Comparison of histologic and radiographic changes of sockets grafted with LPRF and sockets without intervention after tooth extraction.

OBJECTIVES: After tooth extraction, marked resorption occurs in extraction socket walls, leading to functional and esthetic problems in that area. One of the methods introduced to reduce this resorption is the use of platelet derivatives. This study aimed to evaluate the effects of leukocyte and platelet-rich fibrin (L-PRF) on the changes following tooth extraction. MATERIALS AND METHODS: The participants were 24 patients who needed to replace at least one single-rooted tooth with an implant. They were randomly divided into test and control groups. After the tooth extraction, the sockets in the test group received LPRF clots, while in the control group, the sockets were left free of any interventions. CBCT scans were obtained from the extraction site both immediately after the tooth extraction and 8 weeks later. The histologic biopsy was also obtained while the implant site was being prepared 8 weeks after the extraction. RESULTS: The average vertical bone loss in the buccal crest was not significantly different between the two groups (1.67 ± 1.67 in the test group and 2.3 ± 1.36 in the control group; mean difference = - 0.36, 95% CI: - 1.65-0.93, p-value = 0.57). Nor was the difference in resorption of the palatal wall (mean difference = - 0.19, 95% CI: - 1.51.12, p-value = 0.76). The mean ridge width resorption in 25% of the coronal aspect of sockets was also measured in the test (1.30 ± 0.66) and control group (0.58 ± 0.95) (mean difference = 0.73, 95% CI: 0.03-1.42, p-value = 0.04). The new bone formation in histologic view was not statistically different between groups (p-value = 0.15). CONCLUSION: The LPRF neither reduces the rate of ridge resorption in vertical or horizontal dimensions of extraction sockets nor induces more new bone formation. CLINICAL RELEVANCE: This study helps dentists choose the appropriate material for ridge preservation.

Comparison of lateral window and osteotome techniques in sinus augmentation: histological and histomorphometric evaluation.

OBJECTIVE: The aim of this study was to compare the lateral window and osteotome techniques for sinus lifting using histological and histomorphometric methods. MATERIALS AND METHODS: In this clinical trial 10 patients (a total number of 14 sinus areas) who needed implant treatment in the atrophic posterior maxilla were enrolled. In all the cases the residual bone height between the sinus floor and the alveolar crest was less than 5 mm. Sinus augmentation was performed. The treatment modality for a given residual bone height was selected randomly and Bio-Oss was applied in all the cases as the graft material. After a healing period of about 10 months, in all the cases, the implants were placed and biopsies of alveolar crestal bone were obtained at the same time; biopsy specimens were evaluated using histological and histomorphometric methods. Fisher's exact and Mann-Whitney U tests were used to compare distribution of variables in the two groups. Statistical significance was defined at P<0.05. RESULTS: The new bone was located in direct contact with the biomaterial without any gaps. This viable bone consisted of lacunae containing osteocytes. Infiltration of inflammatory cells did not exhibit any significant differences between the two techniques. Foreign body reaction was not observed in any cases. Histomorphometric evaluations demonstrated that The mean values of the new bone in the lateral window and osteotome techniques were 30±6.0 and 25.2±5.2, respectively, with no significant differences between the two groups.. Moreover, the average quantity of residual biomaterial and connective tissue were similar for the two groups. CONCLUSION: The nature and the volume of the new bone in lateral window and osteotome techniques were the same.

Histologic Evaluation of Bone Healing Following Application of Anorganic Bovine Bone and ß-tricalcium Phosphate in Rabbit Calvaria.

OBJECTIVE: Both anorganic bovine bone (ABB) and ß-tricalcium phosphate (ß-TCP) are used in clinical practice as bone substitute materials, but there is limited data comparing these two materials in standardized defects. The aim of this study was to histologically evaluate the effectiveness of ABB and ß-TCP in the healing of experimentally induced bone defects. MATERIALS AND METHODS: Eighteen bone defects were created on the calvaria of six rabbits. In each animal, one defect was left untreated and the other two were filled with ABB and ß-TCP. After one month, histological sections were prepared. Type and vitality of newly formed bone, percentage of new bone formation and residual material, thickness of trabeculae, inflammation and foreign body reaction were assessed. RESULTS: The newly formed osseous tissue was vital in all defects and consisted of woven and lamellar bone. Mean percentages of new bone formation were 30.83±14.29%, 16.83±11.07% and 14.00±8.17% in ß-TCP, ABB and control groups, respectively and the mean percentages of residual biomaterial were 24.17±14.01% and 36.50±8.43% in ß-TCP and ABB groups, respectively. However, the differences were not statistically significant (all ps>0.05). Inflammatory infiltration was statistically higher in ß-TCP compared to the control group (p=0.025), but the difference was not significant between ß-TCP and ABB groups (p=0.083). Trabeculation thickness and foreign body reaction were not statistically different between ß-TCP and ABB groups. CONCLUSION: ß-TCP and ABB were not different with regard to the quantity and quality of newly formed osseous tissue. However, inflammatory infiltration was higher in sites filled with ß-TCP.

