Synthesis of Human Bone Morphogenetic Protein-2 (hBMP-2) in E. coli Periplasmic Space: Its Characterization and Preclinical Testing.

Human BMP-2, a homodimeric protein that belongs to the TGF- ß family, is a recognized osteoinductor due to its capacity of inducing bone regeneration and ectopic bone formation. The administration of its recombinant form is an alternative to autologous bone grafting. A variety of E. coli-derived hBMP-2 has been synthesized through refolding of cytoplasmic inclusion bodies. The present work reports the synthesis, purification, and characterization of periplasmic hBMP-2, obtained directly in its correctly folded and authentic form, i.e., without the initial methionine typical of the cytoplasmic product that can induce undesired immunoreactivity. A bacterial expression vector was constructed including the DsbA signal peptide and the cDNA of hBMP-2. The periplasmic fluid was extracted by osmotic shock and analyzed via SDS-PAGE, Western blotting, and reversed-phase high-performance liquid chromatography (RP-HPLC). The purification was carried out by heparin affinity chromatography, followed by high-performance size-exclusion chromatography (HPSEC). HPSEC was used for qualitative and quantitative analysis of the final product, which showed >95% purity. The classical in vitro bioassay based on the induction of alkaline phosphatase activity in myoblastic murine C2C12 cells and the in vivo bioassay consisting of treating calvarial critical-size defects in rats confirmed its bioactivity, which matched the analogous literature data for hBMP-2.

Synthesis of human bone morphogenetic protein-2 (hBMP-2) in E. coli periplasmic space: its characterization and preclinical testing

Human BMP-2, a homodimeric protein that belongs to the TGF- β family, is a recognized osteoinductor due to its capacity of inducing bone regeneration and ectopic bone formation. The administration of its recombinant form is an alternative to autologous bone grafting. A variety of E. coli-derived hBMP-2 has been synthesized through refolding of cytoplasmic inclusion bodies. The present work reports the synthesis, purification, and characterization of periplasmic hBMP-2, obtained directly in its correctly folded and authentic form, i.e., without the initial methionine typical of the cytoplasmic product that can induce undesired immunoreactivity. A bacterial expression vector was constructed including the DsbA signal peptide and the cDNA of hBMP-2. The periplasmic fluid was extracted by osmotic shock and analyzed via SDS-PAGE, Western blotting, and reversed-phase high-performance liquid chromatography (RP-HPLC). The purification was carried out by heparin affinity chromatography, followed by high-performance size-exclusion chromatography (HPSEC). HPSEC was used for qualitative and quantitative analysis of the final product, which showed >95% purity. The classical in vitro bioassay based on the induction of alkaline phosphatase activity in myoblastic murine C2C12 cells and the in vivo bioassay consisting of treating calvarial critical-size defects in rats confirmed its bioactivity, which matched the analogous literature data for hBMP-2.

Remodeling of cortical and corticocancellous fresh-frozen allogeneic block bone grafts--a radiographic and histomorphometric comparison to autologous bone grafts.

OBJECTIVES: To compare cortical (AL-C) and corticocancellous (AL-CC) fresh-frozen block bone allografts to cortical block bone autografts (AT) used for lateral ridge augmentation in terms of radiographic dimensional maintenance and histomorphometrical graft remodeling. MATERIALS AND METHODS: Twenty-four patients, requiring ridge augmentation in the anterior maxilla prior to implant placement, were treated with AT, AL-C or AL-CC bone blocks (eight patients per graft type). Patients were examined with CBCT prior to, 14 days, and 6-8 months after grafting. Amount of augmentation and dimensional block graft maintenance over time was evaluated by comparing planimetric measurements of the alveolar ridge made on CBCT sections of the augmentation area. During implant installation surgery, 6-8 months after grafting, cylindrical biopsies were harvested perpendicularly to the lateral aspect of the augmented alveolar ridge. The relative volumes of vital and necrotic bone and soft tissues were histomorphometrically estimated. Comparisons among groups and observation times were performed using Friedman test followed by Dunn's post-hoc test. RESULTS: Radiographic evaluation showed that the three types of grafts resulted in a significant increase in alveolar ridge width, with no significant differences among the groups in terms of ridge dimensions at the various observation times. However, significant graft resorption (P = 0.03) was observed in the AL-CC group over time (-8.3 ± 7.1%) compared with the AT and AL-C groups, where a slight increase was observed, on average (1.5 ± 20.6% and 1.3 ± 14.9%, respectively). Histomorphometrical analysis showed that larger amounts of vital bone were found in the biopsies from the AT augmented sites (25.1 ± 11.2%) compared with AL-CC and AL-C augmented sites (9.3 ± 3.8% and 3.9 ± 4.6%, respectively; P ≤ 0.01). AL-CC and AT biopsies had the smallest amount of necrotic bone (38.2 ± 12.1% and 56.7 ± 26.0, respectively) compared with AL-C (83.7 ± 10.8%, P < 0.01) biopsies. AL-CC biopsies showed the largest amount of soft tissues (52.5 ± 11.7%) compared with those from AT (18.1 ± 17.1%, P = 0.03) and AL-C (12.3 ± 8.5%, P < 0.01) sites. CONCLUSIONS: AL block bone graft architecture influences significantly its dimensional incorporation and remodeling. Compared with AT bone graft, a small portion of the AL block consists of vital bone 6-8 months after grafting. Cortical AL blocks seem to show the least amounts of vital bone, while corticocancellous AL blocks seem to undergo more resorption over time.

