Investigation of the intrinsic permeability of ice-templated collagen scaffolds as a function of their structural and mechanical properties.

Collagen scaffolds are widely used in a range of tissue engineering applications, both in vitro and in vivo, where their permeability to fluid flow greatly affects their mechanical and biological functionality. This paper reports new insights into the interrelationships between permeability, scaffold structure, fluid pressure and deformation in collagen scaffolds, focussing in particular on the degree of closure and the alignment of the pores. Isotropic and aligned scaffolds of different occlusivity were produced by ice templating, and were characterised in terms of their structure and mechanical properties. Permeability studies were conducted using two experimental set-ups to cover a wide range of applied fluid pressures. The permeability was found to be constant at low pressures for a given scaffold with more open structures and aligned structures being more permeable. The deformation of scaffolds under high pressure led to a decrease in permeability. The aligned structures were more responsive to deformation than their isotropic equivalents with their permeability falling more quickly at low strain. For isotropic samples, a broad (1 - ɛ)2 dependence for permeability was observed with the constant of proportionality varying with collagen fraction as the starting structures became more occluded. Aligned scaffolds did not follow the same behaviour, with the pores apparently closing more quickly in response to early deformation. These results highlight the importance of scaffold structure in determining permeability to interstitial fluid, and provide an understanding of scaffold behaviour within the complex mechanical environment of the body. STATEMENT OF SIGNIFICANCE: Collagen scaffolds are widely used in tissue engineering applications, for instance to contribute with wound healing. Their permeability to fluid flow, such as water and blood, is important to ensure they perform efficiently when inside the body. The present study reports new insights into the relationships between permeability, scaffold structure, fluid pressure and deformation in collagen scaffolds. It presents in particular the experimental setups used to measure these properties and the result of comparisons between collagen scaffolds with different structures: aligned and isotropic (non-aligned). It indicates quantitative differences in terms of permeability, and the effects of compression on such permeability. The results contribute to the development and understanding of collagen scaffolds and their applications.

Collagen: a network for regenerative medicine.

The basic building block of the extra-cellular matrix in native tissue is collagen. As a structural protein, collagen has an inherent biocompatibility making it an ideal material for regenerative medicine. Cellular response, mediated by integrins, is dictated by the structure and chemistry of the collagen fibers. Fiber formation, via fibrillogenesis, can be controlled in vitro by several factors: pH, ionic strength, and collagen structure. After formation, fibers are stabilized via cross-linking. The final bioactivity of collagen scaffolds is a result of both processes. By considering each step of fabrication, scaffolds can be tailored for the specific needs of each tissue, improving their therapeutic potential.

Structural determinants of hydration, mechanics and fluid flow in freeze-dried collagen scaffolds.

UNLABELLED: Freeze-dried scaffolds provide regeneration templates for a wide range of tissues, due to their flexibility in physical and biological properties. Control of structure is crucial for tuning such properties, and therefore scaffold functionality. However, the common approach of modeling these scaffolds as open-cell foams does not fully account for their structural complexity. Here, the validity of the open-cell model is examined across a range of physical characteristics, rigorously linking morphology to hydration and mechanical properties. Collagen scaffolds with systematic changes in relative density were characterized using Scanning Electron Microscopy, X-ray Micro-Computed Tomography and spherical indentation analyzed in a time-dependent poroelastic framework. Morphologically, all scaffolds were mid-way between the open- and closed-cell models, approaching the closed-cell model as relative density increased. Although pore size remained constant, transport pathway diameter decreased. Larger collagen fractions also produced greater volume swelling on hydration, although the change in pore diameter was constant, and relatively small at ∼6%. Mechanically, the dry and hydrated scaffold moduli varied quadratically with relative density, as expected of open-cell materials. However, the increasing pore wall closure was found to determine the time-dependent nature of the hydrated scaffold response, with a decrease in permeability producing increasingly elastic rather than viscoelastic behavior. These results demonstrate that characterizing the deviation from the open-cell model is vital to gain a full understanding of scaffold biophysical properties, and provide a template for structural studies of other freeze-dried biomaterials. STATEMENT OF SIGNIFICANCE: Freeze-dried collagen sponges are three-dimensional microporous scaffolds that have been used for a number of exploratory tissue engineering applications. The characterization of the structure-properties relationships of these scaffolds is necessary to understand their biophysical behavior in vivo. In this work, the relationship between morphology and physical properties in the dry and hydrated states was investigated across a range of solid concentrations in the scaffolds. The quantitative results provided can aid the design of scaffolds with a target trade-off between mechanical properties and structural features important for their biological activity.

