Measurement of plasma glycoalbumin levels with a monoclonal antibody based ELISA

We have reported the establishment of hybridomas secreting monoclonal antibody that specifically recognizes glycated albumin. The antibody, raised in mice immunized with nonenzymatically glycated albumin isolated from human plasma, recognizes glycated epitopes residing in albumin but not in other plasma proteins, and does not react with unglycated albumin. We now report utilization of this antibody to measure the amount of nonenzymatically glycated albumin in human plasma. When immobilized onto microtiter wells in ELISA format, A717 yields a linear dose-response relationship upon incubation with authentic purified glycoalbumin. This allows construction of standard curves from which the amount of circulating glycoalbumin can be determined. The glycoalbumin level (mean +/- SEM) in 12 samples from nondiabetic subjects was 2.4 +/- 0.22% of total albumin. In normal plasma spiked with known amounts of purified glycoalbumin, values measured with this assay were close to 100% of expected values. Glycoalbumin levels in samples from 25 diabetic subjects ranged from 1.6 to 11.6%, with a mean +/- SEM of 4.5 +/- 1.2%. Glycoalbumin levels in diabetic samples correlated significantly (r = 0.93) with glycohemoglobin values. The ability of this assay to quantitate glycoalbumin, an index of the prevailing blood glucose concentrations over the preceding 2-3 weeks, makes it a favourable candidate for utilization in the clinical setting to monitor glycemic control in diabetic subjects.     

Effect of Oxidation-Modified Fibrinogen on the Formation and Lysis of Fibrin Clot in the Plasma

Turbidimetry studies showed that after addition of thrombin to fresh donor plasma light scatter in the sample increases and slowly attains a plateau. The process of fibrin formation was less intensive in the presence of oxidized fibrinogen. The formation of fibrin clot in lyophilized plasma was characterized by a biphasic kinetic of light scatter, oxidized fibrinogen inhibited both phases of the process. In the presence of streptokinase, oxidized fibrinogen did not modify the kinetics of fibrin clot lysis. Addition of oxidized fibrinogen to plasma reduced optical density of fibrin clot the more intensely the higher was the degree of oxidative modification of fibrinogen.     

Oxidative Modification of Fibrinogen Inhibits Its Transformation into Fibrin under the Effect of Thrombin

Changes in the capacity of fibrinogen subjected to oxidative modification to transform into fibrin under the effect of thrombin and to form a fibrin clot were studied. The effects of oxidized fibrinogen preparations on the clot formation by citrate-treated donor plasma were evaluated by the thrombin time test. Oxidation impaired the capacity of isolated fibrinogen to form a fibrin clot under the effect of thrombin. Addition of oxidized fibrinogen solutions to donor plasma led to prolongation of the plasma clotting time. Maximum addition (33% volume) of oxidized fibrinogen led to a 10-26% prolongation of clotting time in comparison with addition of the same volume of the same solution without fibrinogen. Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 147, No. 2, pp. 160-163, February, 2009     

Assay of fructosamine. Value and limits in diabetology

Fructosamine test using a Nitroblue Tetrazolium (NBT) method offers many advantages: quickness, reproducibility, easy automation and unexpansiveness, but a standardization of the different methods is needed. The results can be expressed in absolute value of equivalent DMF per liter, except in pregnancy where mumol per g of protein is used. The interpretation of the results can be difficult in case of quantitative and/or qualitative proteins abnormalities: icterus and severe chronic renal insufficiency. Fructosamine is significantly higher in diabetic patients. It gives a good correlation with glycated haemoglobin but the provided information is different, concerning a shorter period of 2 to 3 weeks and perhaps more sensitive to recent glycaemic variations. Fructosamine test does not seem to be a good screening test for diabetes and impaired glucose tolerance. The test indications of the assay could be: situations where the dosage of glycated haemoglobin is not interpretable, diabetic pregnancy follow-up and short term evaluation of a therapeutic change on glycaemic control. However, the individual significance of fructosamine concentration remains to be assessed and seems to be less accurate than glycated albumin.     