The ideal timing for experimental cleft lip creation.

Cleft lip and palate (CLP) is the most common congenital defect of the face. Many animal models have been utilized to study embryogenesis and pathogenesis of CLP, including the development of secondary anomalies and consequent deformities. However, the ideal gestational age for surgical creation of lip or palate defects in rat models has never been determined. The aim of the present study is to improve the experimental model utilizing rat fetuses, defining the most appropriate timing for creation of the lip defect model. The study was composed of three groups of fetuses undergoing surgical creation of a lip defect at the left side of the superior lip at 17.5, 18.5, and 19.5 days of gestation. Fetuses were harvested at 21.5 days of gestation (term  =  22 days) and underwent macroscopic and microscopic analyses. We found that the most appropriate moment for lip defect creation was at 19.5 days, given the presence of lip depression at the site of the defect and asymmetry and retraction associated with interruption of the lip and complete reepithelialization of the borders of the defect.

Antenatal steroid and tracheal occlusion restore vascular endothelial growth factor receptors in congenital diaphragmatic hernia rat model.

OBJECTIVE: Investigate the effects of antenatal steroids and tracheal occlusion on pulmonary expression of vascular endothelial growth factor receptors in rats with nitrofen-induced congenital diaphragmatic hernia. STUDY DESIGN: Fetuses were exposed to nitrofen at embryonic day 9.5. Subgroups received dexamethasone or were operated on for tracheal occlusion, or received combined treatment. Morphologic variables were recorded. To analyze vascular endothelial growth factor receptor 1 and vascular endothelial growth factor receptor 2 expression, we performed Western blotting and immunohistochemistry. Morphologic variables were analyzed by analysis of variance and immunohistochemistry by Kruskal-Wallis test. RESULTS: Congenital diaphragmatic hernia decreased body weight, total lung weight, and lung-to-body weight ratio. Tracheal occlusion increased total lung weight and lung-to-body weight ratio (P < .05). Fetuses with congenital diaphragmatic hernia had reduced vascular endothelial growth factor receptor 1 and vascular endothelial growth factor receptor 2 expression, whereas steroids and tracheal occlusion increased their expression. Combined treatment increased expression of receptors, but had no additive effect. CONCLUSION: Vascular endothelial growth factor signaling disruption may be associated with pulmonary hypertension in congenital diaphragmatic hernia. Tracheal occlusion and steroids provide a pathway for restoring expression of vascular endothelial growth factor receptors.

Traque-oclusão intra-útero: técnica cirúrgica assistida à corticoterapia promove aumento nos receptores de VEGF (Vascular Endothelial Growth Factor) em pulmões de fetos de ratas / In utero tracheal occlusion: surgical approach associated to steroid treatment increase VEGF (Vascular Endothelial Growth Factor) receptors in lungs of fetal rats

A hérnia diafragmática congênita (HDC) causa hipoplasia e hipertensãopulmonar e em geral leva a alta morbidade e mortalidade neonatal. Traqueo-oclusão fetal (TO) e corticoterapia pré-natal são alternativas para acelerar o crescimento pulmonar fetal e diminuir a hipoplasia na HDC. A produção de VEGF (Vascular Endothelial Growth Factor) está relacionada com a maturidade pulmonar e sofre alterações na HDC ainda não elucidadas.Materiais e métodos: Seis grupos de 12 fetos de ratos Spreague-Dawley foram comparados: TO, Sham, Controle, TO+Dex, Sham+Dex e Controle+Dex. No dia 18,5º foi realizada TO come sem corticoterapia utilizando dexametasona. No 21,5º dia gestacional os pesos corporal e...

