited numerous differences in their metabolic activities in vitro. Nucleus pulposus cells synthesized fewer proteoglycan and collagen molecules and were less effective in incorporating these into the CM than AF cells. CONCLUSIONS: Intervertebral disc cells, especially NP cells, are extremely sluggish in reforming a CM, a protective shell rich in proteoglycans and collagen molecules. This may help explain why damage to the NP often is accompanied by progressive degeneration of the disc in vivo.     

Healing of the anterior attachment of the rabbit meniscus to bone

In a rabbit model the healing process of the anterior attachment of the medical meniscus was observed during the first 12 weeks after sharp transection and refixation in a tibial bone channel. Evaluations of the healing tissue were histologic analysis, application of immunohistochemical methods to show collagen types and nerve regeneration, and mechanical load to failure tests. Secondary changes to knee joint cartilage, as signs of eventual dysfunction of the refixed meniscus, were evaluated by analysis of proteoglycan fragment concentration in joint fluid and histologic analysis of knee joint articular cartilage and synovium. The healing tissue between the refixed attachment and bone matured from highly cellular, nonspecific granulation tissue at 1 week, to bone, fibrocartilaginous, and fibrous tissues, which at some sites developed an insertion specific tissue arrangement within a 12-week period. However, the irregular interface between the fibrocartilaginous tissue and the underlying bone, which is typical for a normal insertion, was not reestablished. Labeling for collagen Types I and II in the newly formed insertion did not return to normal. In addition a few collagen fibers connected the refixed attachment tissue to bone. New bone formation turned the initially cancellous bone tunnel walls into more solid cortical bone. However, new bone formation did not fill the distal part of the channel. The refixed meniscal attachment underwent necrosis and was revitalized by cell ingrowth from the periphery. Nerve fibers were found in the newly formed insertion by 12 weeks. The failure load at tensile testing never reached more than 20% that of a normal attachment. Degeneration of articular cartilage and increased proteoglycan fragment in the joint fluid were common after this procedure. These data suggest that, despite the focal appearance of insertion specific tissues and healing of collagen fibers to bone, the tissue architecture of a normal meniscal insertion and a normal meniscal joint protective function were not reestablished.     

Alpha 2-macroglobulin of rabbits inhibits the habutobin activity

We have investigated whether alpha 2-macroglobulin (alpha 2M) of rabbits inhibits the activity of habutobin, a thrombin-like enzyme from Trimeresurus flavoviridis venom. Rabbit alpha 2M was purified with ultracentrifugation, gel filtration on Sepharose 6B and ion exchange chromatography on DEAE-Sephacel. Inhibitory effects of rabbit alpha 2M on habutobin was determined by the fibrin forming activity, digestion of A alpha chain of fibrinogen, and the release of fibrinopeptide A from fibrinogen. As a results, purified alpha 2M showed a single band with high molecular weight, around 800,000 mol. wt by means of polyacrylamide gel electrophoresis using PhastSystem. Besides inhibiting amidolytic and caseinolytic activity of porcine trypsin, it has inhibited the activity of habutobin: that is, in the presence of rabbit alpha 2M, fibrin forming activity of habutobin was decreased and habutobin-induced digestion of A alpha chain was inhibited. In addition, rabbit alpha 2M reduced habutobin-induced release of fibrinopeptide A from rabbit fibrinogen.     

A simple in vitro model to study the release kinetics of liposome encapsulated material

A simple in vitro model was developed to study the release kinetics of liposome encapsulated material in the presence of biologic components. Liposomes were embedded in an agarose gel (bottom layer) formed in a glass vial and separated from the receptor compartment buffer by a second layer of agarose gel (top layer). To follow the release of liposomal contents, aqueous space markers differing in molecular weight (from 205 Dalton to 17500 Dalton) were encapsulated. The isotonic buffer in the receptor was completely changed at various time points and the amount of marker released from the agarose matrix containing the liposomes into the receptor medium determined. The release of non-encapsulated markers from the gel followed a time0.5 relationship with about 75% of a 17500 Dalton protein being released from the matrix in 48 h. In the same period, about 7% of the intact liposomes added to the agarose gel appeared in the receptor phase. The release of calcein from various liposome compositions including: (A) egg phosphatidylcholine (EPC)/egg phosphatidylglycerol (EPG) 9:1, (B) dioleoylphosphatidylethanolamine (DOPE)/cholesterylhemisuccinate (CHEMS) 2:1, and (C) dioleoylphosphatidylglycerol (DOPC)/dioleoylphosphatidylglycerol (DOPG) 2:1 was measured. Components of the biological milieu such as serum proteins and calcium influenced release of encapsulated material. This in vitro model is a convenient and reproducible system that permits the study of the release of high molecular weight molecules such as proteins from liposomal formulations in the presence of serum. It may find applications with respect to release of proteins from a variety of colloidal drug delivery systems.     

