Proteomic analysis of calcium-enriched sol-gel biomaterials.

Calcium is an element widely used in the development of biomaterials for bone tissue engineering as it plays important roles in bone metabolism and blood coagulation. The Ca ions can condition the microenvironment at the tissue-material interface, affecting the protein deposition process and cell responses. The aim of this study was to analyze the changes in the patterns of protein adsorption on the silica hybrid biomaterials supplemented with different amounts of CaCl2, which can function as release vehicles. This characterization was carried out by incubating the Ca-biomaterials with human serum. LC-MS/MS analysis was used to characterize the adsorbed protein layers and compile a list of proteins whose affinity for the surfaces might depend on the CaCl2 content. The attachment of pro- and anti-clotting proteins, such as THRB, ANT3, and PROC, increased significantly on the Ca-materials. Similarly, VTNC and APOE, proteins directly involved on osteogenic processes, attached preferentially to these surfaces. To assess correlations with the proteomic data, these formulations were tested in vitro regarding their osteogenic and inflammatory potential, employing MC3T3-E1 and RAW 264.7 cell lines, respectively. The results confirmed a Ca dose-dependent osteogenic and inflammatory behavior of the materials employed, in accordance with the protein attachment patterns.

Osseointegration mechanisms: a proteomic approach.

The prime objectives in the development of biomaterials for dental applications are to improve the quality of osseointegration and to short the time needed to achieve it. Design of implants nowadays involves changes in the surface characteristics to obtain a good cellular response. Incorporating osteoinductive elements is one way to achieve the best regeneration possible post-implantation. This study examined the osteointegrative potential of two distinct biomaterials: sandblasted acid-etched titanium and a silica sol-gel hybrid coating, 70% MTMOS-30% TEOS. In vitro, in vivo, and proteomic characterisations of the two materials were conducted. Enhanced expression levels of ALP and IL-6 in the MC3T3-E1 cells cultured with coated discs, suggest that growing cells on such surfaces may increase mineralisation levels. 70M30T-coated implants showed improved bone growth in vivo compared to uncoated titanium. Complete osseointegration was achieved on both. However, coated implants displayed osteoinductive properties, while uncoated implants demonstrated osteoconductive characteristics. Coagulation-related proteins attached predominantly to SAE-Ti surface. Surface properties of the material might drive the regenerative process of the affected tissue. Analysis of the proteins on the coated dental implant showed that few proteins specifically attached to its surface, possibly indicating that its osteoinductive properties depend on the silicon delivery from the implant.

Bioactive zinc-doped sol-gel coating modulates protein adsorption patterns and in vitro cell responses.

Zinc is an essential element with an important role in stimulating the osteogenesis and mineralization and suppressing osteoclast differentiation. In this study, new bioactive ZnCl2-doped sol-gel materials were designed to be applied as coatings onto titanium. The biomaterials were physicochemically characterized and the cellular responses evaluated in vitro using MC3T3-E1 osteoblasts and RAW264.7 macrophages. The effect of Zn on the adsorption of human serum proteins onto the material surface was evaluated through nLC-MS/MS. The incorporation of Zn did not affect the crosslinking of the sol-gel network. A controlled Zn2+ release was obtained, reaching values below 10 ppm after 21 days. The materials were no cytotoxic and lead to increased gene expression of ALP, TGF-ß, and RUNX2 in the osteoblasts. In macrophages, an increase of IL-1ß, TGF-ß, and IL-4 gene expression was accompanied by a reduced TNF-α liberation. Proteomic results showed changes in the adsorption patterns of proteins associated with immunological, coagulative, and regenerative functions, in a Zn dose-dependent manner. The variations in protein adsorption might lead to the downregulation of the NF-κB pathway, thus explain the observed biological effects of Zn incorporation into biomaterials. Overall, these coatings demonstrated their potential to promote bone tissue regeneration.

The effect of strontium incorporation into sol-gel biomaterials on their protein adsorption and cell interactions.

