Is the snail shell repair process really influenced by eggshell membrane as a template of foreign scaffold?

Biominerals are inorganic-organic hybrid composites formed via self-assembled bottom up processes under mild conditions. Biominerals show interesting physical properties, controlled hierarchical structures and robust remodeling or repair mechanisms. Biological processes associated with biominerals remain to be developed into practical engineering processes. Therefore, the formation of biominerals is inspiring for the design of materials, especially those fabricated at ambient temperatures. The study described herein involves the influence of chicken outer eggshell membrane on the type of calcium carbonate (CaCO3) polymorph deposited on the shell of the land snail Helix aspersa during the repair process after an injury. A piece of snail shell was removed by perforating a hole from the largest body whorl. The operated area was left either uncovered or covered with either a thermoplastic flexible polyolefin-based film Parafilm® or a piece of chicken eggshell membrane. The repaired shells of control and experimental animals were analyzed using SEM, EDS, Raman and FTIR spectroscopies. We found that in the presence of eggshell membrane, the polymorph deposited on the substratum during the first hours resembles calcite, the polymorph present in eggshell normal formation, but at 24 and 48h, when snail mantle cells produced their normal organic matrix (mainly ß-chitin plus proteins and proteoglycans), the polymorph deposited is aragonite, the characteristic polymorph of Helix shell. Therefore, the eggshell membrane influences the type of polymorph, but only in the initial stages of biomineral deposition, before an organic matrix layer is deposited by the snail.

Austromegabalanus psittacus barnacle shell structure and proteoglycan localization and functionality.

Comparative analyzes of biomineralization models have being crucial for the understanding of the functional properties of biominerals and the elucidation of the processes through which biomacromolecules control the synthesis and structural organization of inorganic mineral-based biomaterials. Among calcium carbonate-containing bioceramics, egg, mollusk and echinoderm shells, and crustacean carapaces, have being fairly well characterized. However, Thoraceca barnacles, although being crustacea, showing molting cycle, build a quite stable and heavily mineralized shell that completely surround the animal, which is for life firmly cemented to the substratum. This makes barnacles an interesting model for studying processes of biomineralization. Here we studied the main microstructural and ultrastructural features of Austromegabalanus psittacus barnacle shell, characterize the occurrence of specific proteoglycans (keratan-, dermatan- and chondroitin-6-sulfate proteoglycans) in different soluble and insoluble organic fractions extracted from the shell, and tested them for their ability to crystallize calcium carbonate in vitro. Our results indicate that, in the barnacle model, proteoglycans are good candidates for the modification of the calcite crystal morphology, although the cooperative effect of some additional proteins in the shell could not be excluded.

Breast carcinomas with hyperprolactinemia at the time of diagnosis-clinico-biological association.

AIM: To analyse association between preoperative hyperprolactinemia serum levels and clinical and biological features of breast tumors. METHODS: Serum levels of prolactin were measured in 253 women with invasive breast cancer. Clinical and biological parameters analysed were age, size, lymph node involvement, distant metastasis and immunohistochemical expression of estrogen receptor, progesterone receptor, androgen receptor, bcl-2, p53 and Ki67. RESULTS: In ductal carcinomas hyperprolactinemia were associated with high age (p = 0.017), and with bcl-2 + + + expression (p = 0.017). Furthermore, serum prolactin values were significantly higher in bcl-2 +++ cases vs negative (p = 0.029); the same happened when we considered the positivity threshold of 25 ng/mL (p = 0.015). CONCLUSION: Is possible to detect in 6% of infiltrating ductal breast carcinomas hyperprolactinemia (>25 ng/mL), being associated only with increasing age, but not with other clinical or biological factors; and 2) the most surprising data was the association between prolactinemia (qualitative (>25 ng/mL) and quantitative) and intense bcl-2 tissue expression, which suggests that, probably, this (prolactinemia) is not a sign of worse prognosis and evolution.

The Fanconi anemia family of genes and its correlation with breast cancer susceptibility and breast cancer features.

