Methods of producing three dimensional electrospun scaffolds for bone tissue engineering: A review.

Bone is a dynamic, living tissue that exists and renews itself continuously in a 3D manner. Nevertheless, complex clinical conditions require a bone substitute to replace the defective bone and/or accelerate bone healing. Bone tissue engineering aims to treat bone defects that fail to heal on their own. Electrospinning provides an opportunity to create nano- to micro-fibrous scaffolds that mimic the architecture of the natural extracellular matrix (ECM) with high porosity and large specific surface area. Despite these advantages, traditional electrospun meshes can only provide a 2D architecture for cell attachment and proliferation rather than the 3D attachment in native tissue. Fabrication of 3D electrospun scaffolds for bone tissue regeneration is a challenging task, which has attracted significant attention over the past couple of decades. This review highlights recent strategies used to produce 3D electrospun/co-electrospun scaffolds for bone tissue applications describing the materials and procedures. It also considers combining conventional and coaxial electrospinning with other scaffold manufacturing techniques to produce 3D structures which have the potential to engineer missing bone in the human body.Graphical abstract[Formula: see text].

Testing meshes in a computer model of a laparoscopic ventral hernia repair.

BACKGROUND: The ideal mesh for hernia repair has yet to be found, in addition our knowledge of the biomechanics of the abdominal wall is poor. The aim of this study was to develop a computer model of a laparoscopic ventral hernia repair and to test different meshes in that model at various intra-abdominal pressures. METHODS: Four meshes were tested in a computer model of a ventral hernia. Mechanical failure testing of each mesh was performed in both the longitudinal and transverse directions. A CT scan of a patient with a 5 cm umbilical hernia was used to generate a 3 dimensional model. Meshes were then applied to the model in an intraperitoneal onlay position with a 5 cm overlap. The model was then tested with intraabdominal pressures for standing, coughing and jumping with and without meshes. RESULTS: Meshes varied significantly (p < 0.001) in both rupture force 14.8 (5.6) to 78 (5) n/cm and force in which they changed from elastic to plastic 1.6 (0.1) to 14.2 (0.2) n/cm. When applied to the computer model all significantly reduced the strain on the abdominal wall from 17.5% without mesh to less than 1% with mesh. All meshes prevented the hernia from bulging in the model. CONCLUSIONS: We have developed a computer model of laparoscopic ventral hernia repair based on engineering principles. This model demonstrated that meshes tested significantly reduced the strain on the abdominal wall. Further studies are required to refine this model in order to best simulate the biomechanics of the abdominal wall.

Ultrasound-guided adductor canal block: a cadaver study investigating the effect of a thigh tourniquet.

BACKGROUND: Placement of local anaesthetic within the adductor canal using ultrasonography is an alternative to femoral nerve blocks for postoperative pain relief after knee joint replacement surgery. However, the effect of an inflated thigh tourniquet on the distribution of local anaesthetic within the adductor canal is unknown. The aim of this cadaveric study was to compare the distribution of radio-opaque dye within the adductor canal in the presence or absence of an inflated thigh tourniquet. METHODS: Bilateral ultrasound-guided adductor canal blocks were performed on the thawed lower limbs of five fresh frozen human cadavers. The left and right lower cadaver limbs were randomised to receive or not receive a thigh tourniquet inflated to 300 mm Hg for 1 h. X-rays with iohexol radio-opaque dye were obtained in four views, and fiducial markers inserted as reference points. Virtual editing technology was used to recreate outlines representing the distribution of the radio-opaque dye and superimpose these on anatomical images. RESULTS: Radio-opaque dye was distributed on the medial aspect of the thighs with entire and well circumscribed margins. The majority of the radio-opaque dye was confined within the adductor canal. Superior-inferior dye distribution was 315 mm [95% confidence intervals (CI) 289-342] and 264 mm (95% CI 239-289) in the presence and absence of an inflated thigh tourniquet, respectively (diff 95% CI -80.46 to -22.22, P=0.0081). Image analysis using the recreated radio-opaque outlines suggested that the most proximal point of the radio-opaque dye was 100 mm (95% CI 82-117) or 117 mm (95% CI 62-171) below the inguinal ligament in the presence and absence of an inflated thigh tourniquet, respectively (diff 95% CI -38 to 72, P=0.456). CONCLUSIONS: Application and inflation of thigh tourniquets significantly increased the combined superior-inferior dye distribution within the adductor canal of cadaveric limbs. There was insufficient evidence to suggest significant proximal spread of 25 ml of local anaesthetic to involve the motor branches of the femoral nerve. In some patients, the local anaesthetic may reach the popliteal fossa in close approximation to the sciatic nerve.

