In vivo approach of calcium deficient hydroxyapatite filler as bone induction factor.

Tissue engineering combine biomaterials, cells and biologically active molecules having as a goal create functional tissues; many of the compositions are blends of a polymeric matrix with ceramic fillers, however, reduction of mechanical resistance can be a drawback on ceramic-polymer systems. In this manuscript, we investigate the potential of calcium-deficient hydroxyapatite (CDHA) whiskers, a needle shape bioceramic, to enhance mechanical and osteoconduction properties on the polymeric matrix. For this purpose, PCL scaffolds incorporating CDHA whiskers were produced by combining solvent casting and particulate leaching techniques to develop a composite scaffold that possess mechanical and biological properties which is useful for bone tissue engineering regeneration. We produced CDHA whiskers using alkaline hydrolysis of α-tricalcium phosphate and characterized by XRD, XRF and SEM. PCL/CDHA scaffolds were fabricated with a final porosity of ~70%, quantified by SEM images. Mechanical properties were evaluated by compression test. As an initial test, PCL/CDHA scaffolds were immersed in simulated body fluid to quantify apatite deposition. In vitro and in vivo studies were performed to assess cytotoxicity and bioactivity. CDHA whiskers exhibited a needle-like morphology and a Ca/P ratio equal to calcium deficient hydroxyapatite. The composite scaffolds contained interconnected pores 177 to 350 µm in size and homogeneous ceramic distribution. The addition of CDHA whiskers influences the mechanical results: higher elastic modulus and compressive strength was observed on PCL/CDHA samples. In vitro results demonstrated biocompatibility on PCL and PCL/CDHA films. In vivo data demonstrated cellular infiltration from the surrounding tissue with new bone formation that suggests bioactive potential of CDHA whiskers. Our goal was to produce a scaffold with a potential induction factor and a favorable morphology, which was proved according to this study's findings.

Lack of a strong association between HLA class II, tumour necrosis factor and transporter associated with antigen processing gene polymorphisms and virological response to alpha-interferon treatment in patients with chronic hepatitis C.

The aim of the study was to investigate whether polymorphisms of the HLA class II, tumour necrosis factor (TNF) and transporter associated with antigen processing (TAP) genes influence the response to alpha-interferon in patients with chronic hepatitis C. Twenty-seven sustained responders and 55 non-responders to alpha-interferon monotherapy were investigated. HLA-DRB1, DQA1, DQB1, TNFA, TNFB, TAP1 and TAP2 alleles were determined by PCR-based molecular techniques. Sustained virological response was defined as undetectable serum hepatitis C virus (HCV) RNA for at least 3 years after the end of treatment. Probability (P) values were corrected for the number of alleles tested (Pc). Viral genotype 1b was more frequent in responders than in non-responders (56% vs. 26%, P = 0.009). HLA-DQB1*02 occurred less frequently in responders than in non-responders (14.8% vs. 29%, Pc not significant). HLA-DRB1*11 and DQB1*0602 were found in 22.2% and 9.3% of responders and in 10.9% and 1.8% of non-responders, respectively (Pc not significant). There was no difference in the distribution of TNF alleles in the two groups. Twenty-four (88.8%) responder patients as compared with 34 (61.8%) non-responders were TAP1*0101 homozygous (Pc not significant). Thus, in European Caucasoids with chronic hepatitis C, we could not demonstrate a strong association between HLA class II, TNF, and TAP gene polymorphisms and response to interferon treatment.

Porous and dense poly(L-lactic acid) and poly(D,L-lactic acid-co-glycolic acid) scaffolds: in vitro degradation in culture medium and osteoblasts culture.

The use of bioresorbable polymers as a support for culturing cells has received special attention as an alternative for the treatment of lesions and the loss of tissue. The aim of this work was to evaluate the degradation in cell culture medium of dense and porous scaffolds of poly(L-lactic acid) (PLLA) and poly(D,L-lactic acid-co-glycolic acid) (50:50) (PLGA50) prepared by casting. The adhesion and morphology of osteoblast cells on the surface of these polymers was evaluated. Thermal analyses were done by differential scanning calorimetry and thermogravimetric analysis and cell morphology was assessed by scanning electron microscopy. Autocatalysis was observed in PLGA50 samples because of the concentration of acid constituents in this material. Samples of PLLA showed no autocatalysis and hence no changes in their morphology, indicating that this polymer can be used as a structural support. Osteoblasts showed low adhesion to PLLA compared to PLGA50. The cell morphology on the surface of these materials was highly dispersed, which indicated a good interaction of the cells with the polymer substrate.

Synthesis and characterization of poly(L-lactic acid) membranes: studies in vivo and in vitro.