Fanconi anemia (FA) family of proteins participates in the DNA repair pathway by homologous recombination, and it is currently formed by 13 genes. Some of these proteins also confer susceptibility to hereditary breast and ovarian cancer (HBOC), since FANCD1 is the BRCA2 breast cancer susceptibility gene, and FANCN/PALB2 and FANCJ/BRIP1 explain 2% of non-BRCA1/2 HBOC families. Thus, there is an important connection between FA and BRCA pathways. In a previous case-control association study analysing FANCA, FANCD2 and FANCL, we reported an association between FANCD2 and sporadic breast cancer (BC) risk (OR = 1.35). In order to know whether variants in other FA genes could also be involved in this association, we have extended our study with the rest of FA genes and some others implicated in the BRCA pathway. We have also analyzed the correlation with survival, nodal metastasis and hormonal receptors (ER- and PR-). A total of 61 SNPs in ten FA genes (FANC-B, -C, -D1, -E, -F, -G, -I, -J, -M, -N) and five FA related genes (ATM, ATR, BRCA1, H2AX and USP1) were studied in a total of 547 consecutive and nonrelated sporadic BC cases and 552 unaffected controls from the Spanish population. Association analyses reported marginal statistically significant results with the minor allele of intronic SNPs in three genes: BRCA1, BRCA2/FANCD1, and ATM. Survival association with SNPs on FANCC and BRCA2/FANCD1 genes were also reported. Sub-group analyses revealed associations between SNPs on FANCI and ATM and nodal metastasis status and between FANCJ/BRIP1 and FANCN/PALB2 and PR- status.

Comparative analysis of in vitro proliferative, migratory and pro-angiogenic potentials of bovine fetal mesenchymal stem cells derived from bone marrow and adipose tissue.

Mesenchymal stem cells (MSCs) are found in virtually all tissues, where they self-renew and differentiate into multiple cell types. Cumulative data indicate that MSCs secrete paracrine factors that may play key roles in the treatment of various acute and chronic pathological conditions in diverse animal species including cattle. The aim of the present study was to compare the potentials for proliferation, migration and pro-angiogenesis of bovine fetal BM-MSCs and AT-MSCs under in vitro conditions. Growth curves and population doubling time (PDT) were determined for BM-MSCs and AT-MSCs in order to compare in vitro cell proliferation potentials. The ability of BM-MSCs and AT-MSCs to migrate was evaluated by scratch plate and transwell migration assays. The pro-angiogenic potential of conditioned medium from BM-MSCs and AT-MSCs was compared using an endothelial cell (EC) tubule formation assay. BM-MSCs displayed higher proliferation curves and doubled their populations in fewer days compared to AT-MSCs. No significant differences were detected in the number of migrant cells between BM-MSCs and AT-MSCs; however, a higher migration value was detected for BM-MSCs compared to fibroblasts (FBs), and a higher number of migrant cells were attracted by DMEM supplemented with 5% fetal bovine serum (FBS) compared to stromal cell-derived factor-1 (SDF-1). More tubules of ECs were formed after exposure to concentrated conditioned medium from AT-MSCs compared to BM-MSCs, FBs or DMEM controls. Despite common mesodermal origin, BM-MSCs display higher proliferative capacity and lower pro-angiogenic potential compared to AT-MSCs; however, both cell types possess similar migratory ability.

The inflammatory response recapitulates phylogeny through trophic mechanisms to the injured tissue.

The post-traumatic local acute inflammatory response is described as a succession of three functional phases of possible trophic significance: 1. Nervous or immediate (ischemia-reperfusion); 2. Immune or intermediate (infiltration by inflammatory and bacterial cells) and 3. Endocrine or late (angiogenesis with regeneration and/or cicatrization). Each of these phases emphasizes the trophic role of the mechanisms in the damaged tissue. Hence, the nervous phase is predominated by nutrition by diffusion; in the immune phase trophism is mediated by inflammatory cells and bacteria and, finally, in the endocrine phase, the blood circulation and oxidative metabolism play the most significant nutritive role. Since these trophic mechanisms are of increasing complexity, progressing from anoxia to total specialization in the use of oxygen to obtain usable energy, it could be speculated that they represent the successive reappearance of the stages that take place during the evolution of life on Earth, from ancient times without oxygen. In this sense, the inflammatory response could recapitulate phylogeny through the successive expression of pathophysiologic mechanisms that have a trophic meaning to the injured tissue.

[Preoperative CA15.3 serum levels and cellular proliferation in patients having infiltrating ductal carcinomas of the breast]. / CA15.3 sérico y proliferación celular en carcinomas ductales infiltrantes de mama.