Mechanical behaviour of degradable phosphate glass fibres and composites-a review.

Biodegradable materials are potentially an advantageous alternative to the traditional metallic fracture fixation devices used in the reconstruction of bone tissue defects. This is due to the occurrence of stress shielding in the surrounding bone tissue that arises from the absence of mechanical stimulus to the regenerating bone due to the mismatch between the elastic modulus of bone and the metal implant. However although degradable polymers may alleviate such issues, these inert materials possess insufficient mechanical properties to be considered as a suitable alternative to current metallic devices at sites of sufficient mechanical loading. Phosphate based glasses are an advantageous group of materials for tissue regenerative applications due to their ability to completely degrade in vivo at highly controllable rates based on the specific glass composition. Furthermore the release of the glass's constituent ions can evoke a therapeutic stimulus in vivo (i.e. osteoinduction) whilst also generating a bioactive response. The processing of these materials into fibres subsequently allows them to act as reinforcing agents in degradable polymers to simultaneously increase its mechanical properties and enhance its in vivo response. However despite the various review articles relating to the compositional influences of different phosphate glass systems, there has been limited work summarising the mechanical properties of different phosphate based glass fibres and their subsequent incorporation as a reinforcing agent in degradable composite materials. As a result, this review article examines the compositional influences behind the development of different phosphate based glass fibre compositions intended as composite reinforcing agents along with an analysis of different potential composite configurations. This includes variations in the fibre content, matrix material and fibre architecture as well as other novel composites designs.

Influence of test specimen fabrication method and cross-section configuration on tension-tension fatigue life of PMMA bone cement.

Different cyclic loading modes have been used in in vitro fatigue studies of PMMA bone cement. It is unclear which loading mode is most appropriate from the perspective of the in vivo loading experienced by the cement in a cemented arthroplasty. Also, in different in vitro fatigue studies, different test specimen configurations have been used. The present work considers the influence of test specimen fabrication method (direct moulding vs moulding followed by machining) and cross-section shape (rectangular vs circular) on the tension-tension fatigue performance of two bone cement brands (SmartSet GHV and CMW1), under force control conditions. Two trends were consistent: 1) for each of the cements, for moulded specimens, a longer fatigue life was obtained with circular cross-sectioned specimens and, 2) for either rectangular or circular CMW1 specimens, a longer fatigue life was obtained using machined specimens. A comparison of the present results to those reported in our previous work on fully-reversed tension-compression loading under force control showed that, regardless of the test specimen fabrication method or cross-section configuration used, the fatigue life was considerably shorter under tension-compression than tension-tension loading. This finding highlights the fact that the presence of the compression portion in the loading cycle accelerates fatigue failure.

Effects of test sample shape and surface production method on the fatigue behaviour of PMMA bone cement.