Congenital diaphragmatic hernia (CDH) presents with hypoplastic lungs and usually leads to pulmonary hypertension and high neonatal mortality. Fetal tracheal occlusion (TO) and prenatal corticotherapy are alternatives to accelerate fetal pulmonary growth and decrease hypoplasia in CDH. VEGF (Vascular Endothelial Growth Factor)production and surfactant production by type II pneumocytes are related with pulmonary maturity and are altered in CDH, but little has been described about VEGF receptors. Our objective wasto quantify the receptors of VEGF (VEGFR) and type II pneumocytes, verifying the effects of TO and corticotherapy on normal lungs of fetal rats...

Sperm retrieval techniques in rats with suppressed spermatogenesis by experimental cryptorchidism.

OBJECTIVE: Our aim was to assess the suppression of spermatogenesis and sperm retrieval rate after testicular sperm extraction (TESE) or testicular sperm aspiration (TESA) in adult rats with surgically induced cryptorchidism. METHODS: Adult rats were submitted to TESE and TESA procedures after 15 days of induced cryptorchidism. After spermatozoa retrieval, the testicles were extracted, weighed and a morphological analysis by conventional light microscopy was done. The numbers of spermatozoa retrieved in both TESA and TESE were rated and compared. RESULTS: Histological analysis of the testicles revealed Sertoli cell-only syndrome in 60% of the testicles, and maturation arrest in the remaining cryptorchid testicles. Significant differences were seen in the number of spermatozoa retrieved (P < 0.05) between cryptorchidic and control rats. When sperm retrieval techniques were compared, no differences were detected in the number of spermatozoa obtained (P > 0.05). CONCLUSIONS: It seems that a 15 day period of cryptorchidism is enough to induce spermatogenesis disorders. No differences were detected in the number of spermatozoa retrieved in the right or left testicles, irrespective of the testicular pole. Furthermore, and even more importantly, no differences in the retrieval rate were seen between the two techniques.

Adriamycin-induced fetal hydronephrosis

INTRODUCTION: At the end of pregnancy, the amniotic fluid (AF) depends basically on renal function, corresponding to fetal urine. Changes in AF, especially oligohydramnios, are reported in association with fetal hydronephrosis (FH). The experimental model using adriamycin in pregnant female rats has a teratogenic effect and has been classically employed to study esophageal atresia. Nevertheless, adriamycin promotes FH with high frequency as well. In the present study, using this animal model, we tried to identify the incidence and microscopic changes of FH, as well as its correlation with AF weight. MATERIALS AND METHODS: Eight Spreague-Dawley pregnant female rats received adriamycin 2.2 mg/kg on the 8th and 9th gestational days (considering term gestation = 22 days). Those fetuses that received adriamycin (Adriamycin Group) were compared with fetuses from 2 female rats (Control Group), which received 0.9 percent saline solution. On the 21.5 gestational day, the fetuses were collected by cesarean incision, sacrificed, and examined for macro and microscopic changes in kidneys and ureters. Fetuses with bilateral hydronephrosis formed the Hydronephrosis Group. AF weight was determined as well. RESULTS: Hydronephrosis occurred in 70 (95 percent) of the 74 fetuses in the adriamycin group against none of the 21 fetuses from the control group. The amniotic fluid weight was increased in the adriamycin group in relation to the control group (p < 0.001). The histomorphometric study revealed dilation of the renal pelvis and reduction of renal parenchyma in the hydronephrosis group in relation to the control group. Severe cortical atrophy, cortical tubular atrophy and medullar atrophy were observed in the hydronephrosis group. CONCLUSIONS: Slight renal lesions were in agreement with changes in AF weight, since they suggest that there was production of urine with the maintenance of AF.

Identification and characterization of an antigen recognized by monoclonal antibody TRA 54 in mouse epididymis and vas deferens.

