Craniofacial osteoblast responses to polycaprolactone produced using a novel boron polymerisation technique and potassium fluoride post-treatment

There is no ideal material for craniofacial bone repair at present. The aim of this study was to test the biocompatibility of polycaprolactone (PCL) synthesised by a novel method allowing control of molecular weight and degradation rate, with regard to it being used as matrix for a biodegradable composite for craniofacial bone repair. Human primary craniofacial cells were used, isolated from paediatric skull after surgery. Cell responses were analysed using various assays and antibody staining. Cells attached and spread on the PCL in a similar manner to the Thermanox controls as shown by phalloidin staining of F-actin. Cells maintained the osteoblast phenotype as demonstrated by alkaline phosphatase assay and antibody staining throughout the time points studied, up to 28 days. Cells proliferated on the PCL as shown by a DNA assay. Collagen-1 staining showed extensive production of a collagen-1 containing extracellular matrix, which was also shown to be mineralised by alizarin red staining. Short-term (up to 48 h) attachment studies and long-term (up to 28 days) expression of markers of the osteoblast phenotype have been demonstrated on the PCL. This new method of synthesising PCL shows biocompatibility characteristics that give it potential to be used for craniofacial bone repair.     

Polyamide 6 composite membranes: properties and in vitro biocompatibility evaluation

The aim of the present study was to develop polyamide 6 membrane blended with gelatin and chondroitin sulfate using the phase precipitation method and evaluate its in vitro biocompatibility. Morphology of membranes was studied by laser scanning confocal microscopy which allowed the nondestructive visualization of internal bulk morphology of membranes. Membranes exhibited porous morphology with pores spanning across the membrane width with interconnections at various depths. Membranes showed adequate mechanical properties with tensile strengths of 20.10 +/- 0.64 MPa, % strain of 3.01+/-0.07, and modulus of 1082.50+/-23.50 MPa. In vitro biocompatibility of membranes by direct contact test did not show degenerative effects on NIH3T3 cells and also its leach-out products (LOP), as determined by tetrazolium (MTT) and neutral red uptake (NRU) assay. Mouse peritoneal macrophage cultured in contact with membranes and PTFE control showed comparable expression of activation markers such as CD11b/CD18, CD45, CD14, and CD86 suggesting the membranes' non-activating nature. Membrane LOP did not induce excessive proliferation of mouse splenocytes suggesting its non-antigenic nature. Preliminary blood compatibility of membranes was observed with no detectable hemolysis in static incubation assay. Taken collectively, the present data demonstrate that polyamide 6 composite membranes are biocompatible and prospective candidates for tissue engineering applications.     

A novel dentin bond strength measurement technique using a composite disk in diametral compression

New methods are needed that can predict the clinical failure of dental restorations that primarily rely on dentin bonding. Existing methods have shortcomings, e.g. severe deviation in the actual stress distribution from theory and a large standard deviation in the measured bond strength. We introduce here a novel test specimen by examining an endodontic model for dentin bonding. Specifically, we evaluated the feasibility of using the modified Brazilian disk test to measure the post-dentin interfacial bond strength. Four groups of resin composite disks which contained a slice of dentin with or without an intracanal post in the center were tested under diametral compression until fracture. Advanced nondestructive examination and imaging techniques in the form of acoustic emission (AE) and digital image correlation (DIC) were used innovatively to capture the fracture process in real time. DIC showed strain concentration first appearing at one of the lateral sides of the post-dentin interface. The appearance of the interfacial strain concentration also coincided with the first AE signal detected. Utilizing both the experimental data and finite-element analysis, the bond/tensile strengths were calculated to be: 11.2 MPa (fiber posts), 12.9 MPa (metal posts), 8.9 MPa (direct resin fillings) and 82.6 MPa for dentin. We have thus established the feasibility of using the composite disk in diametral compression to measure the bond strength between intracanal posts and dentin. The new method has the advantages of simpler specimen preparation, no premature failure, more consistent failure mode and smaller variations in the calculated bond strength.     