Altering crystal growth and annealing in ice-templated scaffolds.

The potential applications of ice-templating porous materials are constantly expanding, especially as scaffolds for tissue engineering. Ice-templating, a process utilizing ice nucleation and growth within an aqueous solution, consists of a cooling stage (before ice nucleation) and a freezing stage (during ice formation). While heat release during cooling can change scaffold isotropy, the freezing stage, where ice crystals grow and anneal, determines the final size of scaffold features. To investigate the path of heat flow within collagen slurries during solidification, a series of ice-templating molds were designed with varying the contact area with the heat sink, in the form of the freeze drier shelf. Contact with the heat sink was found to be critical in determining the efficiency of the release of latent heat within the perspex molds. Isotropic collagen scaffolds were produced with pores which ranged from 90 µm up to 180 µm as the contact area decreased. In addition, low-temperature ice annealing was observed within the structures. After 20 h at -30 °C, conditions which mimic storage prior to lyophilization, scaffold architecture was observed to coarsen significantly. In future, ice-templating molds should consider not only heat conduction during the cooling phase of solidification, but the effects of heat flow during ice growth and annealing.

Tailoring chitosan/collagen scaffolds for tissue engineering: Effect of composition and different crosslinking agents on scaffold properties.

Chitosan/collagen (Chit/Col) blends have demonstrated great potential for use in tissue engineering (TE) applications. However, there exists a lack of detailed study on the influence of important design parameters (i.e, component ratio or crosslinking methods) on the essential properties of the scaffolds (morphology, mechanical stiffness, swelling, degradation and cytotoxicity). This work entailed a systematic study of these essential properties of three Chit/Col compositions, covering a wide range of component ratios and using different crosslinking methods. Our results showed the possibility of tailoring these properties by changing component ratios, since different interactions occurred between Chit/Col: samples with Chit-enriched compositions showed a hydrogen-bonding type complex (HC), whereas a self-crosslinking phenomenon was induced in Col-enriched scaffolds. Additionally, material and biological properties of the resultant matrices were further adjusted and tuned by changing crosslinking conditions. In such way, we obtained a wide range of scaffolds whose properties were tailored to meet specific needs of TE applications.

Control of crosslinking for tailoring collagen-based scaffolds stability and mechanics.

We provide evidence to show that the standard reactant concentrations used in tissue engineering to cross-link collagen-based scaffolds are up to 100 times higher than required for mechanical integrity in service, and stability against degradation in an aqueous environment. We demonstrate this with a detailed and systematic study by comparing scaffolds made from (a) collagen from two different suppliers, (b) gelatin (a partially denatured collagen) and (c) 50% collagen-50% gelatin mixtures. The materials were processed, using lyophilisation, to produce homogeneous, highly porous scaffolds with isotropic architectures and pore diameters ranging from 130 to 260 µm. Scaffolds were cross-linked using a carbodiimide treatment, to establish the effect of the variations in crosslinking conditions (down to very low concentrations) on the morphology, swelling, degradation and mechanical properties of the scaffolds. Carbodiimide concentration of 11.5mg/ml was defined as the standard (100%) and was progressively diluted down to 0.1%. It was found that 10-fold reduction in the carbodiimide content led to the significant increase (almost 4-fold) in the amount of free amine groups (primarily on collagen lysine residues) without compromising mechanics and stability in water of all resultant scaffolds. The importance of this finding is that, by reducing cross-linking, the corresponding cell-reactive carboxylate anions (collagen glutamate or aspartate residues) that are essential for integrin-mediated binding remain intact. Indeed, a 10-fold reduction in carbodiimide crosslinking resulted in near native-like cell attachment to collagen scaffolds. We have demonstrated that controlling the degree of cross-linking, and hence retaining native scaffold chemistry, offers a major step forward in the biological performance of collagen- and gelatin-based tissue engineering scaffolds. STATEMENT OF SIGNIFICANCE: This work developed collagen and gelatine-based scaffolds with structural, material and biological properties suitable for use in myocardial tissue regeneration. The novelty and significance of this research consist in elucidating the effect of the composition, origin of collagen and crosslinking concentration on the scaffold physical and cell-binding characteristics. We demonstrate that the standard carbodiimide concentrations used to crosslink collagenous scaffolds are up to 100 times higher than required for mechanical integrity in service, and stability against dissolution. The importance of this finding is that, by reducing crosslinking, the corresponding cell-reactive carboxylate anions (essential for integrin-mediated binding) remain intact and the native scaffold chemistry is retained. This offers a major step forward in the biological performance of tissue engineered scaffolds.