Evaluation of the Effect of Plasma Rich in Growth Factors (PRGF) on Bone Regeneration.

OBJECTIVE: Reconstruction methods are an essential prerequisite for functional rehabilitation of the stomatognathic system. Plasma rich in growth factors (PRGF) offers a new and potentially useful adjunct to bone substitute materials in bone reconstructive surgery. This study was carried out to investigate the influence of PRGF and fibrin membrane on regeneration of bony defects with and without deproteinized bovine bone mineral (DBBM) on rabbit calvaria. MATERIALS AND METHODS: Twelve New Zealand white rabbits were included in this randomized, blinded, prospective study. Four equal 3.3×6.6 mm cranial bone defects were created and immediately grafted with DBBM, PRGF+DBBM, PRGF+fibrin membrane and no treatment as control. The defects were evaluated with histologic and histomorphometric analysis performed 4 and 8 weeks later. RESULTS: Adding PRGF to DBBM led to increased bone formation as compared with the control group in 4- and 8-week intervals. In DBBM and PRGF+fibrin membrane samples, no significant increase was seen compared to the control group. There was also a significant increase in the rate of biodegradation of DBBM particles with the addition of PRGF in the 8-week interval. Neither noticeable foreign body reaction nor any severe inflammation was seen in each of the specimens evaluated. CONCLUSION: Under the limitation of this study, adding PRGF to DBBM enhanced osteogenesis in rabbit calvarias. Applying autologous fibrin membrane in the defects was not helpful.

A new radiopharmaceutical compound (131I-PR81) for radioimmunotherapy of breast cancer: labeling of antibody and its quality control.

PR81 is a monoclonal antibody that binds with high affinity to MUC1, which is over expressed on breast and other tumors. The objective of this study was to evaluate the application of this antibody against MUC1 as a radioimmunotherapeutical agent. Monoclonal antibody (PR81) against MUC1 was prepared, characterized, purified, and labeled with 131I. The immunoreactivity of radiolabeled mAb PR81with MUC1 (the native protein), BSA-P20 (a 20 amino acid corresponding the tandem repeat of MUC1) and MCF7 cell line were performed by RIA. In vitro stability of radiolabeled mAb in human serum was determined by thin layer chromatography (TLC). Cell toxicity and in vitro internalization studies were performed with the MCF7 cell line, and the tissue biodistribution of the radioiodinated PR81 was evaluated in normal BALB/c mice at 4, 24 and 48 hrs. The tumor imaging was performed in BALB/c mice with breast xenograft tumors at 24 and 72 hr after the complex injection. The labeling efficiency was found to be 59.9% ± 7.9%. MAb-131I conjugates showed high immunoreactivity towards MUC1 protein, BSA-P20 and MCF7 cell line. In vitro stability of the labeled product in human serum was found to be more than %50 over 24 hr. Cell toxicity and in vitro internalization studies showed that the mAb-131I conjugate inhibited 80% growth of the MCF7 cultured cell lines in vitro in a high concentration and up to %60 of the conjugate internalized after 24 h. Biodistribution studies were performed in normal BALB/c mice at 4, 24 and 48 hrs post-injection and no important accumulation was observed in vital organs. The tumors were visualized with high sensitivity after 24 and 72 hr in radioimmunoscintographical studies. These results show that the new radiopharmaceutical may be considered as a promising candidate for therapy of breast cancer.

Preparative SDS-PAGE Electroelution for Rapid Purification of Alkyl Hydroperoxide Reductase from Helicobacter pylori.