The use of biodegradable polyesters as temporary structural supports in the recuperation of damaged live tissue is a promising area of research. Poly(L-lactic acid) (PLLA) membranes can act as a support for cell fixation and growth or as a barrier against soft tissues invasion in recuperating bone tissues. In this work, five different types of PLLA membranes, which varied in their polymer-solvent ratio and their content of plasticizer were studied. For the study in vivo, 6 mm diameter disks were inserted subcutaneously in the dorsal region of 15 Wistar rats, and the reactions on rats were studied 15 days later. In another series of experiments the samples were immersed in phosphate buffer, pH 7.4 at 37 degrees C, for 30 days. Membranes without plasticizer were morphologically dense and did not allow cell invasion nor tissue adherence, in contrast to membranes with plasticizer. While porosity enhanced cell fixation and growth, it made the membrane more fragile mechanically when compared to membranes without pores.

Influence of immunogenetic background on the outcome of recurrent hepatitis C after liver transplantation.

In immunocompetent patients, specific human leukocyte antigen (HLA) class II alleles have been associated with the severity of hepatitis C virus (HCV)-related disease, in particular, HLA-DRB1*11 has been found to exert a protective effect. The authors have analyzed the role of HLA class I and II alleles in determining the frequency, timing, and progression of histologically proven recurrent hepatitis C in 89 patients who underwent a liver transplant for HCV-related cirrhosis. In addition, the influence of HLA mismatch between donor and recipient, HCV genotype, and use of steroid pulses was also evaluated. Median patient follow up was 35 months (range 4-119). HLA-DRB1 typing was performed by genomic analysis in all cases. Liver biopsies were obtained routinely and at least at yearly intervals. Histologically proven recurrent hepatitis was observed in 46 patients (52%), 10 patients progressing to stage 5-6 fibrosis in most cases within 2 years after transplant. By univariate analysis, 3 variables, HLA-B14, HLA-DRB1*04, and HLA-DRB1 donor/recipient mismatch, showed a significant effect on time to recurrent hepatitis C disease. These parameters were included in a multivariate regression model along with HCV genotype, treatment with steroid pulses and DRB1*11. HLA-B14, HLA-DRB1*04, and HLA-DRB1 donor/recipient mismatch were confirmed to provide a significant and independent contribution to the risk of hepatitic disease recurrence. As for the severity of the disease, none of the 10 patients with stage 5-6 hepatitis carried the HLA-DRB1*11 allele, in line with what was observed in nontransplant subjects. Our results suggest that in posttransplant recurrent hepatitis C, immunogenetic factors are relevant in determining HCV infection outcome.

Antiviral therapy of HBV- and HCV-induced liver cirrhosis.

Antiviral therapy is generally indicated in patients who have histologic evidence of chronic hepatitis and ongoing viral replication. The aim of treatment is to normalize alanine aminotransferase levels and to eliminate virus replication. Interferon-alfa (IFN-alpha) is the most used agent. The standard treatment regimen for hepatitis B e antigen (HBeAg)-positive cirrhosis is based on IFN-alpha given alone, but the efficacy of new antivirals (famciclovir, lamivudine) with or without IFN-alpha is currently under investigation. Conversely, the therapy of antiHBe-positive cirrhosis is far from being satisfactory. The results of treatment of patients affected by type C cirrhosis with IFN-alpha alone have been disappointing, as 10-15% of treated patients shows a sustained virologic response. Although current evidence suggests that the combination of ribavirin and IFN-alpha might be more efficacious than IFN alone in increasing the response rate in patients in the advanced fibrotic stage, the efficacy of this regimen for patients with well-compensated HCV-related cirrhosis is still unknown and prospective well-designed studies are urgently needed. Patients with decompensated cirrhosis are not generally treated unless they are included in liver transplantation programs. Prospective long-term trials with large sample sizes are needed to determine if responders to IFN-alpha have a low incidence of liver-related complications and hepatocellular carcinoma.

Devices for use as an artificial articular surface in joint prostheses or in the repair of osteochondral defects.

The covering of ultra high molecular weight polyethylene (UHMWPE) and calcium hydroxyapatite (HA)/tricalcium phosphate (TCP) porous solid substrate with polyHEMA hydrogel has been studied aiming at the development of devices to be used as artificial articular surfaces in joint prosthesis or osteochondral repair grafts. Commercial porous UHMWPE was used. Ceramic porous substrate was prepared by load compaction of an HA and TCP powder mixture obtained by aqueous precipitation technique. Two different compaction loads and grain size distribution was used. Polymer particles were added to the powder mixture in order to increase the substrate porosity after the sintering process. The porous substrate was covered with polyHEMA hydrogel by in situ polymerization. Morphological analysis (SEM) showed that a hydrogel layer formed in the porous solid top surface was fixed to the substrate by mechanical interlocking because the porous surface was filled by the hydrogel. After hydrogel covering, the resultant devices showed a decrease in the compressive elastic modulus that was influenced by the porous substrate material.