There is no consensus over the optimal criterion to define the fatigue life of bone cement in vitro. Fatigue testing samples have been made into various shapes using different surface preparation techniques with little attention being paid to the importance of these variations on the fatigue results. The present study focuses on the effect of test sample shape and surface production method on the fatigue results. The samples were manufactured with two cross sectional shapes: rectangular according to ISO 527 and circular according to ASTM F2118. Each shape was produced using two methods: direct moulding of the cement dough and machining from oversized rods. Testing was performed using two different bone cements: SmartSet GHV and DePuy CMW1. At least 10 samples of each category were tested, under fully reversed tension-compression fatigue stress at ±20MPa, to allow for Weibull analysis to compare results. The growth of fatigue cracks was observed by means of the changes in the absorbed energy and apparent modulus. It was found that fatigue crack growth can be altered by the sample shape and production method; however it is also dependent on the chemical composition of the cement. The results revealed that moulded samples, particularly those based on the ASTM F2118 standard, can lead to up to 5.5 times greater fatigue lives compared to the machined samples of the same cement. It is thus essential, when comparing the fatigue results of bone cement, to consider the effect of production method along with the shape of the test sample.

Effects of hydroxyapatite and PDGF concentrations on osteoblast growth in a nanohydroxyapatite-polylactic acid composite for guided tissue regeneration.

The technique of guided tissue regeneration (GTR) has evolved over recent years in an attempt to achieve periodontal tissue regeneration by the use of a barrier membrane. However, there are significant limitations in the currently available membranes and overall outcomes may be limited. A degradable composite material was investigated as a potential GTR membrane material. Polylactic acid (PLA) and nanohydroxyapatite (nHA) composite was analysed, its bioactive potential and suitability as a carrier system for growth factors were assessed. The effect of nHA concentrations and the addition of platelet derived growth factor (PDGF) on osteoblast proliferation and differentiation was investigated. The bioactivity was dependent on the nHA concentration in the films, with more apatite deposited on films containing higher nHA content. Osteoblasts proliferated well on samples containing low nHA content and differentiated on films with higher nHA content. The composite films were able to deliver PDGF and cell proliferation increased on samples that were pre-absorbed with the growth factor. nHA-PLA composite films are able to deliver active PDGF. In addition the bioactivity and cell differentiation was higher on films containing more nHA. The use of a nHA-PLA composite material containing a high concentration of nHA may be a useful material for GTR membrane as it will not only act as a barrier, but may also be able to enhance bone regeneration by delivery of biologically active molecules.

Bioactive ceramic-reinforced composites for bone augmentation.

Biomaterials have been used to repair the human body for millennia, but it is only since the 1970s that man-made composites have been used. Hydroxyapatite (HA)-reinforced polyethylene (PE) is the first of the 'second-generation' biomaterials that have been developed to be bioactive rather than bioinert. The mechanical properties have been characterized using quasi-static, fatigue, creep and fracture toughness testing, and these studies have allowed optimization of the production method. The in vitro and in vivo biological properties have been investigated with a range of filler content and have shown that the presence of sufficient bioactive filler leads to a bioactive composite. Finally, the material has been applied clinically, initially in the orbital floor and later in the middle ear. From this initial combination of HA in PE other bioactive ceramic polymer composites have been developed.

Comparison of two methods of fatigue testing bone cement.

Two different methods have been used to fatigue test four bone cements. Each method has been used previously, but the results have not been compared. The ISO 527-based method tests a minimum of 10 samples over a single stress range in tension only and uses Weibull analysis to calculate the median number of cycles to failure and the Weibull modulus. The ASTM F2118 test regime uses fewer specimens at various stress levels tested in fully reversed tension-compression, and generates a stress vs. number of cycles to failure (S-N) or Wöhler curve. Data from specimens with pores greater than 1mm across is rejected. The ISO 527-based test while quicker to perform, provides only tensile fatigue data, but the material tested includes pores, thus the cement is closer to cement in clinical application. The ASTM regime uses tension and compression loading and multiple stress levels, thus is closer to physiological loading, but excludes specimens with defects obviously greater than 1mm, so is less representative of cement in vivo. The fatigue lives between the cements were up to a factor 15 different for the single stress level tension only tests, while they were only a factor of 2 different in the fully reversed tension-compression testing. The ISO 527-based results are more sensitive to surface flaws, thus the differences found using ASTM F2118 are more indicative of differences in the fatigue lives. However, ISO 527-based tests are quicker, so are useful for initial screening.