BACKGROUND: Alkyl hydroperoxide reductase (AhpC) of Helicobacter pylori is considered as a diagnostic antigen. Therefore, this antigen can be used to detect H. pylori infection by stool immunoassays such as ELISA. The aim of this study was to simplify the AhpC protein purification procedures. METHODS: For whole cell protein extraction, the bacterial cells were ruptured by octly-ß-D glucopyranoside. The isolation and purification of AhpC protein were attempted by various techniques including ammonium sulfate precipitation, dialysis, preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electroelution. RESULTS: A simple method was used for protein purification AhpC protein. One-dimensional preparative gel electrophoresis allows a single and short purification step; the high resolution capacity of this technique leads to a high level of purity of the protein. Moreover, it avoids contamination by other non-specific proteins which often appear during protein purification by column chromatography. CONCLUSION: The present method is simple, rapid and makes it possible to preparate AhpC from H. pylori.

Production and characterization of monoclonal antibody against human serum albumin.

Hybridomas secreting monoclonal antibodies (MAbs) producing stable, specific and high affinity against human serum albumin (HSA) have been established. The aim of the present study was the production of MAbs that will be potentially used in designing immunoassay methods especially immunochromatography assay kit for screening of microalbuminuria (MAU) in the early detection of diabetic and nondiabetic nephropathy. The hybridomas were obtained by fusion of spleen cells from immunized mice with mouse myeloma cell line (SP2). After limiting dilutions three clones producing antibodies were designed as EMRC1-3, which displayed different pattern of fine specificity for HSA and low cross reaction with other proteins as elucidated by inhibition enzyme-linked immunosorbent assay (ELISA). These clones were found to be of immunoglobulin G (IgG) class with k light chain. Subclass determination showed that all three MAbs secreted IgG1 type of antibody. The results of affinity purification for the two selected clones (EMRC1 and EMRC3) displayed high affinity with no cross reactivity with any of the related protein molecules. The stable hybridomas secreting anti-HSA were expanded in 50-mL flasks for large-scale production of the required antibodies. The standard curves were constructed with a sensitivity of 10 pg per well covering up to 100 ng per well. The high binding activity to HSA antigen and having no cross reactivity with other related molecules illustrated the potential application of these antibodies as an immunodiagnostic reagent in designing an immunochromatography assay kit for screening of MAU in diabetic and nondiabetic patients.

A new competitive enzyme linked immunosorbent assay (MRP83-CA15-3) for MUC1 measurement in breast cancer.

A new competitive enzyme linked immunosorbent assay was developed in this study. Monoclonal antibody (PR81) against the tandem repeat of the core protein was prepared, characterized, purified, and conjugated to HRP. This antibody exhibited no cross reactions with proteins such as bovine serum albumin, keyhole limpet homocyanin, human serum albumin, casein, human milk fat globin (HMFG), and peptone. The native cancerous MUC1 protein was purified from ascites fluid of a patient suffering from small cell lung carcinoma by immunoaffinity chromatography and used as a standard preparation in the assay buffer. The standard curve was constructed following a competitive procedure in the range of 0-200 U/mL. The level of MUC1 in normal and cancerous samples was compared following this procedure and using available CA15-3 EIA (Can Ag), as well as LIAISON CA15-3 commercial kits. The correlation coefficient between the procedure reported in this work (MRP83-CA15-3) and CA15-3 EIA (Can Ag) was 0.68 and was 0.95 with the LIAISON CA15-3 kit. We concluded that the present assay can detect MUC1 in breast cancer patients with great sensitivity and accuracy.

Preparation and characterization of a monoclonal antibody against mannoprotein of Candida albicans.

BALB/c mice were immunized via injection with whole cell of Candida albicans serotype A. The spleens were fused with myeloma cells of SP2/0 origin. A mannoprotein-reactive monoclonal antibody (MAb) was selected and characterized by ELISA technique. This MAb reacted with strains of Candida such as C. albicans, C. tropicalis, and C. albicans of the Persian Type Culture Collection (PTCC). However, our antibody did not react with other Candida species such as C. parapsilosis, C. glabrata, C. stellatoidae, C. lusitania, C. krusei, and S. cervisiae. These antibodies also did not recognize extracts of other fungal species such as Aspergillus fumigatus and Aspergillus flavus, and bacterial strains such as Staphylococcus aureus and Pseudomonas aeruginosa. Polyclonal antibody produced in this study could not differentiate the above species and was reactive towards all fungal species mentioned above except bacterial strains of S. aureus and P. aeruginosa. Western blot analysis of ligand affinity-purified mannoproteins of C. albicans wall protein using this MAb showed reactivity toward a single protein band in the region of 55-65 kDa molecular weight. The same antibody, when examined with unpurified C. albicans extract, reacted with a broad band in the region of 55-105 kDa, which we concluded was due to a possible different glycosylation pattern of mannoprotein in crude extract in which the higher molecular weight protein was eliminated by ligand-binding affinity purification.