Preparation of bioabsorbable nerve guide tubes.

The use of bioabsorbable polymers in applications as temporary structural function, recovering damage in live tissues, is a promising research area. Membranes of poly(lactic acid) (PLA) may act as support to adhesion and cellular invasion or as devices for guided tissue regeneration (GTR). In this study, the same casting technique used to prepare membranes was used to prepare PLA tubes. These tubes can be used for tests in nerve guided regeneration (NGR). To improve flexibility of the device, a bioabsorbable plasticizer was added to the polymer. The initial results showed that the proposed technique allowed the preparation of flexible tubes that can be used for NGR.

A randomized, controlled study of thymosin-alpha1 therapy in patients with anti-HBe, HBV-DNA-positive chronic hepatitis B.

No consistently effective therapy is yet available for the treatment of chronic HBsAg, anti-HBe, HBV-DNA-positive hepatitis. A multicenter trial has shown that the response rates are not significantly different when patients with anti-HBe-positive hepatitis are treated with six-month course of thymosin-alpha1 or of interferon-alpha. However, since among these patients, interferon's real efficacy is still debated, with sustained biochemical response achieved in only a few of the treated patients, we conducted this controlled study to investigate the safety and efficacy of thymosin-alpha1 as compared with no treatment. Forty-four chronic hepatitis B virus (HBV) carriers, who were anti-HBe- and HBV-DNA-positive, were randomized, with stratification for the presence of cirrhosis at baseline liver biopsy, to receive either thymosin-alpha1 at a dose of 900 microg/m2 twice a week for six months or no treatment. At entry, both groups of patients were comparable for sex, age, liver histology, ALT, IgM anti-HBc, and HBV-DNA levels. Forty-two patients were followed-up for 20 months (median; range 12-32 months) after completion of therapy: one dropped out, and one developed hepatocellular carcinoma at six months. Thymosin-alpha1 treatment had no side effects. Six months after the end of the therapy, HBV-DNA was negative and ALT had normalized in 14% of treated cases and in 4.5% of control group, while IgM anti-HBc was negative (<0.200) in 14% of the treated patients and in 4.5% of the controls. Among the treated patients, the median ALT levels stayed significantly lower compared to the pretreatment values during the treatment period and six months of follow-up. During the first year, there were six flares of hepatitis in the control group and five among the treated patients (P = NS), yielding a per year average of 0.3 and 0.23 flares per patient, respectively. Among the treated patients, median IgM anti-HBc levels were low with respect to baseline values 4-10 months after treatment started. None became HBsAg negative. In conclusion, these results indicate that, in anti-HBe, HBV-DNA-positive chronic hepatitis B, thymosin-alpha1 therapy alone does not increase the response rate, but may contribute to reduce the immune-mediated liver cell necrosis as indirectly assessed by ALT and IgM anti-HBc levels.

Biomechanical and histological evaluation of hydrogel implants in articular cartilage.

We evaluated the mechanical behavior of the repaired surfaces of defective articular cartilage in the intercondylar region of the rat femur after a hydrogel graft implant. The results were compared to those for the adjacent normal articular cartilage and for control surfaces where the defects remained empty. Hydrogel synthesized by blending poly(2-hydroxyethyl methacrylate) and poly(methyl methacrylate-co-acrylic acid) was implanted in male Wistar rats. The animals were divided into five groups with postoperative follow-up periods of 3, 5, 8, 12 and 16 weeks. Indentation tests were performed on the neoformed surfaces in the knee joint (with or without a hydrogel implant) and on adjacent articular cartilage in order to assess the mechanical properties of the newly formed surface. Kruskal-Wallis analysis indicated that the mechanical behavior of the neoformed surfaces was significantly different from that of normal cartilage. Histological analysis of the repaired defects showed that the hydrogel implant filled the defect with no signs of inflammation as it was well anchored to the surrounding tissues, resulting in a newly formed articular surface. In the case of empty control defects, osseous tissue grew inside the defects and fibrous tissue formed on the articular surface of the defects. The repaired surface of the hydrogel implant was more compliant than normal articular cartilage throughout the 16 weeks following the operation, whereas the fibrous tissue that formed postoperatively over the empty defect was stiffer than normal articular cartilage after 5 weeks. This stiffness started to decrease 16 weeks after the operation, probably due to tissue degeneration. Thus, from the biomechanical and histological point of view, the hydrogel implant improved the articular surface repair.