Quantitation of muscle glycogen synthesis in normal subjects and subjects with non-insulin-dependent diabetes by 13C nuclear magnetic resonance spectroscopy

To examine the extent to which the defect in insulin action in subjects with non-insulin-dependent diabetes mellitus (NIDDM) can be accounted for by impairment of muscle glycogen synthesis, we performed combined hyperglycemic-hyperinsulinemic clamp studies with [13C]glucose in five subjects with NIDDM and in six age- and weight-matched healthy subjects. The rate of incorporation of intravenously infused [1-13C]glucose into muscle glycogen was measured directly in the gastrocnemius muscle by means of a nuclear magnetic resonance (NMR) spectrometer with a 15.5-minute time resolution and a 13C surface coil. The steady-state plasma concentrations of insulin (approximately 400 pmol per liter) and glucose (approximately 10 mmol per liter) were similar in both study groups. The mean (+/- SE) rate of glycogen synthesis, as determined by 13C NMR, was 78 +/- 28 and 183 +/- 39 mumol-glucosyl units per kilogram of muscle tissue (wet weight) per minute in the diabetic and normal subjects, respectively (P less than 0.05). The mean glucose uptake was markedly reduced in the diabetic (30 +/- 4 mumol per kilogram per minute) as compared with the normal subjects (51 +/- 3 mumol per kilogram per minute; P less than 0.005). The mean rate of nonoxidative glucose metabolism was 22 +/- 4 mumol per kilogram per minute in the diabetic subjects and 42 +/- 4 mumol per kilogram per minute in the normal subjects (P less than 0.005). When these rates are extrapolated to apply to the whole body, the synthesis of muscle glycogen would account for most of the total-body glucose uptake and all of the nonoxidative glucose metabolism in both normal and diabetic subjects. We conclude that muscle glycogen synthesis is the principal pathway of glucose disposal in both normal and diabetic subjects and that defects in muscle glycogen synthesis have a dominant role in the insulin resistance that occurs in persons with NIDDM.     

Fibrin(ogen)-independent role of plasminogen activators in acetaminophen-induced liver injury

Hepatic fibrin(ogen) has been noted to occur after acetaminophen (APAP)-induced liver injury in mice. Deficiency in plasminogen activator inhibitor-1 (PAI-1), an endogenous inhibitor of fibrinolysis, increases APAP-induced liver injury in mice. However, the roles of fibrinogen and fibrinolysis in APAP-induced liver injury are not known. We tested the hypothesis that hepatic fibrin(ogen) deposition reduces severity of APAP-induced liver injury. APAP-induced (300 mg/kg) liver injury in mice was accompanied by thrombin generation, consumption of plasma fibrinogen, and deposition of hepatic fibrin. Neither fibrinogen depletion with ancrod nor complete fibrinogen deficiency [via knockout of the fibrinogen alpha chain gene (Fbg(-/-))] affected APAP-induced liver injury. PAI-1 deficiency (PAI-1(-/-)) increased APAP-induced liver injury and hepatic fibrin deposition 6 hours after APAP administration, which was followed by marked hemorrhage at 24 hours. As in PAI-1(-/-) mice, administration of recombinant tissue plasminogen activator (tenecteplase, 5 mg/kg) worsened APAP-induced liver injury and hemorrhage in wild-type mice. In contrast, APAP-induced liver injury was reduced in both plasminogen-deficient mice and in wild-type mice treated with tranexamic acid, an inhibitor of plasminogen activation. Activation of matrix metalloproteinase 9 (MMP-9) paralleled injury, but MMP-9 deficiency did not affect APAP-induced liver injury. The results indicate that fibrin(ogen) does not contribute to development of APAP-induced liver injury and suggest rather that plasminogen activation contributes to APAP-induced liver injury.     

Fibroblast migration in fibrin gel matrices.

In healing wounds and many solid tumors, locally increased microvascular permeability results in extravasation of fibrinogen and its extravascular coagulation to form a fibrin gel, with concomitant covalent cross-linking of fibrin by factor XIIIa. Subsequently, inflammatory cells, fibroblasts, and endothelial cells migrate into the gel and organize it into granulation tissue and later into mature collagenous connective tissue. To gain insight into some of the cell migration events associated with these processes, we developed a quantitative in vitro assay that permits the study of fibroblast migration in fibrin gels. Early passage human or rat fibroblasts were allowed to attach to tissue culture dishes and then were overlaid with a thin layer of fibrinogen that was clotted with thrombin. Fibroblasts began to migrate upwards into the fibrin within 24 hours and their numbers and the distance migrated were quantified over several days. The extent of fibroblast migration was affected importantly by the nature of the fibrin clot. Fibroblasts migrated optimally into gels prepared from fibrinogen at concentrations of -3 mg/ml; ie, near normal plasma fibrinogen levels. Migration was greatly enhanced by extensive cross-linking of the fibrin alpha-chains by factor XIIIa, as occurs when clotting takes place in vivo. When fibrinogen was clotted in Dulbecco's modified Eagle's medium, gamma-chains were cross-linked, but alpha-chain cross-linking was strikingly inhibited, and fibroblasts migrated poorly. Gels prepared from factor XIII-depleted fibrinogen exhibited neither alpha-nor gamma-chain cross-linking and did not support fibroblast migration. Further purification of fibrinogen by anion exchange high pressure liquid chromatography depleted fibrinogen of fibronectin, plasminogen, and other impurities; this purified fibrinogen clotted to form fibrin gels that supported reproducible fibroblast migration.     