OBJECTIVE: To study the possible correlations between the preoperative CA15.3 serum levels and the cellular proliferation, measured by S-phase (SP), in patients having infiltrating ductal carcinomas (IDC) of the breast MATERIAL AND METHODS: The study group included 79 patients with an age ranged between 39 and 86 yrs (64,8 +/- 11,8). Ploidy and S-phase were measured by cytometry (Fascam. Beckton Dikinson. USA) in fresh samples RESULTS: Using as cut-off for SP the value of 7 %, which represents the median obtained previously in 321 patients with IDC (r: 0,8-51,2; 9,3 +/- 7,9; percentiles 25 y 75; 4,3 y 11,8 %), we can observed that the antigenic levels were higher (p:0,015) in the tumors with low SP. These same behavior was noted when 30U/ml was used as cut-off for CA15.3. Likewise, the levels of the tumor marker increased significantly (p:0,007) when the SP moved from < 4,3 % to 7,1 %, to decrease later (p:0,010) when the SP value was comprised between 7,11 % and 11,8 %. The same behavior of this tumor marker in relation to the SP was noted in tumors without axillary involvement tumors, as well as in aneuploid carcinomas. CONCLUSION: a) Release of CA15.3 happens when SP increases to rise the 7,1 % value, to decrease later although that goes on increasing, and b) The same behaviour of this marker with the S-phase was observed in tumors without axillary involvement, as well as in aneuploid carcinomas.

[Preoperative serum CA15.3 and CEA levels and clinical-biological parameters in breast tumors]. / Concentraciones séricas preoperatorias de antígeno carbohidrato 15.3 y antígeno carcinoembrionario y características clínico-biológicas tisulares de los carcinomas mamarios.

OBJECTIVE: To study the possible associations between preoperative serum CEA and CA15.3 levels and different clinical-biological parameters of infiltrating ductal carcinomas of the breast (IDC). PATIENTS AND METHOD: Preoperative serum CA15.3 and CEA levels were determined in 255 and 224 females, respectively, having IDC. We assayed the cytosolic levels of estrogen receptor, pS2, cathepsin D and hyaluronic acid, as well as the levels of epidermal growth factor receptor in cell surfaces. Tumor size, axillary involvement (N), distant metastasis (M), histological grade (HG) and cellular S-phase (SP) were taken into account. RESULTS: 22 IDC were positive for CA15.3 (> 40 U/ml) and they had greater global tumor size (p < 0.001), > 2 cm (p: 0.005) and distant metastasis (p < 0.001). 19 IDC were positive for CEA (> 4 ng/ml) and they had greater global tumor size (p < 0.001), > 2 cm (p < 0.001), > 5 cm (p: 0.052) and global S-phase values (p: 0.062), and were more frequently N + (p: 0.045), > 3N + (p: 0.001) and > 10N + (p < 0.001), M + (p: 0.004), HG3 (p: 0.091) and SP > 7 % (p: 0.006). CONCLUSIONS: Our results led us to the following: In patients having IDC of the breast, preoperative serum CA15.3 levels are associated with greater tumor size and distant metastasis, while pre-treatment CEA serum levels are associated moreover with axillary involvement; we have not observed any correlation between serum levels of both antigens and the hormonal status of the tumor; these results had physiopathological interest but reduced clinical value in pretreatment breast carcinomas due to their low sensitivity of both markers. However, they are useful in the follow-up of the patients when assessing serum concentrations of both markers, since they contribute to knowing the patients' clinical status better.

[Expression of epidermal growth factor receptor (EGFR) in hormone-independent breast cancers]. / La expresión del receptor del factor de crecimiento epidérmico (EGFR) en carcinomas mamarios hormonoindependientes.