Biomaterial properties and biocompatibility in cell culture of a novel self-inflating hydrogel tissue expander

The aim of this study was to investigate the swelling properties and the biocompatibility of a novel tissue expander material. The self-inflating material is a hydrogel consisting of a modified copolymer of methylmethacrylate and N-vinyl-2-pyrrolidone, which takes up water by osmosis. To increase the swelling volume, the primarily neutral gel material was modified by converting it into an ionized gel. To study the swelling and pressure behavior of the material, the anhydrous gel cylinders were equilibrated in distilled water, saline, and sugar solutions. The biocompatibility was investigated in cell culture. We tested the hydrogel eluate after swelling for cytotoxicity and mutagenicity using the cell lines MRC-5 and P3X63 Ag8 653 (Ag8). Furthermore, particles of the material were added to cell cultures to induce foreign body reactions and to verify its influence on monocyte differentiation. The material has a swelling capacity (Q = maximum swelling volume/anhydrous volume) of 5 to 50 depending on the degree of ionization of the polymer network. In this study, two polymer modifications with a swelling equilibrium of Q = 11.1 and 30 in water were tested. The swelling ratio also depends on concentration and ion content of the equilibration medium. The highest swelling capacity was found in water, the lowest in Ringer's solution. The swelling of the anhydrous material with the swelling capacity of Q = 11.1 fits best the average purpose of material properties for tissue expansion and generates a maximal hydrostatic pressure of approximately 235 mmHg. Effects on cell proliferation were detected only at the highest eluate concentration tested (i.e., eluate: culture medium = 1:1), which was far beyond physiological values, whereas mutagenicity was absent. Monocytes neither migrated nor tightly attached to the hydrogel. They neither phagocytose the material nor did they show any sign of a foreign body reaction, e.g., formation of multinucleated giant cells or monocyte proliferation. In the presence of hydrogel material, the differentiation processes of monocytes to macrophages or dendritic cells, respectively, were found to be undisturbed. From these results, we conclude that there is a high biocompatibility of the expander material, which may be a favorable and interesting candidate for further clinical applications.     

In vivo biocompatibility and mechanical study of novel bone-bioactive materials for prosthetic implantation.

Two epoxy materials with or without adhesively bonded hydroxyapatite (HA) coatings were studied for their biocompatibility and mechanical pushout strength using in vivo implantation in the rabbit lower femur for a duration of 10 days to 6 months. Both were two-part epoxies cured at room temperature for 24 h, with material 1 (Ampreg 26; SP Systems Limited, Cowes, UK) postcured at 110 degrees C (Tg approximately 80 degrees C) and Material 2 (CG5052; Ciba Geigy Limited, Cambridge, UK) at 125 degrees C (Tg approximately 120 degrees C). Implantation in dead rabbit bone was performed to provide mechanical baseline levels. Polymethylmethacrylate (PMMA) and conventionally HA-coated titanium alloy (Ti-6Al-4V) were used as control materials. In the biological study, different fluorescent dyes were used to label newly formed bone. After 6 weeks of implantation, results from mechanical pushout tests showed that the interfacial shear strength (ISS) values were significantly higher than for dead bones with each of the different implants (p < .01-.001). HA-coated material 2 showed a significantly higher ISS value than the uncoated material (p < .05) after 6 weeks' implantation. However, the ISS value for the uncoated material 2 was significantly higher than for PMMA controls (p < .05). No significant differences in the ISS values were shown between HA-coated materials 1 and 2 and Ti-6Al-4V on in vivo implantation for 6 weeks. Failure points of the pushout test from the three HA-coated materials were defined by scanning electron microscopy. Specimens implanted with both HA-coated epoxies were fractured within the HA-coatings or the bone, while with HA-coated Ti-6Al-4V cracked between the coating and metal implant. The percentage of bone in contact with the implant surface was obtained by image analysis which showed that there were no significant differences between different materials after short time implantation (up to 6 week). Long-term implantation of the HA-coated material 2 showed that the percentage of bone contact had increased from 52.8+/-1.1% (6 week) to 80.0+/-0.3% (3 months) (p < .01) and remained at 81.0+/-0.8% (6 months). Measurements of bone mineralization rate (BMR) showed that after 3 weeks of implantation, there were no significant differences between PMMA and uncoated materials 1 and 2. After 6 weeks, the BMRs in animals implanted with either HA-coated material 1 or 2 were significantly higher than with HA-coated Ti-6Al-4V (p < .05-.0001 in both cases), but with HA-coated material 2 was lower than with this material uncoated (p < .05-.001). No significant differences were found between the two HA-coated epoxy materials. In addition, there were always lower BMRs during the third week of implantation than other periods regardless of biomaterial implanted. The study indicated that the adhesively bonded HA-coated novel epoxy materials were superior to conventional plasma-sprayed Ti-6Al-4V implants with respect to both BMR and bone integration with the implant surfaces. Adhesively bonded HA-coated epoxy materials had similar ISS values to HA-coated Ti-6Al-4V, but the former failed within the bone and coating, while the latter showed splitting between coating and metal.     