It is known strontium can both inhibit the osteoclast formation and stimulate the osteoblast maturation, so biomaterials containing this element can favour bone structure stabilisation. The addition of Sr to biomaterials could affect their interactions with proteins and cells. Here, a silica-hybrid sol-gel network doped with different amounts of SrCl2 and applied as coatings on titanium discs was examined. in vitro analysis was performed to determine the potential effect of Sr in the coatings, showing enhanced gene expression of osteogenic markers (alkaline phosphatase and transforming growth factor-ß) in MC3T3-E1 incubated with Sr-doped biomaterials. The examination of inflammatory markers (tumour necrosis factor-α and interleukin 10) in RAW 264.7 macrophages revealed an anti-inflammatory potential of these materials. Proteins adsorbed onto the coatings incubated with human serum (3 h at 37 °C) were also analysed; mass spectrometry was used to characterise the proteins adhering to materials with different Sr content. Adding Sr to the coatings increased their affinity to APOE and VTNC proteins (associated with anti-inflammatory and osteogenic functions). Moreover, the proteins involved in coagulation processes, such as prothrombin, were more abundant on the coatings containing Sr than on the base sol-gel surfaces. Correlations between gene expression and proteomic results were also examined.

Complement proteins regulating macrophage polarisation on biomaterials.

One of the events occurring when a biomaterial is implanted in an host is the protein deposition onto its surface, which might regulate cell responses. When a biomaterial displays a compromised biocompatibility, distinct complement pathways can be activated to produce a foreign body reaction. In this article, we have designed different types of biomaterial surfaces to study the inflammation process. Here, we used different concentrations of (3-glycidoxypropyl)-trimethoxysilane (GPTMS), an organically-modified alkoxysilane as a precursor for the synthesis of various types of sol-gel materials functionalizing coatings for titanium implants to regulate biological responses. Our results showed that greater GPTMS surface concentrations induced greater secretion of TNF-α and IL-10 on RAW 264.7 macrophages. When implanted into rabbit tibia, osseointegration decreased with higher GPTMS concentrations. Interestingly, higher deposition of complement-related proteins C-reactive protein (CRP) and ficolin-2 (FCN2), two main activators of distinct complement pathways, was observed. Taking all together, inflammatory potential increase seems to be GPTMS concentration-dependent. Our results show that a greater adsorption of complement proteins can condition macrophage polarization.

Influence of the substrate's hydrophilicity on the in vitro Schwann cells viability.

A series of polymeric biomaterials including poly (methyl acrylate) (PMA), chitosan (CHT), poly(ethyl acrylate) (PEA), poly(hydroxyethyl acrylate) (PHEA), and a series of random copolymers containing ethyl acrylate and hydroxyethyl acrylate monomeric units were tested in vitro as culture substrates and compared for their impact on the proliferation and expansion of Schwann cells (SCs). Immunocytochemical staining assay and scanning electron microscopy techniques were applied to perform a quantitative analysis to determine the correct maintenance of the cultured glial cells on the different biomaterials. The results strongly suggest that cell attachment and proliferation is influenced by the substrate's surface chemistry, and that hydrophobic biomaterials based on PMA, PEA, and the copolymers PEA and PHEA in a narrow composition window are suitable substrates to promote cell attachment and proliferation of SCs in vitro.

Scaffolds of Hyaluronic Acid-Poly(Ethyl Acrylate) Interpenetrating Networks: Characterization and In Vitro Studies.

Hyaluronic acid (HA) provides many advantages to regenerative implants through its bioactive properties, but it also has many limitations as a biomaterial if it is not chemically modified. In order to overcome some of these limitations, HA has been combined with poly(ethyl acrylate) in the form of interpenetrating polymeric networks (IPNs), in which the HA network is crosslinked with divinyl sulfone. Scaffolds of this IPN have been produced through a template-leaching methodology, and their properties have been compared with those of single-network scaffolds made of either PEA or crosslinked HA. A fibroblast cell line has been used to assess the in vitro performance of the scaffolds, revealing good cell response and a differentiated behavior on the IPN surface when compared to the individual polymers. Altogether, the results confirm that this type of material offers an interesting microenvironment for cells, which can be further improved toward its potential use in medical implants.

Influence of scaffold morphology on co-cultures of human endothelial and adipose tissue-derived stem cells.

The interior of tissue engineering scaffolds must be vascularizable and allow adequate nutrients perfusion in order to ensure the viability of the cells colonizing them. The promotion of rapid vascularization of scaffolds is critical for thick artificial constructs. In the present study co-cultures of human endothelial and adipose tissue-derived stem cells have been performed in poly(ethyl acrylate) scaffolds with two different pore structures: grid-like (PEA-o) or sponge-like (PEA-s), in combination with a self-assembling peptide gel filling the pores, which aims to mimic the physiological niche. After 2 and 7 culture days, cell adhesion, proliferation and migration, the expression of cell surface markers like CD31 and CD90 and the release of VEGF were assessed by means of immunocytochemistry, scanning electronic microscopy, flow cytometry and ELISA analyses. The study demonstrated that PEA-s scaffolds promoted greater cell organization into tubular-like structures than PEA-o scaffolds, and this was enhanced by the presence of the peptide gel. Paracrine signaling from adipose cells significantly improved endothelial cell viability, proving the advantageous combination of this system for obtaining easily vascularizable tissue engineered grafts. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1523-1533, 2016.