The effect of fibrin polymerization inhibitors on quantitative measurements of plasma fibrinogen

A number of reports have highlighted discrepancies between the DuPont aca and fibrometer-based methods for quantitating plasma fibrinogen levels. Although many authors have suggested that the presence of fibrin(ogen) degradation products are in some way responsible for the discrepancies, no direct test of this hypothesis has been carried out. In this report, the authors demonstrate that inhibitors of fibrin monomer polymerization have different effects on the aca and fibrometer assays of fibrinogen. Fibrin polymerization inhibitors allow short soluble fibrin polymers to assemble that can scatter light and are therefore detected by the aca method. However, these short polymers do not gel and thus are not detected by the fibrometer. Therefore, the fibrometer method gives lower fibrinogen values than the aca in the presence of fibrin polymerization inhibitors. The authors also assayed plasma from a patient whose fibrinogen did not polymerize normally and therefore was not measurable by the fibrometer assay. The dysfibrinogenemic plasma assayed by the aca method recorded normal fibrinogen levels. In conclusion, the DuPont aca fibrinogen assay is insensitive to alterations of fibrin polymerization whether from inhibitors or a defective fibrinogen molecule.     

Thrombin activatable fibrinolysis inhibitor (TAFI) at the interface between coagulation and fibrinolysis

The thrombin-catalysed conversion of plasma fibrinogen into fibrin and the development of an insoluble fibrin clot are the final steps of the coagulation cascade during haemostasis. A delicate balance between coagulation and fibrinolysis determines the stability of the fibrin clot. Thrombin Activatable Fibrinolysis Inhibitor (TAFI) plays an important role in this process. TAFI is activated by thrombin and protects the fibrin clot against lysis. The role of TAFI in bleeding and thrombotic disorders is discussed as well as its novel emerging role in wound healing and inflammation.     

Preliminary study of the fibrin structure in hypertensive, dyslipidemic and type 2 diabetic patients

Individuals with hypertension, dyslipidemia or diabetes are at a higher risk to suffer cardiovascular disease than other people; while impaired fibrin structure/function may contribute to further raise the cardiovascular risk in the former. The purpose of this work was to study the fibrin network and fibrin degradation properties in hypertensive (HT) patients, pharmacologically treated, 124 +/- 11 mmHg, systolic blood pressure, and 70 +/- 10 mmHg, diastolic blood pressure, n = 12; metabolic dyslipidemic patients (DL), cholesterol: 5.7 +/- 1.5 mmol/L, n = 10; patients with type 2 diabetes mellitus (T2D), fasting plasma glucose: 8.8 +/- 2.2 mmol/L, n = 10; and a control group of healthy individuals, n = 9. The fibrinogen concentration was determined by the gravimetric method. Fibrin network formation and porosity were assessed by turbidity and permeation techniques, respectively; fibrin elastic properties were evaluated by compaction and fibrin lysis, by turbidity after addition of external tPA prior to plasma clotting. Fibrinogen concentration was significantly higher only in T2D patients (p = 0.004), compared to the control group. The fibrin polymerization and lysis processes were similar for all patient and control groups. Permeation was significantly slower in DL and T2D patients, p = 0.022 and 0.0002, respectively, whereas the compaction coefficient was significantly smaller in T2D patients, p = 0.0015. Our results suggest that the fibrin structure was altered in DL and T2D patients, probably due to the increased cholesterol and glycation, respectively.     