OBJECTIVE: To determine the expression of epidermal growth factor receptor (EGFR) in ER-negative and Pg-R negative infiltrating ductal carcinomas of the breast (IDC) and to analyze the possible relationship between the EGFR positivity and some clinico-biological parameters of tumors. MATERIAL AND METHODS: EGFR was measured by a single point radioligand assay in the cell surfaces of 115 ER-negative and Pg-R-negative (< 10 fmol/mg prot.) IDC. We measured also the cytosolic concentrations of pS2, cathepsin D, tissue-type plasminogen activator (t-PA) and hyaluronic acid (AH), as well as the levels of AH in cell surfaces. Tumor size, axillary involvement, distant metastasis, histological grading, ploidy and S-phase (SP) were taken account. RESULTS: Using as cut-off for EGFR a value of 5 fmol/mg prot., we can observed that IDCs -EGFR + had greater global values of S-phase (p: 0.005) and were more frequently metastastatic (p: 0.004), SP > 7 % (p < 0.001) and SP > 14 % (p: 0.077); likewise, they were lower frequently pS2-positive (p < 0.01) and t-PA-positive (p < 0.01). During the follow up time (median 85 months), the number of recurrences was higher in EGFR-positive than in EGFR- negative tumors (14/41 frente a 1/29; p: 0.002), but there was not differences in the number of deaths by the tumor. CONCLUSIONS: 1) The EGFR-positivity in ER-negative and PgR-negative IDCs is associated with distant metastasis, greater cellular proliferation (SP), lower positivity for pS2 and t-PA and greater number of recurrences. 2) Using 10 fmol/mg prot. as cut-off, we observed the same findings, except the change in cellular proliferation. 3) Our findings support the possible use of EGFR as a prognostic parameter in those breast carcinomas.

[Cytosolic pS2 levels and cellular proliferation in ER-positive and PgR-positive infiltrating ductal carcinomas of the breast]. / pS2 citosólica y proliferación celular en carcinomas ductales infiltrantes de mama hormonodependientes.

OBJECTIVE: The trefoil factor 1 (TFF1/pS2) is an estrogen-induced molecule in breast tumours. We wanted to study its expression in ER+ and PgR+ infiltrating ductal carcinomas of the breast (IDCs), and to correlate it with other clinical-biological parameters and the outcome. MATERIAL AND METHODS: Cytosolic pS2 levels were measured using an IRMA (CIS. Biointernational. France) in 170 tumors. Likewise we determined the cytosolic levels of cathepsin D and tissue-type plasminogen activator (t-PA), as well as the concentrations of the epidermal growth factor receptor (EGFR), erbB2 oncoprotein, CD44v5 and CD44v6 on cell surfaces. Also the tumour size, histological grade (HG), axillary lymph node involvement, distant metastasis, ploidy, DNA index and of cellular synthesis phase (SP) was taken in account. RESULTS: The pS2-positive (> 5 ng/mg prot.) tumours showed higher concentrations of cathepsin D (p: 0.0043) and t-PA (p: 0.0089) than the pS2-negative ones. Likewise, they were less frequently HG3 (p: 0.0231), SP > 7 % (p: 0.0005) and SP > 14% (p:0.0014). During the follow-up time (r: 1-147; 50,1+/-31,7; median 37 months) the pS2-positive tumors showed a less number of recurrences (5/101 vs 6/69; p: 0.059) but not of deaths by the tumor (1/101 vs 2/69). CONCLUSIONS: These results support an inverse relationship between pS2 positivity and cellular proliferation in IDCs and suggest a new role of this protein (different of the hormone dependence) in the biology of these breast carcinomas, while further studies will be required to establish the impact of this finding on their outcome.

Histological grade in breast cancer: association with clinical and biological features in a series of 229 patients.

In order to study the association of histological grade (HG) with specific clinical and biological parameters which may influence the clinical behavior of infiltrating ductal carcinomas of the breast (IDC), we analyzed in 229 tissue samples the cytosolic concentrations of estrogen receptor (ER), progesterone receptor (PR), pS2, cathepsin D, hyaluronic acid (HA) and tissue-type plasminogen activator (t-PA), as well as those of the erbB2 oncoprotein, epidermal growth factor receptor (EGFR), HA, CD44v5 and CD44v6 in the cell membrane fraction. Likewise, we considered size, ploidy, S-phase fraction and axillary node involvement as variables of the study. The transition from HG1 to HG2 and from HG2 to HG3 was accompanied by a number of common features: global increase in size, greater number of tumors >2.0 cm, decrease in membrane hyaluronic acid concentrations, increased cell proliferation (S-phase >7%) and greater aneuploidy. Other events observed during the transition from HG2 to HG3 were a decrease in ER, PR, t-PA and cytosolic hyaluronic acid. These results led us to consider that HG is associated with certain clinical-biological changes that may help explain its value as a prognostic factor in breast carcinomas.

Auxiliary liver transplantation in the pig without portal blood: a study of a new technique for application to the heterotopic hepatic xenotransplant.