Fabrication, characterization, and biocompatibility of single-walled carbon nanotube-reinforced alginate composite scaffolds manufactured using freeform fabrication technique

Composite polymeric scaffolds from alginate and single-walled carbon nanotube (SWCNT) were produced using a freeform fabrication technique. The scaffolds were characterized for their structural, mechanical, and biological properties by scanning electron microscopy, Raman spectroscopy, tensile testing, and cell-scaffold interaction study. Three-dimensional hybrid alginate/SWCNT tissue scaffolds were fabricated in a multinozzle biopolymer deposition system, which makes possible to disperse and align SWCNTs in the alginate matrix. The structure of the resultant scaffolds was significantly altered due to SWCNT reinforcement, which was confirmed by Raman spectroscopy. Microtensile testing presented a reinforcement effect of SWCNT to the mechanical strength of the alginate struts. Ogden constitutive modeling was utilized to predict the stress-strain relationship of the alginate scaffold, which compared well with the experimental data. Cellular study by rat heart endothelial cell showed that the SWCNT incorporated in the alginate structure improved cell adhesion and proliferation. Our study suggests that hybrid alginate/SWCNT scaffolds are a promising biomaterial for tissue engineering applications.     

Physical and biological properties of a novel siloxane adhesive for soft tissue applications

The aim of this study was to investigate the adhesive properties of an in-house aminopropyltrimethoxysilane-methylenebisacrylamide (APTMS-MBA) siloxane system and compare them with a commercially available adhesive, n-butyl cyanoacrylate (nBCA). The ability of the material to perform as a soft tissue adhesive was established by measuring the physical (bond strength, curing time) and biological (cytotoxicity) properties of the adhesives on cartilage. Complementary physical techniques, X-ray photoelectron spectroscopy, Raman and infrared imaging, enabled the mode of action of the adhesive to the cartilage surface to be determined. Adhesion strength to cartilage was measured using a simple butt joint test after storage in phosphate-buffered saline solution at 37 degrees C for periods up to 1 month. The adhesives were also characterised using two in vitro biological techniques. A live/dead stain assay enabled a measure of the viability of chondrocytes attached to the two adhesives to be made. A water-soluble tetrazolium assay was carried out using two different cell types, human dermal fibroblasts and ovine meniscal chondrocytes, in order to measure material cytotoxicity as a function of both supernatant concentration and time. IR imaging of the surface of cartilage treated with APTMS-MBA siloxane adhesive indicated that the adhesive penetrated the tissue surface marginally compared to nBCA which showed a greater depth of penetration. The curing time and adhesion strength values for APTMS-MBA siloxane and nBCA adhesives were measured to be 60 s/0.23 MPa and 38 min/0.62 MPa, respectively. These materials were found to be significantly stronger than either commercially available fibrin (0.02 MPa) or gelatin resorcinol formaldehyde (GRF) adhesives (0.1 MPa) (P < 0.01). Cell culture experiments revealed that APTMS-MBA siloxane adhesive induced 2% cell death compared to 95% for the nBCA adhesive, which extended to a depth of approximately 100-150 microm into the cartilage surface. The WST-1 assay demonstrated that APTMS-MBA siloxane was significantly less cytotoxic than nBCA adhesive as an undiluted conditioned supernatant (P < 0.001). These results suggest that the APTMS-MBA siloxane may be a useful adhesive for medical applications.     