Type 2 diabetes mellitus and its renal complications in relation to apolipoprotein E gene polymorphism

The apolipoprotein E (APOE) epsilon2 allele is reported to be associated with greater risk of renal impairment in type 2 diabetes. Relationships among APOE polymorphisms, renal impairment, and biochemical parameters were explored. A prospective study of 405 consenting Chinese type 2 diabetic patients [mean age +/- standard deviation (SD): 59.2 +/- 10.3 years] without advanced complications at entry was conducted. APOE genotyping and measurement of plasma biomarkers of oxidative stress and antioxidants were performed at entry. HbA1C, plasma glucose, lipids, creatinine, urine albumin/creatinine, and blood pressure were measured at entry and at up to 4 years of follow-up. APOE allelic frequencies were in Hardy-Weinberg equilibrium. Odds ratios of albuminuria at entry and/or during follow-up for different APOE groups were not significantly different. The non-epsilon2 (epsilon3/3, epsilon3/4, epsilon4/4) group had significantly greater plasma ascorbate (51.6 +/- 20.1 mumol/L) than the epsilon2 (epsilon2/2, epsilon2/3) group (44.5 +/- 16.2 mumol/L, P = 0.021), but higher plasma ascorbate levels did not seem to decrease the risk of renal impairment in the non-epsilon2 group. Baseline plasma lipid-standardized alpha-tocopherol levels were least in epsilon2 subjects with persistent albuminuria (3.6 +/- 1.1 mumol/mmol of total cholesterol plus triglycerides, P = 0.008) compared with epsilon2 subjects who had no albuminuria at entry or during follow-up (4.5 +/- 0.8 mumol/mmol of total cholesterol plus triglycerides). The APOE epsilon2 allele does not seem to be associated with increased risk of renal impairment in Chinese type 2 diabetic patients. Plasma lipid-standardized alpha-tocopherol may play a role in determining risk of renal dysfunction in type 2 diabetes.     

含重组碱性成纤维细胞生长因子培养基中3T3成纤维细胞在血纤维蛋白凝块上生长的可能性（英文）

目的：重组碱性成纤维细胞生长因子对培养条件下３Ｔ３成纤维细胞在血纤维蛋白凝块上生长的可能研究。方法：采用Ｇｉｅｍｓａ染色和ＭＴＴ检测法,经过电镜扫描,分段对比观察,研究３Ｔ３细胞在血纤维蛋白凝块上的生长情况。结果：ｒｈｂＦＧＦ促进细胞生长产维持细胞存活的最佳浓度是１００ｎｇ／ｍｌ,在含有１００ｎｇ／ｍｌ的低血清培养基中细胞可在血纤维蛋白凝块生长。经４８ｈ培养以后仍有大量的细胞存活。结论：３Ｔ３成纤     

An evaluation of currently available methods for plasma fibrinogen

Fibrinogen levels in a variety of clinical plasma samples were assessed concurrently by several methods. Technics that appeared to provide the best improvements over the basic varieties of methods for fibrinogen assay were used. Results were correlated against a reference method based on Ancrod clottable fibrinogen and calibrated by ultraviolet absorbance with alkaline solutions of carefully dried fibrin standard. The best correlations with the reference method were achieved by an immunologic method using the Centrifichem principle and by heat precipitation with quantitation by packing in microhematocrit tubes. A modified clot opacity method also gave acceptable results. The turbidimetric ammonium sulfate and sodium sulfite precipitation methods correlated less well with the reference method, and in particular, the sodium sulfite technic gave high apparent fibrinogen levels with jaundiced plasmas. Neither of the turbidimetric methods were useful for fibrinogen levels below 50 mg/dl. The thrombin time method showed excellent sensitivity to fibrinogen, even at very low fibrinogen levels, but did not correlate well with the reference method.     

The Receptor for the Globular “Heads” of C1q, gC1q-R, Binds to Fibrinogen/Fibrin and Impairs Its Polymerization