Ionic solutes impact collagen scaffold bioactivity.

The structure of ice-templated collagen scaffolds is sensitive to many factors. By adding 0.5 wt% of sodium chloride or sucrose to collagen slurries, scaffold structure could be tuned through changes in ice growth kinetics and interactions of the solute and collagen. With ionic solutes (sodium chloride) the entanglements of the collagen molecule decreased, leading to fibrous scaffolds with increased pore size and decreased attachment of chondrocytes. With non-ionic solutes (sucrose) ice growth was slowed, leading to significantly reduced pore size and up-regulated cell attachment. This highlights the large changes in structure and biological function stimulated by solutes in ice-templating systems.

The effects of scaffold architecture and fibrin gel addition on tendon cell phenotype.

Development of tissue engineering scaffolds relies on careful selection of pore architecture and chemistry of the cellular environment. Repair of skeletal soft tissue, such as tendon, is particularly challenging, since these tissues have a relatively poor healing response. When removed from their native environment, tendon cells (tenocytes) lose their characteristic morphology and the expression of phenotypic markers. To stimulate tendon cells to recreate a healthy extracellular matrix, both architectural cues and fibrin gels have been used in the past, however, their relative effects have not been studied systematically. Within this study, a combination of collagen scaffold architecture, axial and isotropic, and fibrin gel addition was assessed, using ovine tendon-derived cells to determine the optimal strategy for controlling the proliferation and protein expression. Scaffold architecture and fibrin gel addition influenced tendon cell behavior independently in vitro. Addition of fibrin gel within a scaffold doubled cell number and increased matrix production for all architectures studied. However, scaffold architecture dictated the type of matrix produced by cells, regardless of fibrin addition. Axial scaffolds, mimicking native tendon, promoted a mature matrix, with increased tenomodulin, a marker for mature tendon cells, and decreased scleraxis, an early transcription factor for connective tissue. This study demonstrated that both architectural cues and fibrin gel addition alter cell behavior and that the combination of these signals could improve clinical performance of current tissue engineering constructs.

The process of EDC-NHS Cross-linking of reconstituted collagen fibres increases collagen fibrillar order and alignment.

We describe the production of collagen fibre bundles through a multi-strand, semi-continuous extrusion process. Cross-linking using an EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), NHS (N-hydroxysuccinimide) combination was considered. Atomic Force Microscopy (AFM) and Raman spectroscopy focused on how cross-linking affected the collagen fibrillar structure. In the cross-linked fibres, a clear fibrillar structure comparable to native collagen was observed which was not observed in the non-cross-linked fibre. The amide III doublet in the Raman spectra provided additional evidence of alignment in the cross-linked fibres. Raman spectroscopy also indicated no residual polyethylene glycol (from the fibre forming buffer) or water in any of the fibres.

Nanoindentation analysis of αtricalcium phosphate-poly(lactide-co-glycolide) nanocomposite degradation.