Hydrophilic surface modification of acrylate-based biomaterials.

Acrylic polymers have proved to be excellent with regard to cell adhesion, colonization and survival, in vitro and in vivo. Highly ordered and regular pore structures thereof can be produced with the help of polyamide templates, which are removed with nitric acid. This treatment converts a fraction of the ethyl acrylate side groups into acrylic acid, turning poly(ethyl acrylate) scaffolds into a more hydrophilic and pH-sensitive substrate, while its good biological performance remains intact. To quantify the extent of such a modification, and be able to characterize the degree of hydrophilicity of poly(ethyl acrylate), poly(ethyl acrylate) was treated with acid for different times (four, nine and 17 days), and compared with poly(acrylic acid) and a 90/10%wt. EA/AAc copolymer (P(EA-co-AAc)). The biological performance was also assessed for samples immersed in acid up to four days and the copolymer, and it was found that the incorporation of acidic units on the material surface was not prejudicial for cells. This surface modification of 3D porous hydrophobic scaffolds makes easier the wetting with culture medium and aqueous solutions in general, and thus represents an advantage in the manageability of the scaffolds.

Schwann-cell cylinders grown inside hyaluronic-acid tubular scaffolds with gradient porosity.

Cell transplantation therapies in the nervous system are frequently hampered by glial scarring and cell drain from the damaged site, among others. To improve this situation, new biomaterials may be of help. Here, novel single-channel tubular conduits based on hyaluronic acid (HA) with and without poly-l-lactide acid fibers in their lumen were fabricated. Rat Schwann cells were seeded within the conduits and cultured for 10days. The conduits possessed a three-layered porous structure that impeded the leakage of the cells seeded in their interior and made them impervious to cell invasion from the exterior, while allowing free transport of nutrients and other molecules needed for cell survival. The channel's surface acted as a template for the formation of a cylindrical sheath-like tapestry of Schwann cells continuously spanning the whole length of the lumen. Schwann-cell tubes having a diameter of around 0.5mm and variable lengths can thus be generated. This structure is not found in nature and represents a truly engineered tissue, the outcome of the specific cell-material interactions. The conduits might be useful to sustain and protect cells for transplantation, and the biohybrids here described, together with neuronal precursors, might be of help in building bridges across significant distances in the central and peripheral nervous system. STATEMENT OF SIGNIFICANCE: The paper entitled "Schwann-cell cylinders grown inside hyaluronic-acid tubular scaffolds with gradient porosity" reports on the development of a novel tubular scaffold and on how this scaffold acts on Schwann cells seeded in its interior as a template to produce macroscopic hollow continuous cylinders of tightly joined Schwann cells. This cellular structure is not found in nature and represents a truly engineered novel tissue, which obtains as a consequence of the specific cell-material interactions within the scaffold.

New semi-biodegradable materials from semi-interpenetrated networks of poly(ϵ-caprolactone) and poly(ethyl acrylate).

Semi-degradable materials may have many applications. Here poly(ethyl acrylate) and poly(ϵ-caprolactone) were combined as semi-interpenetrated networks, and thoroughly characterized in terms of final composition, interactions between components, wettability, and mechanical properties. PCL modulates the mechanical properties of the PEA elastomeric network. Cultures of fibroblasts and adipose-tissue derived stem cells showed excellent biological performance of the materials. The results are relevant for applications seeking materials leaving a permanent supporting skeleton after the partial degradation, as in patches for cardiac regeneration or in abdominal wall meshes.

Fibrinolytic activity in laparoscopic cholecystectomy.