Spermatozoa in testicular fluid are known to have weak forward motility and cannot fertilize eggs. The epididymis is known to participate in sperm maturation leading fertilization, but little is known about the specific epididymal molecules involved in the modification of sperm. In this study, we characterized the new pattern of expression of an antigen previously identified in testicular germ cells by monoclonal antibody (mAb) TRA 54. This antigen is expressed in epididymal and vas deferens epithelial cells in mice older than 24 days but not during younger developmental stages. Evaluation by immunohistochemistry shows that antigen expression is limited to the cytoplasm of a specific cell population of epithelia along the epididymal regions and vas deferens of adult mice. The molecules synthesized and released by epididymal and vas deferens epithelia into their lumen seem to bind on spermatozoa moving down through the ducts. Immunoblot analysis showed that the molecules recognized by mAb TRA 54 in testis and epididymis were similar and share a common epitope involving carbohydrate domains. Interestingly, the antigens identified in epididymal and vas deferens epithelial cells were expressed independently of testicular germ cells and are produced in an androgen-dependent manner. Finally, the molecules recognized by mAb TRA 54 seem to play an important role in spermatogenesis, as well as in epididymal function related to spermatozoa maturation and ability to fertilize.

Esophageal atresia and other visceral anomalies in a modified Adriamycin rat model and their correlations with amniotic fluid volume variations.

The Adriamycin rat model (ARM) has been used to produce visceral malformations in fetuses to explain the mechanisms of foregut division. The models vary in the dosage of Adriamycin (ADR) and in the number of applications. Our study of a modified ARM using 2.2 mg/kg of ADR for 2 days only, intraperitoneally in pregnant rats, is presented. A total of 81 fetuses were obtained with this model from the ADR group, 74 (91%) alive. Uretero-hydronephrosis (UHN) was observed in 70 fetuses (95%), esophageal atresia (EA) in 68 (92%), duodenal atresia (DA) in 68 (92%), bladder hypoplasia (BH) in 67 (90%), plus other malformations. In evaluating amniotic fluid (AF) volume of the fetuses with EA with tracheo-esophageal fistula (TEF) (group I) and EA without TEF (group II), both associated with bilateral UHN when compared with the control group (group III), groups I and II showed higher AF volume in groups I and II than the control group (group III) did ( p=0.0001). In conclusion, ARM was adequate to produce EA and other visceral malformations. The use of ADR in a higher dosage for a shorter period of time produced better results than those presented in previous literature. The increase of AF volume obtained in fetuses presenting EA plus bilateral UHN strongly suggests, despite ureteral dilatation (urinary obstruction), that a malformed communication may exist between the urinary system and the amniotic cavity, permitting the existence of polyhydramnios that is due to digestive obstruction such as EA and DA.

Adriamycin-induced fetal hydronephrosis.

INTRODUCTION: At the end of pregnancy, the amniotic fluid (AF) depends basically on renal function, corresponding to fetal urine. Changes in AF, especially oligohydramnios, are reported in association with fetal hydronephrosis (FH). The experimental model using adriamycin in pregnant female rats has a teratogenic effect and has been classically employed to study esophageal atresia. Nevertheless, adriamycin promotes FH with high frequency as well. In the present study, using this animal model, we tried to identify the incidence and microscopic changes of FH, as well as its correlation with AF weight. MATERIALS AND METHODS: Eight Spreague-Dawley pregnant female rats received adriamycin 2.2 mg/kg on the 8th and 9th gestational days (considering term gestation = 22 days). Those fetuses that received adriamycin (Adriamycin Group) were compared with fetuses from 2 female rats (Control Group), which received 0.9% saline solution. On the 21.5 gestational day, the fetuses were collected by cesarean incision, sacrificed, and examined for macro and microscopic changes in kidneys and ureters. Fetuses with bilateral hydronephrosis formed the Hydronephrosis Group. AF weight was determined as well. RESULTS: Hydronephrosis occurred in 70 (95%) of the 74 fetuses in the adriamycin group against none of the 21 fetuses from the control group. The amniotic fluid weight was increased in the adriamycin group in relation to the control group (p < 0.001). The histomorphometric study revealed dilation of the renal pelvis and reduction of renal parenchyma in the hydronephrosis group in relation to the control group. Severe cortical atrophy, cortical tubular atrophy and medullar atrophy were observed in the hydronephrosis group. CONCLUSIONS: Slight renal lesions were in agreement with changes in AF weight, since they suggest that there was production of urine with the maintenance of AF.