Synthesis, characterization, and biocompatibility of novel injectable, biodegradable, and in situ crosslinkable polycarbonate-based macromers

A series of novel self-crosslinkable and biodegradable polymers, poly(hexamethylene carbonate-fumarate) and poly(hexamethylene carbonate) diacrylate, and their amphiphilic copolymers with polyethylene glycol, poly(ethylene glycol fumarate-co-hexamethylene carbonate-fumarate) (PEGF-co-PHMCF), were developed for tissue engineering using propylene oxide as an acid scavenger. The synthesized polymers are white, which makes them more suitable for self-crosslinking via photopolymerization. These novel polymers were fully characterized using nuclear magnetic resonance spectroscopy, Fourier-transform infrared spectroscopy, gel permeation chromatography, differential scanning calorimetry, and rheometry. The cytocompatibility of the photocrosslinked networks were evaluated by [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. These polymers can be used as precursors to prepare polymer networks and scaffolds with controlled hydrophilicity, biodegradability, and mechanical characteristics. Results obtained suggest that these polymers are potentially useful as injectable and photocrosslinkable materials for cell delivery, tissue engineering, and drug delivery applications.     

Human tooth culture: a study model for reparative dentinogenesis and direct pulp capping materials biocompatibility

In a previous work, based on an in vitro entire tooth culture model of human immature third molars, we demonstrated that perivascular progenitor cells can proliferate and migrate to the injury site after pulp exposure. In this work, we investigated the differentiation of cells after direct capping with biomaterials classically used in restorative dentistry. Histological staining after direct pulp capping with Calcium Hydroxide XR(R) or MTA revealed early and progressive mineralized foci formation containing BrdU-labeled sequestered cells. The molecular characterization of the matrix and the sequestered cells by immunohistochemistry (Collagene type I, Dentin sialoprotein, and Nestin) clearly demonstrates that these areas share common characteristics of the mineralized matrix of reparative dentin formed by odontoblast-like cells. This reproduces some features of the pulp responses after applying these materials in vivo and demonstrates that the entire tooth culture model reproduces a part of the early steps of dentin regeneration in vivo. Its future development may be useful in studying the effects of biomaterials on this process.     

Silver coated materials for external fixation devices: in vitro biocompatibility and genotoxicity.

The most significant complication in external fixation is pin tract infection causally related to the highly adaptive ability of bacteria to colonise the surfaces of "inert" biomaterials or of adjacent damaged tissue cells. The hypothesis that coating a pin with a silver-containing compound will decrease bacterial colonisation and/or pin tract infection has been confirmed in other studies in vitro and in vivo experiments. In this work, biocompatibility of silver-coated orthopaedic external fixation pins was compared with stainless steel controls in an in vitro study. Human peripheral blood lymphocytes were used to assess the possible genotoxic effect of silver, studying the frequency of sister-chromatid exchanges and micronuclei while fibroblasts (NIH 3T3) and osteoblast-like cells were used for cytotoxicity and cytocompatibility studies. These studies have shown that silver is neither genotoxic nor cytotoxic as compared to stainless steel, a material in wide use as a metal implant. At 4 days cells cultured on the silver-coated material evidenced good cell spreading and a higher cell count with respect to the uncoated material. It appears that the addition of silver onto implantable medical devices could be beneficial when specific biological properties, such as antibacterial behaviour, are required. Based on these and the previous bacterial studies it seems like the toxicity towards bacteria was quite a bit greater than that towards the human cells.     

The biocompatibility and osteoconductive activity of a novel hydroxyapatite/collagen composite biomaterial, and its function as a carrier of rhBMP-2

A hydroxyapatite/type I collagen (HAp/Col) composite, in which the hydroxyapatite nanocrystals align along the collagen molecules, has been prepared. The biocompatibility, osteoconductive activity, and efficacy as a carrier of recombinant human bone morphogenetic proteins (rhBMPs) of this novel biomaterial were examined. The composite material was implanted in the backs of Wistar rats, and specimens were collected for histological observations until week 24. In a second experiment, other samples of the composite material (5 x 5 x 10 mm3) were drilled and immersed in a solution of rhBMP-2 (0, 200, 400 microg/mL), and subsequently grafted in radii and ulnae in beagle dogs. As a control, three unfilled holes were left in one radius and ulna. X-ray images were prepared, and specimens collected for histological observation at weeks 8 and 12. Histological findings of the composites grafted in rats showed that the surface of the material was eroded as a result of macrophage infiltration. X-ray images and histological findings for the composites implanted in dogs support the idea that HAp/ Col has a high osteoconductive activity and is able to induce bone-remodeling units. In cases where the implants are grafted at weight bearing sites, treatment with rhBMP-2 at a dose of 400 microg/mL may be useful to shorten the time needed until bone union has occurred.     

