Effect of autologous platelet-rich plasma in combination with bovine porous bone mineral and bio-guide membrane on bone regeneration in mandible bicortical bony defects.

OBJECTIVE: Growth factors contained in platelet-rich plasma (PRP) can induce osteoblast differentiation in certain studies, whereas in others, osteogenesis of PRP on mandible bone defects has not been proved clinically. The aim of the study was to investigate the effect of autologous PRP on the osteogenic potential of combining bovine porous bone mineral (BPBM) and bio-guide membrane (BGM) in promoting mandible bicortical bony defects in rabbits. METHODS: One circular mandible bicortical bony defects were created in each of 54 rabbits, which were divided into 3 groups: group 1: 18 of the defects were left unfilled as a negative control; group 2: 18 of the defects were grafted with autologous PRP and BPBM/BGM; group 3: 18 of the defects were grafted with BPBM/BGM without PRP. Animals were killed at 4, 8, and 12 weeks after operation. Harvested tissue and specimens were evaluated histologically and radiographically, and metabolized observation was performed. Histological parameters associated with osteoblast activities, bone trabecula, neovascularization, newly formed mineralized bone, rudimental grafts and connective tissue formation were measured. Densities of the bones at 4, 8, and 12 weeks were studied by radiographic. The bone defect closure ratio was measured at 12 weeks. The bone metabolized parameter alkaline phosphatase was also measured and compared between 4, 8, and 12 weeks. RESULTS: The platelet concentration of PRP is 4.19- to 4.43-fold to that of the whole blood. Histological analysis showed new bone formation at all therapeutic sites including BPBM/BGM grafts with or without PRP. A statistically significant difference in new bone formation between group PRP/BPBM/BGM and group BPBM/BGM was observed. Untreated defects of group control showed the less bone regeneration. There was significant difference of bone density between group PRP/BPBM/BGM and control, and group BPBM/BGM and control, at 4, 8, and 12 weeks postoperative. There were more bone defects filling, and the grafts were absorbed at 12 weeks of group PRP/BPBM/BGM compared with group BPBM/BGM. Defects treated with PRP/BPBM/BGM demonstrated significantly increased activity of osteoblasts, enhanced amount of mitochondria and rough endoplasmic reticulum in osteoblasts, and increased concentration of alkaline phosphatase at 4, 8, and 12 weeks compared with those treated with BPBM/BGM and control group. Complete closure ratio of bone defects treated with PRP/BPBM/BGM (50%) was significantly increased compared with that treated with BPBM/BGM (16.6%). CONCLUSIONS: The study suggested that PRP combination of BPBM and BGM had significant therapeutic effects on mandible bicortical bony defects of rabbits. The effects are associated with the high concentration of platelet in PRP and the porous configuration of BPBM. Although we cannot reveal the detailed statistical relationship of PRP on promoting BPBM/GBM osteoinductive effects, PRP demonstrated superior results of bone regeneration.

Serum thymidine kinase 1 concentration as a prognostic factor of chemotherapy-treated non-Hodgkin's lymphoma patients.

PURPOSE: This study was performed to examine possible use of thymidine kinase 1 concentration in serum (STK1) for prognosis of non-Hodgkin's lymphoma patients following chemotherapy treatment. METHODS: The STK1 levels of 37 patients were determined by enhanced chemiluminescent dot-blot assay on the day before chemotherapy, and on day 1 and day 28 after start of the treatment. The specificity and sensitivity was evaluated by Western blot with anti-TK1 IgY antibody and by receiver operating characteristic (ROC) analysis. RESULTS: Western blot and ROC analysis of TK1 in serum showed high specificity and sensitivity. The mean STK1 level of the non-Hodgkin's lymphoma patients was significantly higher compared to healthy persons (p < 0.001). The mean STK1 level increased significantly (p < 0.001) on day 1 and then declined, reaching on day 28 values corresponding to those of healthy persons. The mean STK1 values before treatment and at 1 and 28 days after start of the treatment also correlated significantly with the clinical response (CR, PR and NR) and five-year survival. CONCLUSION: Although the number of patients was limited in this study, TK1 in serum might possess an important reference value in the evaluation of treatment and prognosis of non-Hodgkin's lymphoma following chemotherapy.

[Establishment of Quantitative Assay for Human Erythrocyte Pyrimidine 5'-Nucleotidase Content by Double Antibody Sandwich ELISA]

For exploring pathogenesis of pyrimidine 5'-nucleotidase (P5'N) deficiency, a quantitative assay method for human erythrocyte pyrimidine 5'-nucleotidase was established. The specific substrate uridine monophosphate (UMP) of P5'N was used as ligand. The UPM-ADH-Sepharose 4B affinity column was prepared. P5'N of human erythrocyte was purified by ammonium sulfate fractionation and precipitation, ion chromatography and affinity chromatography. Rabbit anti-human P5'N antibody was acquired by immunizing rabbits with purified P5'N. Using rabbit anti-human antibody as the coated anti-body and HRP-rabbit anti-human antibody as demonstrated anti-body, the double antitody sandwich ELISA for quantitative assay of human erythrocyte P5'N was established after square rank trial, antigen blocked trial and antigen substituted test. Results showed that the titer of rabbit anti-human erythrocyte was 1:4 and the sensitivity of double antibody sandwich ELISA was 5 ng/ml. Its blocking rate was more than 95% and the rate of substitution less than 30%. The content of P5'N was (71.77 + 10.98) ng/mg NHP in normal human erythrocyte. A new ELISA method for quantitative determination of human erythrocyte P5'N was first established. It not only had high specificity and sensitivity but also could assay the minimun content of P5'N as 5 - 20 ng/ml. It could be a suitable method for large sample in clinics.

[Microsequencing, analysis of molecular weight and amino acid composition for pyrimidine 5'-nucleotidase I of human erythrocytes].

To further explore the mechanism of congenital pyrimidine 5'-nuleotidase I (P5'N-I) deficiency, on the basis of purification of the protein, the molecular weight and amino acid composition were analysed by mass-spectrograph and amino-acid analyzer, microsequencing and bioinformation analysis of P5'N-I were performed after it was hydrolysed by trypsin. The results showed that three fractions were found in the purified P5'N-I and their molecular weights were 26,952.9, 55,476 and 110,938, respectively. The sequence from one of the peptide fragments was I-E-G-P-T-I-R-Q-I-E. The homologous sequence was not found after comparision with the ten-amino-acid sequence in GenBank by blast procedure. Amino acid analysis indicated that P5'N-I was composed of 18 amino acids at least, and 243 amino acid residues. In conclusion, the enzyme might be an allosteric enzyme, there might be homologous dimer or tetramer in physiological status of normal human erythrocyte, the microsequence could be designed as the probe for fishing the genes of interest. The composition of amino acid might be an important information in determination of its protein primary structure.