An auxiliary liver transplantation surgical technique in the pig, in which the graft only receives blood flow via the hepatic artery since an end-to-side portacaval shunt is performed in the donor animal prior to the transplantation, is described. The functional impairment of the receptor liver was done by a ligature of the choledoch, provoking death by sepsis in six of the transplanted animals at 8.16 +/- 6.61 days post transplantation. The auxiliary liver never suffered atrophy and prevented jaundice. It can be concluded that this simple surgical technique could be applied to the heterotopic xenotransplantation when the complications inherent in the immunological aggression are eliminated.

[The cytosolic hyaluronic acid level defines several clinico-biological properties of CD44v5-positive infiltrating ductal carcinoma of the breast]. / La concentración de ácido hialurónico citosólico define ciertas propiedades clinicobiológicas del carcinoma ductal infiltrante de mama CD44v5 positivo.

BACKGROUND: The hyaluronic acid (HA) is a ligand of CD44 adhesion molecule. In this work, we study if the cytosolic level of this proteoglycan can modulate certain clinical-biological properties at CD44v5-positive infiltrating ductal carcinomas (IDC) of the breast. PATIENTS AND METHODS: We have assayed, by a radioligand method, the cytosolic level of hyaluronic acid in 127 IDC. Likewise, cytosolic levels of estrogen receptors (ER), progesterone receptors (PR), pS2, cathepsin D and tissue.-type plasminogen activators (t-PA) have been dossified, as well as those of epidermal growth factor receptor (EGFR) at cell surfaces. The menopausal status, tumor size, axillary lymph node involvement, histological grade, ploidy and S-phase have also been taken into account. RESULTS: HA positive (> 4800 ng/mg prt., which represents the median value obtained with 252 tumors) carcinomas had higher levels of PR (p = 0.035) and t-PA (p = 0.000), whereas HA negative showed a higher frequency of a tumor size > 2 cm (p = 0.015), aneuploidy (p = 0.015) and S-phase > 14% (p = 0.019), as well as histological grade 3 which reached statistical significance (p = 0.062), all of which were indicators of a worse behaviour and evolution. CONCLUSIONS: Our results suggest that, as it also happens with that of the cell surface, cytosolic HA levels seems to modulate certain clinical-biological features of CD44v5-positive infiltrating ductal carcinomas of the breast. Likewise, they can help us to explain the discordant results described at the literature concerning its practical value when each of them are considered separately.

[Tuberculosis epidemiology in the Cervo (Lugo) health area]. / Epidemiología de la tuberculosis en el área de salud de Cervo (Lugo).

From 1994 to 1995, 80 cases which belonged to 78 patients were diagnosed. The rate was 56.6 cases/100,000 inhabitants/year in 1994 and 51.2 in 1995. The ratio male/female was 2:1 for the two years. Diagnose in hospital was 73% and in health centre 27%. Acid-fast stain and/or positive culture of M. Tuberculosis (MT) and another mycobacteria were obtained in 82.5% cases. The MT resistances were about 3.6. It was usually placed in lungs in 69.2%. The 5.1% patients were positive for HIV. Only one patient died (1.8%) who suffered from a miliary form. The favorite administered treatment (78.2%) was six months with three drugs. The adenosine deaminase (ADA) was discriminate for 80% of pleuritis. The average stay was 17.6 days. Important differences in each town councils rates.

[Study of the cytosolic concentrations of the tissue poly-peptide specific (TPS) antigen in infiltrating ductal carcinomas of the breast. Positive relationship with hormone dependency and negative with cellular proliferation]. / Estudio de las concentraciones citosolicas del antígeno polipeptídico específico tisular (TPS) en carcinomas ductales infiltrantes de mama. Relación positiva con la hormonodependencia y negativa con la proliferación celular.