A novel technique for sacropelvic fixation using image-guided sacroiliac screws: a case series and biomechanical study

In this study, we sought to assess the safety and accuracy of sacropelvic fixation performed with image-guided sacroiliac screw placement using postoperative computed tomography and X-rays. The sacroiliac screws were placed with navigation in five patients. Intact specimens were mounted onto a six-degrees-of-freedom spine motion simulator. Long lumbosacral constructs using bilateral sacroiliac screws and bilateral S1 pedicle and iliac screws were tested in seven cadaveric spines. Nine sacroiliac screws were well-placed under an image guidance system (IGS); one was placed poorly without IGS with no symptoms. Both fixation techniques significantly reduced range of motion (P < 0.05) at L5–S1. The research concluded that rigid lumbosacral fixation can be achieved with sacroiliac screws, and image guidance improves its safety and accuracy. This new technique of image-guided sacroiliac screw insertion should prove useful in many types of fusion to the sacrum, particularly for patients with poor bone quality, complicated anatomy, infection, previous failed fusion and iliac harvesting.     

Mechanical characterization and biocompatibility of a novel reinforced fascia patch for rotator cuff repair

To provide mechanical augmentation for rotator cuff repair, it is necessary (though perhaps not sufficient) that scaffolds have tendon-like material and suture retention properties, be applied to the repair in a surgically appropriate manner, and maintain their mechanical properties for an acceptable period of time following surgery. While allograft fascia lata has material, structural, and biochemical properties similar to tendon tissue, its poor suture retention properties abrogates its potential as an augmentation device. The goal of this work was to design a novel reinforced fascia patch with suture retention and stiffness properties adequate to provide mechanical augmentation for rotator cuff repair. Fascia was reinforced by stitching with PLLA or PLLA/PGA polymer braids. Reinforced fascia patches had a maximum construct load greater than (or equal to) the suture retention properties of human rotator cuff tendon (～250N) at time zero and after in vivo implantation for 12 weeks in a rat subcutaneous model. The patches were able to withstand the 2500 loading cycles projected for the early post-operative period. The patches also demonstrated biocompatibility with the host using a rat abdominal wall defect model. These studies suggest the potential use of reinforced fascia patches to provide mechanical augmentation, minimize tendon retraction and possibly reduce the incidence of rotator cuff repair failure.     

The biocompatibility of novel starch-based polymers and composites: in vitro studies

Studies with biodegradable starch-based polymers have recently demonstrated that these materials have a range of properties. which make them suitable for use in several biomedical applications, ranging from bone plates and screws to drug delivery carriers and tissue engineering scaffolds. The aim of this study was to screen the cytotoxicity and evaluate starch-based polymers and composites as potential biomaterials. The biocompatibility of two different blends of corn-starch, starch ethylene vinyl alcohol (SEVA-C) and starch cellulose acetate (SCA) and their respective composites with hydroxyapatite (HA) was assessed by cytotoxicity and cell adhesion tests. The MTT assay was performed with the extracts of the materials in order to evaluate the short-term effect of the degradation products. The cell morphology of L929 mouse fibroblast cell line was also analysed after direct contact with polymers and composites for different time periods and the number of cells adhered to the surface of the polymers was determined by quantification of the cytosolic lactate dehydrogenase (LDH) activity. Both types of starch-based polymers exhibit a cytocompatibility that might allow for their use as biomaterials. SEVA-C blends were found to be the less cytotoxic for the tested cell line, although cells adhere better to SCA surface. The cytotoxicity test also revealed that SCA and SEVA-C composites have a similar response to the one obtained for SCA polymer. Scanning electron microscopy (SEM) analysis showed that cells were much more spread on the SCA polymer and LDH measurements showed a higher number of cells on this surface.     

Alginate-poly-L-lysine microcapsule biocompatibility: a novel RT-PCR method for cytokine gene expression analysis in pericapsular infiltrates.