Effect of zinc ions on fibrin network structure

In addition to calcium, other physiologically important divalent cations (magnesium and zinc) are known to influence fibrin formation and structure. We have studied the effect of different concentrations (0-20 micromol/l) of zinc ions (Zn2+) in the absence and presence of calcium on the gel structure formed in purified fibrinogen-enzyme systems. For that purpose, we used turbidity measurement, liquid permeation and confocal three-dimensional microscopy of the gel as well as sodium dodecyl sulphate (SDS)-gel electrophoresis. The results of turbidity measurements indicated that the clotting time decreased with increasing concentrations of Zn2+. The fiber mass: length ratio (mu) values showed that the porosity of the gels increased in a concentration-dependent manner, i.e. at higher concentrations of Zn2+, larger pores with thicker fibrin fibers were formed. Three-dimensional microscopy data of the gels were in good agreement with the mu data. On SDS-gel electrophores of reduced fibrin, no cross-linking was observed in the presence of zinc ions only (without the addition of calcium ions), nor were D-D dimer bands observed in non-reduced plasmin digested fibrin samples in the presence of zinc ions only. The above results show that zinc changes the fibrin gel structure and that this effect appears to be independent of calcium.     

Pathologic fibrin formation and cold-induced clotting of membrane oxygenators during cardiopulmonary bypass

In 1,800 patients undergoing cardiac surgery over a 2-year period, 11 incidents of abnormal inlet pressure elevations occurred before the membrane oxygenators. In 3 patients, the oxygenators had to be changed during cardiopulmonary bypass. This complication was found to be caused by fibrin formation possibly secondary to precipitation of fibrinogen with other coagulation factors in the heat exchangers of the oxygenators during the cooling phase. Large amounts of fibrin were demonstrated in the heat exchanger of the oxygenators. After careful washing of the apparatus, plasmin was added and fibrin was detected by measuring D-dimer levels. In heat exchangers from uneventful operations, only trace amounts of fibrin were found. Because there were no cold agglutinins demonstrated in the patients before surgery, cryoprecipitation studies were performed soon after surgery. When the patients' plasma samples were studied at different temperatures, from 37 degrees C down to 3 degrees C, cryoprecipitates or a gel (in 1 patient only) were formed. This indicated that there might be something abnormal with regard to fibrinogen-fibrin formation. The study patients were therefore investigated after the acute phase of the operation had ended for various coagulation factors, as well as for fibrin gel network characteristics. The results were compared with those of a control group (n = 10) with uneventful operations. There were no differences between the groups with regard to levels of coagulation factors VII and VIII and von Willebrand factor, although they were increased in both groups. The mean levels of coagulation inhibitors, antithrombin and Protein S, were slightly lower in the study patients. All of these patients had a highly pathologic, ie, tight fibrin gel network, except for the patient in whose sample a gel formed, despite being treated with aspirin or oral anticoagulants. The network was also tighter in some of the controls (v middle-aged reference individuals), although it was significantly tighter in the patients. It is concluded that some individuals who have an increased tendency to form tighter fibrin gel networks might be at increased risk for a severe complication during cardiac surgery performed under hypothermia.     

Human procarboxypeptidase U, or thrombin-activable fibrinolysis inhibitor, is a substrate for transglutaminases. Evidence for transglutaminase-catalyzed cross-linking to fibrin

Procarboxypeptidase U (EC 3.4.17.20) (pro-CpU), also known as plasma procarboxypeptidase B and thrombin-activable fibrinolysis inhibitor, is a human plasma protein that has been implicated in the regulation of fibrinolysis. In this study, we show that pro-CpU serves as a substrate for transglutaminases. Both factor XIIIa and tissue transglutaminase catalyzed the polymerization of pro-CpU and the cross-linking to fibrin as well as the incorporation of 5-dimethylaminonaphthalene-1-sulfonyl cadaverine (dansylcadaverine), [14C]putrescine, and dansyl-PGGQQIV. These findings show that pro-CpU contains both amine acceptor (Gln) and amine donor (Lys) residues. The amine acceptor residues were identified as Gln2, Gln5, and Gln292, suggesting that both the activation peptide and the mature enzyme participate in the cross-linking reaction. These observations imply that transglutaminases may mediate covalent binding of pro-CpU to other proteins and cell surfaces in vivo. In particular, factor XIIIa may cross-link pro-CpU to fibrin during the latter part of the coagulation cascade, thereby helping protect the newly formed fibrin clot from premature plasmin degradation. Moreover, the cross-linking may facilitate the activation of pro-CpU, stabilize the enzymatic activity, and protect the active enzyme from further degradation.     