The 33-kDa cellular C1q binding protein, designated gC1q-R was previously shown to bind a number of plasma proteins involved in the coagulation and kinin systems. This study demonstrates the interaction between recombinant gC1q-R and fibrinogen. Using enzyme-linked immunosorbent assays, biotinylated gC1q-R was found to bind to microplate-immobilized fibrinogen in a manner which was specific and inhibited by excess soluble fibrinogen or polyclonal antibodies directed against either gC1q-R or fibrinogen. Moreover, gC1q-R inhibited fibrin polymerization in a dose-dependent manner. Reptilase induced fibrin clot formation was completely inhibited by gC1q-R at a 2:1 molar ratio (gC1q-R:fibrinogen), and repolymerization of thrombin induced fibrin monomers was similarly abrogated. At equivalent molar concentrations, gC1q-R appeared to be a more potent inhibitor of fibrin polymerization than fibrinogen, a well-known inhibitor. Moreover, in the presence of both gC1q-R and soluble fibrinogen, the effect of each inhibitor on fibrin polymerization was additive. When plasmin derived fibrinogen degradation products, including the C-terminal D domain (D-100) or the N-terminal E domain, were immobilized on microtiter plates, gC1q-R bound to fibrinogen fragment D-100, but not to fragment E. Further digestion of fibrinogen fragment D-100 by plasmin to fragment D-60 resulted in loss of gC1q-R binding. Thus, gC1q-R binds to the D domain of fibrinogen/fibrin, and the carboxyterminal segment of at least the fibrinogen/fibrin γ chain appears important for this interaction. These observations may suggest a potential role for gC1q-R in modulating fibrin formation particularly at local sites of immune injury or inflammation.     

Quantitative evaluation of the fibrin clot extension on different implant surfaces: an in vitro study

The aim of the present study was a quantitative evaluation of the in vitro fibrin clot extension on different implant surfaces. Forty-five disk-shaped commercially pure Grade 2 titanium samples with three different surface topographies (machined, DPS, and Plus) were used in the present study. For the quantitative evaluation of the fibrin clot, 30 specimens were used (10 per group); human whole blood was employed. Venous blood was drawn from three healthy adult volunteers, and 0.2 mL were immediately dropped onto the surface of each specimen. Contact time was 5 min at room temperature; then the samples were rinsed with saline solution and fixed in a buffered solution of glutaraldehyde and paraformaldehyde. Samples were washed again with buffer and dehydrated in an ascending alcohol series. Specimens belonging to all groups were observed under SEM at a magnification of 1000x. From each sample, 50 random micrographs were collected in .tif format with an N x M 1024 x 768 grid of pixels. Quantitative analysis of fibrin clot extension showed the following results: in machined samples fibrin clot extension was 345987.2 +/- 63747.7 pixels(2) (mean +/- SD), in DPS samples fibrin clot extension was 375930.9 +/- 54726.86 pixels(2) (mean +/- SD), and in Plus samples, fibrin clot extension was 612333.6 +/- 46268.42 pixels(2) (mean +/- SD). With ANOVA it was possible to find that there were significant differences among the groups. The Tukey test revealed that the extension of the fibrin clot of Plus samples was statistically higher compared to both machined and DPS samples. The results of this in vitro study indicate that there is a correlation between implant surface morphology and fibrin clot extension. Improvement in surface microtexture complexity seems to determine the formation of a more extensive and three dimensionally complex fibrin scaffold. Further investigations are necessary to explain in more detail the mechanisms that regulate the fibrin clot formation on different implant surfaces.     

The receptor for the globular "heads" of C1q, gC1q-R, binds to fibrinogen/fibrin and impairs its polymerization

The 33-kDa cellular C1q binding protein, designated gC1q-R was previously shown to bind a number of plasma proteins involved in the coagulation and kinin systems. This study demonstrates the interaction between recombinant gC1q-R and fibrinogen. Using enzyme-linked immunosorbent assays, biotinylated gC1q-R was found to bind to microplate-immobilized fibrinogen in a manner which was specific and inhibited by excess soluble fibrinogen or polyclonal antibodies directed against either gC1q-R or fibrinogen. Moreover, gC1q-R inhibited fibrin polymerization in a dose-dependent manner. Reptilase induced fibrin clot formation was completely inhibited by gC1q-R at a 2:1 molar ratio (gC1q-R:fibrinogen), and repolymerization of thrombin induced fibrin monomers was similarly abrogated. At equivalent molar concentrations, gC1q-R appeared to be a more potent inhibitor of fibrin polymerization than fibrinogen, a well-known inhibitor. Moreover, in the presence of both gC1q-R and soluble fibrinogen, the effect of each inhibitor on fibrin polymerization was additive. When plasmin derived fibrinogen degradation products, including the C-terminal D domain (D-100) or the N-terminal E domain, were immobilized on microtiter plates, gC1q-R bound to fibrinogen fragment D-100, but not to fragment E. Further digestion of fibrinogen fragment D-100 by plasmin to fragment D-60 resulted in loss of gC1q-R binding. Thus, gC1q-R binds to the D domain of fibrinogen/fibrin, and the carboxyterminal segment of at least the fibrinogen/fibrin gamma chain appears important for this interaction. These observations may suggest a potential role for gC1q-R in modulating fibrin formation particularly at local sites of immune injury or inflammation.     