Production and characterization of a new antibody specific for the mutant EGF receptor, EGFRvIII, in Camelus bactrianus.

EGFRvIII is the type III deletion mutant form of the epidermal growth factor receptor (EGFR) with transforming activity. This tumor-specific antigen is ligand independent, contains a constitutively active tyrosine kinase domain and has been shown to be present in a number of human malignancies. In this study, we report the production and characterization of camel antibodies that are directed against the external domain of the EGFRvIII. Antibodies developed in camels are smaller (i.e. IgG2 and IgG3 subclasses lack light chains) than any other conventional mammalian antibodies. This property of camel antibodies makes them ideal tools for basic research and other applications such as tumor imaging and cancer therapy. In the present study, camel antibodies were generated by immunization of camelids (Camelus bactrianus and Camelus dromedarius) with a synthetic 14-amino acid peptide corresponding to the mutated sequence of the EGFR, tissue homogenates of several patients with human glioblastoma, medulloblastoma and aggressive breast carcinoma, as well as EGFR-expressing cell lines. Three subclasses of camel IgG [conventional (IgG1, 160 kD) and heavy chain-only antibodies (IgG2 and IgG3, 90 kD)] were separated by their different binding properties to protein A and protein G affinity columns. The anti-EGFRvIII peptide antibodies from immunized camels were purified further using the EGFRvIII synthetic peptide affinity column. The purified anti-EGFRvIII peptide camel antibodies selectively bound to the EGFRvIII peptide and affinity-purified EGFRvIII from malignant tissues and detected a protein band of 140 kD from malignant tissues by Western blot. Affinity analysis showed that the antibodies from C. bactrianus and C. dromedarius reacted with peptide and antigen purified from a small cell lung cancer ascitic fluid with affinities of 2 x 10(8) and 5 x 10(7)M(-1) to the same extent, respectively. Since the functional antigen-binding domain of the anti-EGFRvIII antibodies in camels is much simpler and located only on the heavy chains of proteins, we are currently developing recombinant and smaller versions of the variable domain of these naturally occurring heavy-chain antibodies (V(HH)) for use in tumor imaging and cancer therapy.

Production of monoclonal antibody, PR81, recognizing the tandem repeat region of MUC1 mucin.

A monoclonal antibody (MAb) was generated by immunizing BALB/c mice with homogenized breast cancerous tissues. This antibody (PR81) was found to be of IgG(1) class and subclass, containing kappa light chain. PR81 reacted with either the membrane extracts of several breast cancerous tissues or the cell surface of some MUC1 positive cell lines (MCF-7, BT-20 and T-47D) tested by enzyme immunoassay and for MCF-7 by immunofluorescence method. PR81 also reacted with two synthetic 27 and 16-amino acid peptides, TSA-P1-24 and A-P1-15, respectively, which included the core tandem repeat sequence of MUC1. However, this antibody did not react with a synthetic 14 amino acid peptide that has no similarity with tandem repeat found in MUC1. The generated antibody had good and similar affinities (2.19 x 10(8) M(-1)) toward TSA-P1-24 and A-P1-15, which are mainly shared in the hydrophilic sequence of PDTRPAP. Through Western blot analysis of homogenized breast tissues, PR81 recognized only a major band of 250 kDa. This band is stronger in malignant tissue than benign and normal tissues.

Preparation and characterization of monoclonal antibody against digoxin.

Mouse-mouse hybridoma cell lines producing stable, highly specific and with good affinity monoclonal antibody (MAb) against the cardiac glycoside digoxin were established. Balb/c mice were immunized via injection of digoxin-3'-bovine serum albumin (BSA). The spleens of which were fused with myeloma cells of SP2/0 origin. Three clones designated as BBA, MBE, and BMG producing good antibodies displayed different patterns of fine specificity for digoxin and low cross-reaction with several digoxin analogues as elucidated by inhibition enzyme-linked immunosorbant assay (ELISA). All three MAbs were of the same class and subclass (IgG(1)). Affinity purification was performed for the selected clone BBA displaying the highest affinity and nearly no cross-reactivity with any of the structurally related molecules. Ultrafiltered concentrated hybrid cell supernatant was also purified by polyethylene glycol (PEG) 6000 precipitation for large-scale preparation and coated onto the wells of microtiter plates. The standard curve was constructed with a sensitivity of 10 pg/well covering up to 10 ng/well.