The aim of this study was to examine the fibrinolytic activity in laparoscopic cholecystectomy (LC) to determine whether changes occur that might indicate a greater risk of thrombosis. The study was carried out in 20 patients who had undergone laparoscopic surgery for cholelithiasis without complications. The average age was 59.4 years (34-77 years). Seventy-five percent were women. The mean operating time was 70 minutes (35-120 minutes). Pneumoperitoneum at 14 mm Hg was maintained in all patients, and they were in 30 degrees reverse Trendelenburg position. Postoperative mobilization was obtained before 24 hours, and patients were discharged 48 hours after surgery. The control group was composed of 12 patients, evenly distributed by age, sex, and length of surgery, who had undergone Bassini herniorrhaphy without complications or relapses. The following hemostatic parameters were studied: plasma fibrinolytic activity (PFA), euglobulin fibrinolytic activity (EFA), tissue-type plasminogen activator (t-PA), fast-acting plasminogen activator inhibitor-1 (PAI-1), and D-dimer (D-D). Samples were obtained at the following times: (1) under basal conditions the day before surgery, (2) preoperatively, (3) at the end of surgery, (4) 24 hours after surgery, and (5) on the seventh day following surgery. No patient had clinical manifestations of thromboembolic disease immediately after surgery or during an average follow-up period of 16 months (range 15-18 months). Analysis of the results of global fibrinolysis showed that fibrinolytic activity was enhanced only in the postoperative period (third sample) of the LC patients. The fraction of euglobulins enhances fibrinolytic activity in both groups in the third sample with regard to the other determinations; the LC patients showed a higher degree of significance (p<0.005). A significant increase of postoperative t-PA in both groups was found, being more significant in the LC group (p<0.005). In the PAI-1 values, no significant differences existed between either determinations or groups. A significant increase in D-dimer (p<0.05) occurred in the immediate postoperative period (third sample) and 24 hours later (fourth sample), returning to normal basal values on the seventh day. No significant differences were found between the two groups. These results seem to indicate that LC produces an increase in the fibrinolytic activity in plasma as a result of the liberation of tissue plasminogen activator from the venous endothelium, which could indicate hypocoagulability during the immediate postoperative period and, therefore, signify less thrombotic risk for patients undergoing this procedure.

Roux-en-Y syndrome after surgical treatment of alkaline reflux gastritis.

OBJECTIVE: to evaluate the possible existence of the so-called <> in a group of 21 patients who underwent surgery to correct postoperative alkaline reflux gastritis. METHODS: the study group consisted of 15 men and 6 women (mean age 39.2 years). All had undergone Billroth II subtotal gastrectomy (20 for ulcer and 1 for gastric cancer). Alkaline diversion was done with the Roux-en-Y technique 50-60 cm away from the gastrojejunal anastomosis. In all patients bilateral truncal vagotomy at the hiatus was also done. No mechanical alterations in the gastrojejunal anastomosis were found during surgery. Mean follow-up period was 8.2 years (range 6.5-10.7 years), during which clinical, radiological and endoscopic studies were obtained. Gammagraphic study of gastric remnant emptying was done on postoperative day 30. RESULTS: none of the patients had clinical, radiological or endoscopic manifestations that indicated the presence of Roux-en-Y syndrome. Gammagraphic studies of gastric remnant emptying did not demonstrate significant differences between preoperative (T1/2: 7.3 min) and postoperative values (T1/2: 10.1 min). CONCLUSIONS: we found no evidence of disturbances in gastric remnant emptying after Roux-en-Y gastrojejunostomy to treat postoperative alkaline gastric reflux.

[Acute cholecystitis: an evaluation of the factors that determine the start of surgical treatment]. / Colecistitis aguda: valoración de los factores que condicionan el inicio del tratamiento quirúrgico.

The authors studied 100 acute cholecystitis treated between 1984 and 1990. In 71.4% of the cases it was associated with gallstones and 28.6% were primary acalculous cholecystitis. Two percent were postoperative. 77 patients underwent surgery. 14.3% needed an emergency operation due to acute abdominal syndrome and sepsis. In the remaining patients, the surgical procedure was performed days or weeks later according to the course of the disease, the surgeons criteria, and family and social-labour conditions. Based on these criteria, 31.7% had surgery during the first week, 23.8% in the second and 44.5% in the third or later. Cholecystectomy was the surgical procedure performed in 98.7% of the cases. Morbidity rate was 11.6% and mortality 3.9%. Analysis of morpho-clinical grades has been done in relation with the type of cholecystitis, the clinical symptoms and the course of the disease. Finally the different factors used to argue for an early or delayed surgical treatment are discussed.

[Carcinoma in a long-standing Zenker's diverticulum]. / Carcinoma en divertículo de Zenker de larga evolución.