The internal mechanical property characteristics as functions of position and degradation time of PLGA(50:50)-αTCP nanocomposites of varying ceramic-polymer ratios degraded in an aqueous medium have been assessed using depth-sensing nanoindentation. The addition of nanoparticulate αTCP increases the elastic modulus of undegraded specimens from 3.72 ± 0.12 GPa for pure PLGA(50:50) samples to 7.23 ± 0.16 GPa recorded for undegraded 40 wt.% TCP nanocomposites. Additionally αTCP incorporation decreases the viscoelastic loss tangent from 0.189 ± 0.040 measured for pure undegraded PLGA(50:50) to an average of 0.091 ± 0.006 for undegraded ceramic-polymer composites. No variation in viscosity for the composites with ceramic loading was evidenced. The stiffening effect of αTCP addition closely conforms to the lower Hashin-Shtrikman bounds demonstrating that an evenly dispersed nano-filler is the least amenable ceramic configuration to enhance the mechanical properties of PLGA-αTCP nanocomposites. The mechanical property evolution for all composite types in an aqueous degradation medium is dominated by material hydration which effects reduced material stiffness and increased specimen viscosity generating a core-periphery mechanical property distribution in terms of elastic modulus and viscoelastic phase angle. The mechanical property core-periphery structure correlates strongly with the core-periphery density structure characterized using X-ray microtomography. Hydrated regions exhibit significant reductions in elastic modulus and viscosity increases which are typical of elastomers.

Understanding anisotropy and architecture in ice-templated biopolymer scaffolds.

Biopolymer scaffolds have great therapeutic potential within tissue engineering due to their large interconnected porosity and biocompatibility. Using an ice-templated technique, where collagen is concentrated into a porous network by ice nucleation and growth, scaffolds with anisotropic pore architecture can be created, mimicking natural tissues like cardiac muscle and bone. This paper describes a systematic set of experiments undertaken to understand the effect of local temperatures on architecture in ice-templated biopolymer scaffolds. The scaffolds within this study were at least 10mm in all dimensions, making them applicable to critical sized defects for biomedical applications. It was found that monitoring the local freezing behavior within the slurry was critical to predicting scaffold structure. Aligned porosity was produced only in parts of the slurry volume which were above the equilibrium freezing temperature (0°C) at the time when nucleation first occurs in the sample as a whole. Thus, to create anisotropic scaffolds, local slurry cooling rates must be sufficiently different to ensure that the equilibrium freezing temperature is not reached throughout the slurry at nucleation. This principal was valid over a range of collagen slurries, demonstrating that by monitoring the temperature within slurry during freezing, scaffold anisotropy with ice-templated scaffolds can be predicted.

Novel porous scaffolds of pH responsive chitosan/carrageenan-based polyelectrolyte complexes for tissue engineering.

Polyelectrolyte complexes (PECs) represent promising materials for drug delivery and tissue engineering applications. These substances are obtained in aqueous medium without the need for crosslinking agents. PECs can be produced through the combination of oppositely charged medical grade polymers, which include the stimuli responsive ones. In this work, three-dimensional porous scaffolds were produced through the lyophilization of pH sensitive PECs made of chitosan (CS) and carrageenan (CRG). CS:CRG molar ratios of 1:1 (CSCRG1), 2:1 (CSCRG2), and 3:1 (CSCRG3) were used. The chemical compositions of the PECs, as well as their influence in the final structure of the scaffolds were meticulously studied. In addition, the pH responsiveness of the PECs in a range including the physiological pH values of 7.4 (simulating normal physiological conditions) and 4.5 (simulating inflammatory response) was assessed. Results showed that the PECs produced were stable at pH values of 7.4 and under but dissolved as the pH increased to nonphysiological values of 9 and 11. However, after dissolution, the PEC could be reprecipitated by decreasing the pH to values close to 4.5. The scaffolds obtained presented large and interconnected pores, being equally sensitive to changes in the pH. CSCRG1 scaffolds appeared to have higher hydrophilicity and therefore higher water absorption capacity. The increase in the CS:CRG molar ratios improved the scaffold mechanical properties, with CSCRG3 presenting the higher compressive modulus under wet conditions. Overall, the PEC scaffolds appear promising for tissue engineering related applications that require the use of pH responsive materials stable at physiological conditions.

A design protocol for tailoring ice-templated scaffold structure.