INTRODUCTION: The tissue-specific polypeptide antigen (TPS) is an epitope of the tissue polypeptide antigen (TPA) which is defined by the M3 monoclonal antibody and is related to cytokeratin 18. Several groups have demonstrated its value as a useful parameter in the follow-up of some tumors. This work has aimed to study the TPS cytosolic levels in infiltrating ductal carcinomas of the breast (IDC) and their possible correlations with other clinical-biological parameters. PATIENTS AND METHODS: The TPS was determined by means of an immunoradiometric assay (Beki Diagnostics. Sweden). Other parameters included in the study were the estrogen receptors (ER), progesterone receptors (PR), pS2, cathepsin D, tissue-type plasminogen activator (t-PA), tumor size, axillary lymph node involvement, distant metastases, histological grade, ploidy and S-phase. RESULTS: The TPS cytosolic levels ranged from 1.8 to 606.3 KU/mg prt. (median 110.2) and had a significant correlation with the ER (r: 0.721), PR (r: 0.287), cathepsin D (r: 0.550) and t-PA (r:0.436). The TPS positive (> 110.2 KU/mg prt.) carcinomas had higher levels of ER (p: 0.001), PR (p: 0.021), pS2 (p: 0.058), cathepsin D (p: 0.000) and t-PA (p: 0.053) than the TPS negative tumors. When the IDC were classified according to S-phase values, we observed that the positive cases (S-phase > 8.1%, which represents the median value of all carcinomas) had lower levels of TPS (p: 0.046) than the negative tumors. Likewise, the GoG1 cellular fraction correlated positively and significantly with the TPS cytosolic levels (p: 0.000). CONCLUSIONS: Based on our results, we suggest that there is a positive correlation between the TPS cytosolic levels and hormone-dependence parameters, as well as an inverse correlation between these and the cellular proliferation parameters. Based on the above, we consider that it is worthwhile to carry out further studies on cytosolic TPS in order to investigate its possible value as a prognostic parameter in breast carcinomas.

Only in patients with hormone-dependent breast infiltrating ductal carcinomas, CA15.3 serum levels are inversely correlated with the immunohistochemical expression of Bcl2.

OBJECTIVE: To study possible association between serum CA15.3 levels and immunohistochemical expression of Bcl2 in women affected by infiltrating ductal breast carcinomas. MATERIALS AND METHODS: Two hundred and fifty consecutives women with breast infiltrating ductal carcinomas, aged between 37 and 83 years were included in this study. Serum CA15.3 was determined by electro-chemoluminescence assay (ECLIA-Elecsys 170 Roche). Immunohistochemical staining on tissue sections of 4-5 microns was done by the EnVision method with a heat-induced antigen retrieval step. Antibody used for Bcl2 was (124, Dako, dilution 1/150). Bcl2 expression was assessed as negative (-), weak positive (+) or strong positive (++). RESULTS: In the study group, serum CA15.3 concentrations ranged between 1 and 1743 U/ml, with 25, 50 and 75 percentiles of 12.7, 17.6 and 24.3 U/ml respectively. Serum CA15.3 concentrations were higher in Bcl2 negative cases than in Bcl2 + and Bcl2 ++. We found statistically significant differences between subgroups Bcl2 negative and Bcl2 ++ (p=0.044), between Bcl2 + Bcl2 ++ (p=0.039) and between Bcl2 ++ and Bcl2 -/+ (p=0.013). When we considered 25 U/mL as the threshold of positivity, antigen values>25 U/ml were more frequent in tumors Bcl2- than in Bcl2 ++ (20/52 vs 29/170, p=0.001). The same behavior was observed when comparing the subgroups -/+ with ++ (p=0.001). A very important aspect of our work was that this CA15.3 behavior in relation to the immunohistochemical expression of Bcl2 was maintained in hormone-dependent tumors (ER+), but not in hormone-independent ones. CONCLUSIONS: The results led us to the following consideration: In women affected by infiltrating ductal breast carcinomas, serum levels of CA15.3 associated inversely, both qualitatively and quantitatively, with the immunohistochemical expression of Bcl2, but this fact exists only in hormone-dependent tumors.

Evaluation of a multilayered chitosan-hydroxy-apatite porous composite enriched with fibronectin or an in vitro-generated bone-like extracellular matrix on proliferation and diferentiation of osteoblasts.