Bioactive composites for bone tissue engineering.

One of the major challenges for bone tissue engineering is the production of a suitable scaffold material. In this review the currently available composite material options are considered and the methods of production and assessing the scaffolds are also discussed. The production routes range from the use of porogens to produce the porosity through to controlled deposition methods. The testing regimes include mechanical testing of the produced materials through to in vivo testing of the scaffolds. While the ideal scaffold material has not yet been produced, progress is being made.

Absorption and release of protein from hydroxyapatite-polylactic acid (HA-PLA) membranes.

OBJECTIVES: The aim of this study was to investigate the kinetics of protein interactions with a novel hydroxyapatite-polylactide (HA-PLA) composite membrane material. METHODS: Trilayer PLA and HA-PLA composite membranes reinforced with PLA fibres were used to absorb and release protein which was measured by a BioRad assay. The proteins used were fetal calf serum and bovine serum albumin. PLA and HA-PLA composite films were manufactured to test permeability. RESULTS: Maximal protein absorption was seen within 5min of treating materials; a nearly 8-fold increase in total absorption was seen with HA-containing composites compared to those without HA. These also exhibited a more gradual and sustained release of protein for periods of up to 96h, for example at 24h protein concentrations released were 2.20+/-2.80 and 0.49+/-5.38microg/ml for membranes with and without HA respectively. In addition low pressure and temperature used during production of membranes also allowed greater and more sustained protein release. HA-PLA composite films also showed marked increased permeability compared to plain PLA films, for example after 24h PLA only films 3.64+/-1.01microg/ml, PLA film with 25% HA: 44.99+/-35.61microg/ml, PLA film with 75% HA: 153.12+/-65.57microg/ml. CONCLUSIONS: The results demonstrate that these composite membranes rapidly absorb protein and that the absorbed protein is released slowly for periods of up to 96h, dependent on constituents of the material and the manufacturing conditions. Incorporation of HA into these membranes was the key factor for improved protein kinetics and membrane permeability.

In vitro biocompatibility of hydroxyapatite-reinforced polymeric composites manufactured by selective laser sintering.

The selective laser sintering (SLS) technique was used to manufacture hydroxyapatite-reinforced polyethylene and polyamide composites as potential customized maxillofacial implants. In vitro tests were carried out to assess cellular responses, in terms of cell attachment, morphology, proliferation, differentiation, and mineralized nodule formation, using primary human osteoblast cells. This study showed that the SLS composite processed was biocompatible, with no adverse effects observed on cell viability and metabolic activity, supporting a normal metabolism and growth pattern for osteoblasts. Positive von Kossa staining demonstrated the presence of bone-like mineral on the SLS materials. Higher hydroxyapatite content composites enhanced cell proliferation, increased alkaline phosphatase activity, and produced more osteocalcin. The present findings showed that SLS materials have good in vitro biocompatibility and hence demonstrated biologically the potential of SLS for medical applications.

Effect of ultrasound on the setting characteristics of glass ionomer cements studied by Fourier transform infrared spectroscopy.

OBJECTIVE: To investigate the effect of ultrasound (US) application, US staring time and US duration on the setting of glass ionomer cement (GIC) by using Attenuated Total Reflectance Fourier Transform Infrared (ATR/FTIR) spectrometer. METHODS: Two conventional GICs, Fuji IX Fast and Ketac Molar were studied. US application was started at 30 s or 40 s after mixing and was applied for times between 15 and 55 s on samples of two different thicknesses. The samples were analysed using ATR/FTIR. RESULTS: US accelerated the curing process in both cements, US needed to be applied for more than 15 s. Both Fuji IX and Ketac Molar showed increased setting on increasing the US application duration from 15 s to 55 s. Increased setting of the GICs was produced when US application started 40 s after mixing rather than 30 s after mixing. CONCLUSIONS: The significant findings of the study include that US application accelerated the setting processes, by accelerating the formation of the acid salts. The salt formation increased with increase time of US application. The effect of application of US to setting GICs is influenced by time of the start of application of the US. The effects appear to material specific, with Ketac Molar showing a greater effect than Fuji IX.