Biomechanical and histological evaluation of hydrogel implants in articular cartilage

We evaluated the mechanical behavior of the repaired surfaces of defective articular cartilage in the intercondylar region of the rat femur after a hydrogel graft implant. The results were compared to those for the adjacent normal articular cartilage and for control surfaces where the defects remained empty. Hydrogel synthesized by blending poly(2-hydroxyethyl methacrylate) and poly(methyl methacrylate-co-acrylic acid) was implanted in male Wistar rats. The animals were divided into five groups with postoperative follow-up periods of 3, 5, 8, 12 and 16 weeks. Indentation tests were performed on the neoformed surfaces in the knee joint (with or without a hydrogel implant) and on adjacent articular cartilage in order to assess the mechanical properties of the newly formed surface. Kruskal-Wallis analysis indicated that the mechanical behavior of the neoformed surfaces was significantly different from that of normal cartilage. Histological analysis of the repaired defects showed that the hydrogel implant filled the defect with no signs of inflammation as it was well anchored to the surrounding tissues, resulting in a newly formed articular surface. In the case of empty control defects, osseous tissue grew inside the defects and fibrous tissue formed on the articular surface of the defects. The repaired surface of the hydrogel implant was more compliant than normal articular cartilage throughout the 16 weeks following the operation, whereas the fibrous tissue that formed postoperatively over the empty defect was stiffer than normal articular cartilage after 5 weeks. This stiffness started to decrease 16 weeks after the operation, probably due to tissue degeneration. Thus, from the biomechanical and histological point of view, the hydrogel implant improved the articular surface repair.

Semiquantitative assessment of IgM antibody to hepatitis B core antigen and prediction of the severity of chronic hepatitis B.

Controversial results have been reported concerning the correlation between serum levels of IgM antibodies to hepatitis B core antigen (IgM HBcAb) and the histological activity of chronic hepatitis B. In this study, paired serum samples and liver biopsies were collected from 200 consecutive chronic hepatitis B patients (mean age 39.2 +/- 0.8 years; M:F 154:46; 41 hepatitis B e antigen (HBeAg) positive) and tested for IgM HBcAb using a semiquantitative highly sensitive assay (IMx CORE-M(R)). The severity of liver disease was assessed by the Ishak score, grading the necroinflammatory activity (by using the histology activity index, HAI) and staging the fibrosis. The index values of IgM HBcAb were significantly different among patients with mild (HAI < or = 6), moderate (HAI 7-12) and severe (HAI > or = 13) necroinflammatory activity but the stage of fibrosis was unrelated to the IgM HBcAb. According to the index value of IgM HBcAb, we selected three groups of patients: Group A included 36 patients with an index value below 0.200; Group B, 99 patients with an index value between 0.200 and 0.500; and Group C, 65 patients with an index value over 0.500. The mean HAI grading in Group A was 5.3 +/- 0.4, in Group B it was 7.4 +/- 0.3 and in Group C it was 8.9 +/- 0.4 (f = 16.5, P < 0.0001). A mild HAI grading was observed in 77.8% of Group A, in 47.5% of Group B and in 23.1% of Group C patients; conversely, severe grading was detected in 0% of Group A, in 11.1% of Group B and in 18.5% of Group C patients (P < 0.0001). An index value of IgM HBcAb below 0.200 was 75% predictive of a mild necroinflammatory activity (29% sensitivity and 91.6% specificity) and ruled out a severe activity. Therefore, the quantitative assessment of IgM HBcAb appears to be a useful clinical tool in the prediction of the necroinflammatory activity of chronic hepatitis B. A serum index value of IgM HBcAb consistently below 0.200 could be considered a surrogate marker of remission of hepatitis B virus-induced liver disease.

Human leukocyte antigen class II and III alleles and severity of hepatitis C virus-related chronic liver disease.