The authors report a case of carcinoma arising in a longstanding Zenker's diverticulum, in a 66 years old man that refused surgical treatment for 25 years. Three months prior to his admission to our hospital, an exacerbation of his dysphagia, which became severe, was observed; and so was regurgitation, with passage to the airway during swallowing or when asleep. The esophagram showed the diverticulum without images suggestive of neoplasm, and with spilling of barium into the tracheobronchial tree. Esophagoscopy was refused by the patient. After surgical diverticulectomy, a thickened area in the inferior portion of the diverticular body was observed, which was histologically reported as a squamous cell carcinoma with pearl formation, involving only the diverticular wall. Complementary radiotherapy with TCT was administered over the esophagus, mediastinum and supraclavicular lymphatic areas, with a total dose of 5000 Cgy. Concomitant chemotherapy with Mitomycin and 5-Fluorouracil was administered. After a 2 year follow-up, the patient is completely asymptomatic. We discuss etiopathogenic factors, clinical manifestations, diagnostic procedures, and therapeutic possibilities.

[Risk factors of peptic ulcer hemorrhage]. / Estudio de factores de riesgo de la hemorragia por úlcera péptica.

A retrospective analysis is made of the risk factors in 229 patients admitted to hospital with hemorrhage from peptic ulcer. The factors that have been studied are: 1) age. 2) type of ulcer lesion. 3) associated disease. 4) antiinflammatory intake. 5) prior ulcer symptoms. 6) intensity and outcome of the bleeding episode. 7) endoscopic findings. 8) treatment modality. 9) mortality. The analysis of these factors tries to establish, by means of the square chi test with Yates correction, the possible relations between the factors, to determine which ones will have a pronostic value. From the results obtained we conclude that the factors with the highest pronostic importance are: 1) With respect to the severity of the bleeding episode, antiinflammatory intake and duodenal location of the ulcer lesion. 2) With respect to the need for urgent surgical treatment, antiinflammatory intake and prior ulcer symptoms. 3) With respect to mortality, severity, persistence and recurrence of bleeding, and the need for urgent surgical treatment. Finally, it is important to mention the absence of pronostic value, with respect to mortality, of advanced age and the endoscopic findings of active and/or recent bleeding.

[Blood gastrin changes in the late postoperative period after proximal gastric vagotomy]. / Alteraciones de la gastrinemia en el postoperatorio tardío tras vagotomía gástrica proximal.

Changes in serum gastrin levels in the late postoperative period have been studied in 24 patients with non-stenotic duodenal ulcer who underwent proximal gastric vagotomy. Twenty healthy volunteers were used as a control group. Serum gastrin levels were determined under basal conditions and after a high protein meal stimulation. Both measurements were done in the preoperative, early postoperative (12th day) and late postoperative periods (X = 5.5 yrs.). Regarding basal serum gastrin levels, the results show mean values of 46.2 pg/ml in the preoperative, 61.6 pg/ml in the early postoperative, 73.9 pg/ml in the late postoperative and 51 pg/ml in the control group. Early and late postoperative period values show statistical significant differences when compared with preoperative values (p < 0.05), but not with the control group ones. Stimulated gastrin levels show mean values of 75.7 pg/ml in the preoperative, 99.1 pg/ml in the early postoperative, 134.1 pg/ml in the late postoperative and 73.4 pg/ml in the control group. Late postoperative values show statistical significant differences when compared with preoperative and early postoperative values (p < 0.05), and also when compared with the control group (p < 0.05). Possible causes and the physiopathological effects of these variations are discussed.

[Determination of basal and stimulated blood gastrin in duodenal ulcer]. / Valoración de la gastrinemia basal y estimulada en la úlcera duodenal.

Serum gastrin levels were measured under basal conditions and after hyperproteic meal stimulation in 24 patients with non-stenotic duodenal ulcer, 78% of them were males with a mean age of 36.4 years. Results were compared with those obtained in 20 volunteers. Basal gastrin levels in patients with duodenal ulcer 46.2 +/- 17.5 pg/ml did not show any significant statistical differences when compared with those in the control group (51.01 +/- 28.1 pg/ml). After meal stimulation gastrin levels at different time intervals, were similar in patients with duodenal ulcer and in the control group. We conclude that serum gastrin does not seem to play a relevant pathogenic role in the development of duodenal ulcer; its measurement is of no value as a biological marker of duodenal ulcer disease.