Nucleation and growth of apatite on NaOH-treated PEEK, HDPE and UHMWPE for artificial cornea materials.

The skirt of an artificial cornea must integrate the implant to the host sclera, a major failure of present devices. Thus, it is highly desirable to encourage the metabolic activity of the cornea by using more bioactive, flexible skirt materials. Here we describe attempts to increase the bioactivity of polyether ether ketone (PEEK), high-density polyethylene (HDPE) and ultra-high molecular weight polyethylene (UHMWPE) films. The effectiveness of different strength NaOH pre-treatments to initiate apatite deposition on PEEK, HDPE and UHMWPE is investigated. We find that exposure of PEEK, HDPE and UHMWPE films to NaOH solutions induces the formation of potential nuclei for apatite (calcium phosphate), from which the growth of an apatite coating is stimulated when subsequently immersing the polymer films in 1.5 strength Simulated Body Fluid (SBF). As immersion time in SBF increases, further nucleation and growth produces a thicker and more compact apatite coating that can be expected to be highly bioactive. Interestingly, the apatite growth is found to also be dependent on both the concentration of NaOH solution and the structure of the polymer surface.

Characterization and dynamic mechanical analysis of selective laser sintered hydroxyapatite-filled polymeric composites.

Selective laser sintering (SLS) is a manufacturing technique which enables the final product to be made directly and rapidly, without tooling or additional machining. For biomedical applications, SLS permits the fabrication of implants and scaffolds with complex geometry accurately and economically. In this study, hydroxyapatite-reinforced polyethylene and polyamide composites were fabricated using SLS. The SLS samples were characterized in terms of their internal structure, morphology, and porosity. The mechanical properties were examined by dynamic mechanical analysis. The effects of SLS processing conditions, including particle size and laser power, were investigated, and the results were compared with conventional compression-molded and machined specimens. The internal structure of sintered samples was porous, with open interconnected pores, and the pore size was up to 200 microm. Particle size and laser energy play a key role in the final density and mechanical properties of the sintered components. In the parameter range used, the use of smaller particles produced higher density and stiffness, and the laser-induced energy could also be varied to optimize the manufacturing process. This study demonstrated that high-HA-content reinforced polymer composite can be successfully manufactured by SLS with controlled porosity features.

Effect of filler surface morphology on the impact behaviour of hydroxyapatite reinforced high density polyethylene composites.

Instrumented falling weight impact tests have been carried out to characterize the impact behaviour of hydroxyapatite reinforced high-density polyethylene composite (HA-HDPE) in order to use this biomaterial in skull implants. The effects of HA filler surface morphology and volume fraction on the fracture toughness were studied, and fracture mechanism investigated. Impact resistance was found to be markedly improved by using a sintered grade HA filler with smooth particle surface instead of spray dried grade HA with rough surface. SEM examination of impacted fracture surfaces revealed that the improvement of impact resistance was due to the stronger interfacial bonding between smooth HA particles and HDPE polymer matrix compared with that between rough HA and HDPE, which results in more energy absorption during impact and hence better fracture resistance.

Fabrication of porous bioactive structures using the selective laser sintering technique.