Biocompatibility and immunology in the encapsulation of islets of Langerhans (bioartificial pancreas)

Successful transplantation of encapsulated islets (bioartificial pancreas) would circumvent problems of islet availability and rejection in the treatment of insulin-dependent diabetes with biological organ replacement. Alginates are widely used as a hydrogel matrix or membrane for immunoprotected transplantation. A major problem in the use of diffusion-based devices is the biocompatibility of the material used. The foreign body reaction after implantation of empty microcapsules into different compartments in rats, dogs and pigs is evaluated in this article. However, biocompatibility of the bioartificial pancreas has three different aspects: reaction of the entrapped islet to the encapsulation technique and material; reaction of the recipient against the incorporated device ( = foreign body reaction); and finally the reaction of the recipient against the encapsulated islet ( = immunology of bioartificial pancreas). It is obvious from different experiments that even if foreign body reactions (reactions against material) are almost abolished the recipient may react against material released from the encapsulated islet. In conclusion, transplantation of encapsulated islets induces various morphological reactions (i.e. inflammation and fibrosis) as a result of a variety of donor and recipient related factors. Therefore, the use of an adequate animal model that reflects the human situation is essential for progress in the development of a bioartificial pancreas.     

Fibrin in human plasma: gel architectures governed by rate and nature of fibrinogen activation

The porosity, fiber dimension and architecture of fibrin gels formed in recalcified plasma on addition of thrombin are, within a certain range of thrombin concentrations, determined by the initial rate of fibrinogen activation. Furthermore, the initial network formed in this range creates the scaffold into which subsequently activated fibrinogen molecules are deposited. Change in thrombin concentration that occurs during gelation, as a result of indigenous thrombin generation in plasma, does not qualitatively alter this scaffold. The formation of the networks obeys a more complex rule when low amounts of thrombin are added or with recalcified plasma without added thrombin. These networks are tighter than would be expected from the initial rate of fibrinogen activation. In this case an extremely porous network is probably formed initially, followed by formation of a secondary, superimposed network of a less porous architectural quality. The latter structure appears to be governed by the rate of indigenous generation in plasma of thrombin-like enzymes in combination with the particular type of fibrinmonomers being produced. In addition our findings establish the rules for proper determination of gel structures in clinical plasma samples. The sequelae of a variety of clot structures that may be formed in vivo are discussed.     

Immuno-isolation of xenogenic islands of Langerhans in a tissue engineered autologous cartilage capsule

Islet transplantation is a potential cure for diabetes mellitus. The major problem for clinical application remains the prevention of transplant rejection without major side effects. Broad application in early disease will make the usage of xenogeneic tissue necessary. Immunoisolation is an experimental strategy to prevent rejection, by separating the transplanted allogeneic or xenogeneic cells from the host immune system using a barrier device. Current methods of immunoisolation use artificial, not completely inert materials as barrier devices and induce an unwanted foreign body reaction. Using recipient own cells for encapsulation the foreign body reaction could be prevented. This study describes a new method of encapsulation of islets of Langerhans within a capsule of chondrocytes, which may serve as an immunoisolation barrier utilizing the immunoprivileged properties of the chondrocyte matrix and demonstrates the functional survival of the encapsulated islets in vivo.     

Role of fibrinopeptide B release: comparison of fibrins produced by thrombin and Ancrod

The gelation time, opacity, light scattering, and elastic moduli of human fibrin gels clotted in the presence of thrombin, Ancrod, and Reptilase have been compared. At low ionic strength lateral association to thick fibers is observed in all cases. At all ionic strengths thrombin fibrin forms thicker fibers than does Ancrod fibrin. We have demonstrated that an increase in the extent of lateral association is linked to an increase in its velocity and to a decrease in the gelation time. One may consider the removal of fibrinopeptide B to act as a switch: after it is removed fibrin assembles rapidly to thick fibers and gelation is fast; but when this peptide is still attached, there is a slow assembly of thin fibers, and gelation, especially of dilute fibrin, is delayed. We believe that this delay is critical for the complete digestion by plasmin of fibrin formed during in vivo defibrination with Ancrod and of fibrin produced by very small amounts of thrombin (which would still contain fibrinopeptide B), and that slow release of fibrinopeptide B is part of a control mechanism for the regulation of fibrin formation and the prevention of intravascular coagulation.     