High Sensitivity Micro-Elastometry: Applications in Blood Coagulopathy

Highly sensitive methods for the assessment of clot structure can aid in our understanding of coagulation disorders and their risk factors. Rapid and simple clot diagnostic systems are also needed for directing treatment in a broad spectrum of cardiovascular diseases. Here we demonstrate a method for micro-elastometry, named resonant acoustic spectroscopy with optical vibrometry (RASOV), which measures the clot elastic modulus (CEM) from the intrinsic resonant frequency of a clot inside a microwell. We observed a high correlation between the CEM of human blood measured by RASOV and a commercial thromboelastograph (TEG), (R = 0.966). Unlike TEG, RASOV requires only 150 μL of sample and offers improved repeatability. Since CEM is known to primarily depend upon fibrin content and network structure, we investigated the CEM of purified clots formed with varying amounts of fibrinogen and thrombin. We found that RASOV was sensitive to changes of fibrinogen content (0.5–6 mg/mL), as well as to the amount of fibrinogen converted to fibrin during clot formation. We then simulated plasma hypercoagulability via hyperfibrinogenemia by spiking whole blood to 150 and 200% of normal fibrinogen levels, and subsequently found that RASOV could detect hyperfibrinogenemia-induced changes in CEM and distinguish these conditions from normal blood.     

New method to prepare autologous fibrin glue on demand

Fibrin-based sealants are commonly employed to arrest bleeding after surgery. Usually, fibrinogen obtained from pooled human plasma is used to prepare sealants, with attendant risk of blood-borne infections. Availability of autologous fibrinogen would eliminate this risk. To prepare autologous fibrin sealant, fibrinogen was precipitated from human plasma using protamine. Under optimal conditions (10-mg/mL protamine and 22 degrees C), 96 +/- 4% of clottable fibrinogen was recovered by a simple and inexpensive technique. Nearly 50% of the plasma factor XIII was also recovered with the fibrinogen. Using bovine thrombin, the fibrinogen was clotted (1) in a specially designed mold to measure tensile strength and (2) in a lap joint between 2 aortic vessel strips to measure adhesion strength. Tensile and adhesion strengths increased with increasing fibrinogen concentration, and they were increased by the addition of calcium chloride. The addition of aprotinin and -aminocaproic acid to the fibrinogen concentrate before clotting had no effect on the mechanical properties of the clots. After adding thrombin to sealant containing 15-mg/mL fibrinogen, maximum tensile strength was achieved in 1-5 min, and maximum adhesion strength was reached in 5-15 min. For the sealant with 30-60-mg/mL fibrinogen and added calcium, the tensile strength was equivalent to that of the commercial fibrin sealant Tisseel. The adhesion strength of sealant with 30-60-mg/mL fibrinogen exceeded the adhesive strength of Tisseel under identical conditions. Autologous fibrin sealant is an attractive alternative to commercial sealants. It can be readily prepared from 5-mL plasma or more and exhibits mechanical properties equivalent to those of the leading commercial sealant.     

The behavior of human mesenchymal stem cells in 3D fibrin clots: dependence on fibrinogen concentration and clot structure

Natural biopolymers such as human fibrin are appealing to tissue engineers, because fibrin is biocompatible, bioresorbable, and essential in normal wound healing. There have been numerous studies to date to develop a fibrin-based injectable cell delivery system, albeit with varying success. We propose that the outcome of fibrin cell delivery can, in part, be attributed to the relative concentrations of fibrinogen and thrombin solutions (i.e., formulations) and the structure of the final 3D fibrin clot. Formulation-dependent proliferation of human mesenchymal stem cells (hMSCs) within 3D fibrin clots was investigated in vitro. Our results indicate that hMSCs are viable in all fibrin sealant formulations investigated, and proliferation rates vary with fibrin formulations. Furthermore, the fibrinogen solution, not thrombin, was found to have a more dominant role on hMSC proliferation, with dilute fibrinogen solutions promoting greater hMSC proliferation. Confocal and electron microscopy reveal formulation dependence on 3D fibrin clot structure, with dilute fibrinogen solutions yielding more open, homogeneous microstructures. This study suggests that the concentrations of fibrinogen and thrombin solutions must be carefully considered for cell delivery because they affect 3D fibrin clot structure and cell proliferation.