Hydroxyapatite, a ceramic with which natural bone inherently bonds, has been incorporated into a polymer matrix to enhance the bioactivity of implant materials. In order to manufacture custom-made bioactive implants rapidly, selective laser sintering has been investigated to fabricate hydroxyapatite and polyamide composites and their properties investigated. One objective of this research was to identify the maximum hydroxyapatite content that could be incorporated into the matrix, which was sintered at various parameters. The study focused on investigating the control of porosity and pore size of the matrix by manipulating the selective laser sintering parameters of the laser power and laser scan speed. The interception method was used to analyse the internal porous morphology of the matrices which were cross-sectioned through the vertical plane. Most notably, all structures built demonstrated interconnection and penetration throughout the matrix. Liquid displacement was also used to analyse the porosity of the matrices. The laser power showed a negative relationship between porosity and variation in parameter values until a critical power value was reached. However, the same relationship for laser scan speed matrices was inconsistent. The effects of the laser power and laser scanning speed on the features of porous structures that could influence cell spreading, proliferation, and bone regeneration are presented.

Effect of iodixanol particle size on the mechanical properties of a PMMA based bone cement.

Iodixanol (IDX) is a water soluble opacifier widely used in radiographical examinations of blood vessels and neural tissue, and it has been suggested as a potential contrast media in acrylic bone cement. The effect of the iodixanol particle size on the polymerisation process of the bone cement, the molecular weight, and the quasi-static mechanical properties have been investigated in this article. The investigation was performed using radiolucent Palacos powder mixed with 8 wt% of iodixanol with particle sizes ranging from 3 to 20 microm MMD, compared with commercial Palacos R (15 wt% ZrO2) as control. Tensile, compressive and flexural tests showed that smaller particles (groups with 3, 4, and 5 microm particles) resulted in significantly lower mechanical properties than the larger particles (groups with 15, 16, and 20 microm particles). There was no difference in molecular weight between the groups. The thermographical investigation showed that the IDX cements exhibit substantially lower maximum temperatures than Palacos R, with the 4 microm IDX group having the lowest maximum temperature. The isothermal and the constant rate differential scanning calorimetry (DSC) did not show any difference in polymerisation heat (DeltaH) or glass transition temperature (Tg) between radiolucent cement, or cement containing either IDX, or ZrO2. The findings show that the particle size for a bone cement containing iodixanol should be above 8 microm MMD.

In vitro osteoblastic response to 30 vol% hydroxyapatite-polyethylene composite.

Hydroxyapatite-reinforced high-density polyethylene (HA-HDPE) composite, as a bone replacement material, has successfully been used clinically as middle ear prostheses and orbital floor implants. The aim of this study was to examine its in vitro biocompatibility in order to develop a further application, that is, as skull reconstruction implants. Human osteoblast cells isolated from femoral heads and crania were used to determine the biological response of the composites. HA-HDPE composites (30 vol %) with two grades of HA filler that had different surface morphologies were selected for this in vitro assessment. The results showed that HA-HDPE composite was bioactive and supported osteoblast attachment, proliferation, and differentiation. The composite with rough-surfaced HA filler demonstrated slightly better cellular response than the composite with smooth-surfaced HA filler. Although osteoblastic cells derived from skull showed an overall slower response compared with those from femoral heads, these in vitro results show that HA-HDPE composite potentially could be used as a skull implant.

2-Dimensional MEMS dielectrophoresis device for osteoblast cell stimulation.

A fixed microelectrode device for cell stimulation has been designed and fabricated using micro-electro-mechanical systems (MEMS) technology. Dielectrophoretic forces obtained from non-uniform electric fields were used for manipulating and positioning osteoblasts. The experiments show that the osteoblasts experience positive dielectrophoresis (p-DEP) when suspended in iso-osmotic culture medium and exposed to AC fields at 5 MHz frequency. Negative dielectrophoresis (n-DEP) is obtained at 0.1 MHz. The viability of osteoblasts under dielectrophoresis has been investigated. The viability values for cells exposed to DEP are nearly three times higher than the control values, indicating that dielectrophoresis may have an anabolic effect on osteoblasts.