Modelling biological gel contraction by cells: mechanocellular formulation and cell traction force quantification

Traction forces developed by most cell types play a significant role in the spatial organisation of biological tissues. However, due to the complexity of cell-extracellular matrix interactions, these forces are quantitatively difficult to estimate without explicitly considering cell properties and extracellular mechanical matrix responses. Recent experimental devices elaborated for measuring cell traction on extracellular matrix use cell deposits on a piece of gel placed between one fixed and one moving holder. We formulate here a mathematical model describing the dynamic behaviour of the cell-gel medium in such devices. This model is based on a mechanical force balance quantification of the gel visco-elastic response to the traction forces exerted by the diffusing cells. Thus, we theoretically analyzed and simulated the displacement of the free moving boundary of the system under various conditions for cells and gel concentrations. This model is then used as the theoretical basis of an experimental device where endothelial cells are seeded on a rectangular biogel of fibrin cast between two floating holders, one fixed and the other linked to a force sensor. From a comparison of displacement of the gel moving boundary simulated by the model and the experimental data recorded from the moving holder displacement, the magnitude of the traction forces exerted by the endothelial cell on the fibrin gel was estimated for different experimental situations. Different analytical expressions for the cell traction term are proposed and the corresponding force quantifications are compared to the traction force measurements reported for various kind of cells with the use of similar or different experimental devices.     

Adhesions: pathogenesis and prevention-panel discussion and summary

This article summarizes the discussions of the faculty and chairpersons on four major topics on postsurgical adhesions examined at the symposium, "Adhesions: Pathogenesis and Prevention". These topics are: 1) clinical significance; 2) pathogenesis; 3) research status and directions; and 4) recommendations for reduction or prevention. Abdominal postsurgical adhesions develop following trauma to the mesothelium, which is damaged often by surgical handling and instrument contact, foreign materials such as sutures and glove dusting powder, desiccation, and overheating. Postoperative adhesions occur after most surgical procedures and can result in serious complications, including intestinal obstruction, infertility, and pain. A long-term and unpredictable problem, postoperative adhesions impact the surgical workload and hospital resources, resulting in considerable health care expenditures. Although understanding of the pathogenesis of adhesions has improved recently, the molecular mechanisms involved continue to be delineated. Adhesions result from the normal peritoneal wound healing response and develop in the first five to seven days after injury. Adhesion formation and adhesion-free re-epithelialization are alternative pathways, both of which begin with coagulation which initiates a cascade of events resulting in the buildup of fibrin gel matrix. If not removed, the fibrin gel matrix serves as the progenitor to adhesions by forming a band or bridge when two peritoneal surfaces coated with it are apposed. The band or bridge becomes the basis for the organization of an adhesion. Protective fibrinolytic enzyme systems of the peritoneum, such as the plasmin system, can remove the fibrin gel matrix. However, surgery dramatically diminishes fibrinolytic activity. The pivotal events determining whether the pathway taken is adhesion formation or re-epithelialization are therefore the apposition of two damaged surfaces and the extent of fibrinolysis. Research in postsurgical adhesion formation and prevention abounds in a variety of avenues of investigation, including: 1) identification on a molecular level of the components involved in adhesiogenesis and their interactions; 2) clarification of the role of fibrin and fibrinolysis in adhesion formation; 3) standardization of design in preclinical and clinical studies of adhesion formation and prevention; 4) delineation of the relationship between adhesion formation and adhesive complications; and 5) elucidation of efficient, site-specific methods of prophylactic drug delivery. Currently, it seems logical to focus preventive research on development of barriers, fibrinolytic drugs, and selected agents such as phospholipids. The major strategies for adhesion prevention or reduction are adjusting surgical practice and applying adjuvants. Surgeons should adjust their major practices by: 1) becoming aware of the potential adhesive complications of a procedure; 2) minimizing the invasiveness of surgery; and 3) minimizing surgical trauma, ischemia, exposure to intestinal contents, introduction of foreign material into the body, and the use of talc- or starch-containing gloves. Available adjuvants include a newly developed by hyaluronic acid-phosphate-buffered saline solution applied intraoperatively to protect peritoneal surfaces from indirect surgical trauma and three mechanical barriers. One of these, a bioresorbable membrane consisting of hyaluronic acid and carboxymethylcellulose, has demonstrated efficacy and safety in both general and gynecological surgery. The other two barriers, one made of expanded polytetrafluoroethylene and one developed from oxidized regenerated cellulose, are indicated only for use in gynecological surgery.     