The use of extracellular matrix (ECM) molecules from tissues is an interesting way to induce specific responses of cells grown onto composite scaffolds to promote adhesion, proliferation and differentiation. There have been several studies on the effects on cell proliferation and differentiation of osteoprogenitor cells cultured onto composites, either adding some ECM molecules or grown in the presence of growth factors. Other studies involve the use of osteoblasts cultured on a three-dimensional (3D) matrix, enriched with ECM molecules produced by the same cells grown previously inside the composite. Here, the effect of enrichment of a novel multilayered chitosan-hydroxyapatite composite with ECM molecules produced by osteoblasts, or the addition of 25 or 50 µg/ml fibronectin to the composite, on proliferation and differentiation of osteoblasts cultured on these composites was studied. The results showed an increase in the number of osteoblasts from day 1 of culture, which was higher in the group grown onto composites enriched with the highest concentration of fibronectin or with ECM molecules produced naturally by osteoblasts cultured previously on them, when compared with the control group. However, this increment tended to decline in all groups after day 7 of culture, the day when they reached the highest peak of proliferation. Differentiation expressed as alkaline phosphatase activity followed the proliferation pattern of the cells cultivated on the scaffolds. The results demonstrate the potential offered by these enriched 3D multilayered composites for improving their ability as bone grafting material.

Epoxy putty external skeletal fixation in a tibiotarsal fracture of a wild choroy parakeet (Enicognathus leptorhynchus) / Fixação externa com massa epóxi em uma fratura tibiotársica de um periquito choroy selvage (Enicognathus leptorhynchus)

Tibiotarsal fractures are common in birds because in most birds this is the longest, most exposed bone in the leg. Transverse fractures are most common and rotational and shear forces must be stabilized in order to achieve good bone regeneration. A 230g male Slender-billed Parakeet or Choroy parakeet (Enicognathus leptorhynchus), with more than five years of age, was received with non-weight bearing lameness with 24 hours duration. X-rays were taken, and these revealed a closed, complete, non-comminuted transverse fracture of the distal diaphysis of the left tibiotarsal bone. Fixation was planned with 10-minute fast-setting epoxy putty. In order to assess the temperature of polymerization of the epoxy dough and the possibility of heat-bone necrosis, the temperature was recorded every 30 seconds for 12 minutes with three different amounts of the epoxy material in an ex vivo test. The temperature of the pieces reached a peak of 50-60ºC, where the highest peak corresponds to the highest amount of material. When approximately 6g of putty were used, the peak temperature reached only 51ºC. This peak changed to 58ºC when 4 times more epoxy was mixed and measured. If the temperature of the pins inserted in the bone exceeds 70ºC, bone necrosis could occur. In light of these results, the fracture was treated with 6 g of epoxy putty that was allowed to polymerize over a 1A 2/2 external skeletal fixation, with 1-mm pins bent at 90º and joined together with cerclage wire. At 6 weeks after surgery the bird had formed a good primary bone callus, and the external fixators were therefore removed. With this approach a satisfactory recovery of the patient was achieved with normal use of the affected limb.

Fraturas tibiotársicas são comuns em aves, pois, na maioria das aves, o tibiotarso é o osso mais longo e exposto nesses animais. Fraturas transversais são mais comuns, sendo que as forças de rotação e cisalhamento devem ser estabilizadas de modo a alcançar uma boa regeneração óssea. Um periquito macho do tipo psitacídeo de 230g, com mais de 5 anos, foi recebido apresentando claudicação a 24 horas não devido a transferência de carga. Os exames radiográficos revelaram uma fratura fechada completa, não triturada, transversal de diáfise distal do osso esquerdo tibiotársico. A fixação foi feita com massa epóxi de endurecimento rápido (10 minutos). A fim de avaliar a temperatura de polimerização da massa epóxi e a possibilidade de necrose desses ossos, a temperatura foi registrada a cada 30 segundos durante 12 minutos, com três quantidades diferentes de epóxi em teste in vivo. A temperatura das amostras fragmentadas atingiu um pico de 50-60ºC, em que o pico mais elevado corresponde à maior quantidade de massa epóxi. Quando foram utilizados cerca de 6g de massa de vidraceiro, o pico de temperatura foi de apenas 51ºC. Esse pico foi alterado para 58ºC quando se aumentou a quantidade de massa epóxi quatro vezes. Se a temperatura dos pinos inseridos no osso for superior a 70ºC, pode ocorrer necrose do osso. À luz desses resultados, a fratura foi tratada com 6g de massa epóxi deixada polimerizar sobre uma fixação externa, em configurações 1A 2/2 usando pinos de 1mm, dobrados em 90º e unidos com fios de cerclage. Após 6 semanas da cirurgia, o pássaro tinha formado um calo ósseo primário, sendo os fixadores externos removidos. Com essa abordagem, conclui-se que houve a restauração das funções normais do membro e total recuperação da ave.