Hepatitis C outcome is likely related both to viral factors and host's immune responses. We correlated the severity of liver disease with human leukocyte antigen (HLA) genes (C4A, C4B, TNFA, TNFB, DRB1, DRB3, DRB4, DRB5, DQA1, DQB1, TAP1, and TAP2) in three groups of subjects: 99 patients with chronic hepatitis, 41 asymptomatic carriers, and 179 uninfected controls. Patients with grade/stage 3 to 4 hepatitis significantly differentiated for their low frequency of alleles TNFB*1, DRB1*1104, and DRB3*03, which had a protective role, and high frequency of allele DRB1*1001, which was associated with disease severity. HLA-DRB*11 subtypes were differentially distributed: DRB1*1104 was most frequent in carriers, whereas DRB1*1101 was more frequent in patients. The TAP1C,2A haplotype was also underrepresented in patients with respect to controls. Finally, a decrease of heterozygous subjects was observed in patients with respect to carriers at the DQB1 locus. Multivariate analysis by correspondence analysis and multiple logistic regression indicated that age, sex, and hepatitis C virus (HCV) type were the strongest risk factors; however, some immunogenetic variables (TNFB*1, DRB1*1104, and DRB3*03) showed an independent contribution, especially in comparing patients with extreme manifestations of disease. The involvement of different genes in various HLA subregions suggests that anti-HCV responses are modulated by a complex gene interplay rather than by single alleles.

Association between HLA class II alleles and protection from or susceptibility to chronic hepatitis C.

BACKGROUND/AIMS: Recent studies have suggested that the course of chronic hepatitis C may be influenced by the immunogenetic background of the host. Specifically, HLA-DR11 (5) has been associated with less advanced hepatitis C virus (HCV)-related liver disease. The aim of the present study was to investigate whether HLA-DRB1*11 subtypes or HLA-DQA1 and DQB1 genes might be associated with protection from or susceptibility to chronic HCV infection, histological severity of HCV-induced liver disease and infecting HCV genotype. METHODS: Ninety-nine unrelated outpatients with histologically documented chronic hepatitis C were studied and their allele frequencies were compared with those of 179 ethnically matched controls and with those of 41 HCV RNA-positive patients with persistently normal aminotransferase levels (HCV carriers). HLA-DQ types and HLA-DRB1*11 subtypes were determined by polymerase chain reaction gene amplification with sequence specific primers. RESULTS: None of 10 DQA1 or 12 DQB1 alleles was significantly associated with susceptibility to or protection from chronic HCV infection or with histological staging or with HCV genotype. However, analysis of DQA1-DQB1 combinations showed that DQA1*0201-DQB1*0201 combination was significantly more frequent in patients compared to controls, both in cis (26.3% vs 16.2%, p = 0.04, odds ratio = 1.8, 95% confidence interval, 0.96-3.5) and in trans (12.1% vs. 1.1%, p = 0.0001, OR = 12.2, 95% CI, 2.6-113.7). HCV carriers did not differ from controls or from patients in the frequency of DQA1-DQB1 combinations. The extended haplotype DRB1*1104, DQA1*0501, DQB1*0301 was seen significantly less frequently in patients than in controls (8% vs 22.3%, p = 0.0025, OR = 0.31, 95% CI, 0.12-0.7) or HCV-RNA carriers (8% vs 26.8%, p = 0.003, OR = 0.24, 95% CI, 0.08-0.73). CONCLUSIONS: Immunogenetic factors may play a role in determining both protection from and susceptibility to chronic hepatitis C, the trans-dimer DQA1*0201-DQB1*0201 predisposing to and the DRB1*1104, DQA1*0501, DQB1*0301 haplotype protecting from chronic hepatitis C.

Are alanine aminotransferase, hepatitis B virus DNA or IgM antibody to hepatitis B core antigen serum levels predictors of histological grading in chronic hepatitis B?

Paired sera and liver biopsies from 105 patients with chronic hepatitis B virus infection (34 HBeAg positive and 71 anti-HBe positive) were studied to investigate the relation between the degree of histological activity and alanine aminotransferase (ALT), hepatitis B virus DNA (HBV-DNA) or IgM antibody to hepatitis B core antigen (IgM anti-HBc) levels. ALT levels were significantly higher in patients with piecemeal necrosis (155 +/- 124 vs 75 +/- 42, p = 0.0017), but there were no differences in the ALT values of patients with or without intralobular necrosis. ALT values were within normal range in 29% of 31 patients without versus 15% of 65 with piecemeal necrosis (p = 0.19). Serum HBV-DNA levels were not related to the grade of lobular or portal/periportal activity in HBeAg-positive patients. Anti-HBe-positive subjects with piecemeal necrosis had higher HBV-DNA levels (34 +/- 93 vs 4 +/- 6, p = 0.01). IgM anti-HBc indexes were significantly higher in patients with intralobular necrosis (0.635 +/- 0.600 vs 0.356 +/- 0.367, p = 0.0005) or piecemeal necrosis (0.671 +/- 0.633 vs 0.321 +/- 0.219, p = 0.0002). In summary, since serum IgM anti-HBc-IMx indexes can reflect the grade of histological activity, the quantitative assessment of this antibody could be useful for non-invasive monitoring of hepatocellular damage in chronic hepatitis B.