Transplantation of microencapsulated islets of Langerhans is impaired by a pericapsular host reaction that eventually induces graft failure. We are studying the role of cytokines in the pathogenesis of this reaction, using the model of alginate-polylysine microcapsule implantation in rat epididymal fat pads. The objectives were: (1) to develop a method to measure, by semiquantitative PCR, TGF-beta1 gene expression in fat pad pericapsular infiltrates, and (2) to use this method to evaluate TGF-beta1 gene expression 14 days after microcapsule implantation. TGF-beta1 mRNA level was significantly higher in pericapsular infiltrate cells than in nonimplanted tissue cells and saline-injected tissue cells (p < 0.0001 and p < 0.01, respectively). There was no significant difference between the TGF-beta1 mRNA levels of the two types of controls (p = 0.0945). These results suggest that TGF-beta1 plays a role in the pathogenesis of the pericapsular reaction. The method developed can be used to study the role of other fibrogenic cytokines potentially involved. This will shed light on the mechanisms underlying the pericapsular reaction and will serve as a basis for the development of strategies to control this reaction.     

Quality of alveolar bone--Structure-dependent material properties and design of a novel measurement technique

The evidence for the efficiency of clinical methods used to assess the quality of alveolar bone in terms of a density measure prior to and during dental implant surgery is limited. The aim of this paper is to describe the biomechanical background which can be used as a basis for determining the bone quality by measuring the elastic properties of the bone and to design a novel device for the determination of the bone quality during dental implant surgery. Applying material mechanical equations for porous and cellular structured models, the elastic material properties (modulus of elasticity) of cellular and cortical bone as porous structures were approximated over the whole range of relative bone mineral density of trabecular and cortical bone. Based on a circular disc with a central hole reflecting a horizontal cross-section of an implant socket, the mechanical effects of expanding the central hole were studied. Subsequently, the clinical situation of a socket prepared for the placement of a dental implant (depth: 10?mm; diameter 3.5?mm) was simulated using three-dimensional (3D) finite element analysis. A loading device (thickness: 3.5?mm) was placed in the trabecular part of the socket and expanded, while the resulting pressure was recorded and used for the calculation of an elastic modulus. Finite element analysis revealed that it was possible to estimate the bone quality by applying the measurement technique proposed. Maximum deviations of 6% of the experimentally determined elastic modulus from the setpoint elastic modulus were found. Measuring the internal pressure in a drill hole, e.g., in an implant socket caused by a defined expansion of a rotational symmetric loading device, could be used for establishing a clinically meaningful test system for the objective classification of alveolar bone.     

18q21 Rearrangement and trisomy 3 in extranodal B-cell lymphomas: a study using a fluorescent in situ hybridisation technique

t(11;18)(q21;q21) Translocation and trisomy 3 are the most common chromosomal aberrations reported in low-grade mucosa-associated lymphoid tissue (MALT) lymphoma. The current study aims to investigate the frequency of these chromosomal aberrations in a series of 52 extranodal B-cell lymphomas. The tumours were categorised into three histological grades: grade 1 (low-grade lymphoma of MALT type), grade 2 [diffuse large B-cell lymphoma (DLBCL) with MALT component] and grade 3 (DLBCL without MALT component). Fluorescence in situ hybridisation analyses on paraffin tissue sections were performed using a locus-specific probe for the 18q21 region and a centromeric probe for chromosome 3. The 18q21 rearrangement was detected in 9 of 40 (23%) cases, including 7 of 23 (30%) grade-1 and 2 of 11 (18%) grade-3 tumours. Amplification of the 18q21 region was detected in 10 of 40 (25%) cases, and trisomy 3 was detected in 9 of 34 (26%) cases. Amplification of the 18q21 region may be an important alternative pathogenetic pathway in MALT lymphoma and was found almost exclusively in tumours without 18q21 rearrangement. Our study showed that tumours with 18q21 rearrangement and 18q21 amplification develop along two distinct pathways, and the latter was more likely to transform into high-grade tumours upon acquisition of additional genetic alterations, such as trisomy 3. Trisomy 3 was more frequently found in coexistence with 18q21 abnormalities, suggesting that it was more likely to be a secondary aberration.