Characterization of ovomucoid-specific T-cell lines and clones from egg-allergic subjects

Three-dimensional gel culture systems represent conditions that mimic the differentiated state of mesenchymal cells in vivo. We examined gel contraction, cell growth, and phenotypic modulation of rabbit arterial SMC in three-dimensional gel culture. The gel contraction rate was dependent on the collagen type; that is, the contraction by freshly isolated SMC was faster and more pronounced in type I collagen than in type III collagen. In contrast, the phenotypic modulation of SMC was independent of collagen type. The major portion of cells in both type I and III collagens with growth factors underwent transition from a contractile (G0 phase) to a synthetic phenotype (G1B phase), but this transition was clearly delayed compared with that on collagens. The cells had hardly begun DNA synthesis in either collagen type and failed to proliferate even after 10 days of culture. These results indicate that collagen type is important in gel contraction by vascular SMC, while the organization of collagen fibrils (two-dimensional vs three-dimensional) is more critical in the phenotypic transition and proliferation of these cells. However, the more specific organization of extracellular matrix than the collagen gel culture system may be necessary to maintain the contractile phenotype of SMC.     

Fibrin deposition in squamous cell carcinomas of the larynx and hypopharynx

Extravascular fibrin deposition is frequently observed within and around neoplastic tissue and has been implicated in various aspects of tumor growth. The distribution of fibrin deposits was investigated in squamous cell carcinomas representing different stages of tumor progression of the larynx (n = 25) and hypopharynx (n = 9) by immunofluorescent techniques. Double and treble labelings were used to detect fibrinogen and fibrin in combination with marker antigens for tumor cells (cytokeratin), endothelial cells (von Willebrand factor), macrophages (recognized by KiM7), as well as factor XIII subunit A (FXIIIA) and tenascin (an embryonic extracellular matrix protein newly expressed during tumorigenesis). All tissue samples showed specific staining for fibrinogen/fibrin. Fibrin deposition was localized almost exclusively in the connective tissue compartment of tumors with characteristic accumulation at the interface of connective tissue and the tumorous parenchyma. In certain tumor samples showing highly invasive characteristics, fibrin deposits were observed in close association with tumor blood vessels in the tumor cell nodules. The overlapping reactions with polyclonal antibody to fibrinogen/fibrin and monoclonal antibody to fibrin indicate the activation of the coagulation cascade resulting in in situ thrombin activation and fibrin formation. Fibrin was crosslinked and stabilized by FXIIIA as revealed by urea insolubility test. Accumulation of phagocytozing macrophages detected by Ki M7 monoclonal antibody could be seen in areas of fibrin deposition. The blood coagulation factor XIIIA was detected in and around the cells labeled with Ki M7 antibody. Tenascin and fibrin deposits were found in the same localization in the tumor stroma and in association with tumor blood vessels within the tumor cell nodules. Neither fibrin nor tenascin were detected in the histologically normal tissue adjacent to tumors. The close association between fibrin deposits and macrophage accumulation strongly suggests the active participation of tumor-associated macrophages in the formation of stabilized intratumoral fibrin that facilitates tumor matrix generation and tumor angiogenesis.     

Effect of demineralized bone matrix on bone growth within a porous HA material: a histologic and histometric study

Coralline hydroxyapatite (cHA) is an osteoconductive material currently being used as a bone graft substitute. Created by the hydrothermal conversion of the calcium carbonate skeleton of coral to hydroxyapatite, this material has a porous structure similar to cancellous bone. Addition of demineralized bone matrix (DBM) would conceivably create a composite with both osteoconductive and osteoinductive properties. This pilot study evaluated the healing of rabbit cranial defects that had been filled with cHA or cHA augmented with a DBM gel formed by adding glycerol to the DBM particulate. Data from these were then compared to unfilled defects from a previous study. Results indicated enhancement of new bone formation and an increase in the rate of healing in the defects filled with the cHA-DBM gel composite. Further studies are warranted.     

Meniscal regeneration in the long-term after total meniscectomy?

A cohort of 39 patients (28 male, 11 female) that had undergone total meniscectomy as adolescents (mean age 16 years) underwent FISP 3D Magnetic Resonance Imaging at a mean follow up of 30 years. The presence of meniscal tissue was assessed by two independent observers blinded to the operation details. The volume of any meniscal tissue present was calculated. A posterior horn remnant was seen in 57% of medial and 45% of lateral meniscectomy cases. The mean volume of an operated medial meniscal remnant was 0.29 mL compared with a mean volume of 1.15 mL for an intact medial meniscus. The mean volume of an operated lateral meniscal remnant was 0.30 mL compared with 1.07 mL for an intact lateral meniscus. We have shown that the incidence of incomplete excision of the posterior horn is more common after total medial meniscectomy, and that at a mean follow up of 30 years there is no convincing in vivo MRI evidence of long-term meniscal regeneration.