In this paper, we show, for the first time, the key link between scaffold architecture and latent heat evolution during the production of porous biomedical collagen structures using freeze-drying. Collagen scaffolds are used widely in the biomedical industry for the repair and reconstruction of skeletal tissues and organs. Freeze-drying of collagen slurries is a standard industrial process, and, until now, the literature has sought to characterize the influence of set processing parameters including the freezing protocol and weight percentage of collagen. However, we are able to demonstrate, by monitoring the local thermal events within the slurry during solidification, that nucleation, growth and annealing processes can be controlled, and therefore we are able to control the resulting scaffold architecture. Based on our correlation of thermal profile measurements with scaffold architecture, we hypothesize that there is a link between the fundamental freezing of ice and the structure of scaffolds, which suggests that this concept is applicable not only for collagen but also for ceramics and pharmaceuticals. We present a design protocol of strategies for tailoring the ice-templated scaffold structure.

Effect of fiber crosslinking on collagen-fiber reinforced collagen-chondroitin-6-sulfate materials for regenerating load-bearing soft tissues.

Porous collagen-glycosaminoglycan structures are bioactive and exhibit a pore architecture favorable for both cellular infiltration and attachment; however, their inferior mechanical properties limit use, particularly in load-bearing situations. Reinforcement with collagen fibers may be a feasible route for enhancing the mechanical characteristics of these materials, providing potential for composites used for the repair and regeneration of soft tissue such as tendon, ligaments, and cartilage. Therefore, this study investigates the reinforcement of collagen-chondroitin-6-sulfate (C6S) porous structures with bundles of extruded, reconstituted type I collagen fibers. Fiber bundles were produced through extrusion and then, where applicable, crosslinked using a solution of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide. Fibers were then submerged in the collagen-C6S matrix slurry before being lyophilized. A second 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide crosslinking process was then applied to the composite material before a secondary lyophilization cycle. Where bundles had been previously crosslinked, composites withstood a load of approximately 60 N before failure, the reinforcing fibers remained dense and a favorable matrix pore structure resulted, with good interaction between fiber and matrix. Fibers that had not been crosslinked before lyophilization showed significant internal porosity and a channel existed between them and the matrix. Mechanical properties were significantly reduced, but the additional porosity could prove favorable for cell migration and has potential for directing aligned tissue growth.

Synthetic collagen fascicles for the regeneration of tendon tissue.

The structure of an ideal scaffold for tendon regeneration must be designed to provide a mechanical, structural and chemotactic microenvironment for native cellular activity to synthesize functional (i.e. load bearing) tissue. Collagen fibre scaffolds for this application have shown some promise to date, although the microstructural control required to mimic the native tendon environment has yet to be achieved allowing for minimal control of critical in vivo properties such as degradation rate and mass transport. In this report we describe the fabrication of a novel multi-fibre collagen fascicle structure, based on type-I collagen with failure stress of 25-49 MPa, approximating the strength and structure of native tendon tissue. We demonstrate a microscopic fabrication process based on the automated assembly of type-I collagen fibres with the ability to produce a controllable fascicle-like, structural motif allowing variable numbers of fibres per fascicle. We have confirmed that the resulting post-fabrication type-I collagen structure retains the essential phase behaviour, alignment and spectral characteristics of aligned native type-I collagen. We have also shown that both ovine tendon fibroblasts and human white blood cells in whole blood readily infiltrate the matrix on a macroscopic scale and that these cells adhere to the fibre surface after seven days in culture. The study has indicated that the synthetic collagen fascicle system may be a suitable biomaterial scaffold to provide a rationally designed implantable matrix material to mediate tendon repair and regeneration.

Biomimetic collagen scaffolds with anisotropic pore architecture.

Sponge-like matrices with a specific three-dimensional structural design resembling the actual extracellular matrix of a particular tissue show significant potential for the regeneration and repair of a broad range of damaged anisotropic tissues. The manipulation of the structure of collagen scaffolds using a freeze-drying technique was explored in this work as an intrinsically biocompatible way of tailoring the inner architecture of the scaffold. The research focused on the influence of temperature gradients, imposed during the phase of crystallisation of collagen suspensions, upon the degree of anisotropy in the microstructures of the scaffolds produced. Moulding technology was employed to achieve differences in heat transfer rates during the freezing processes. For this purpose various moulds with different configurations were developed with a view to producing uniaxial and multi-directional temperature gradients across the sample during this process. Scanning electron microscopy analysis of different cross-sections (longitudinal and horizontal) of scaffolds revealed that highly aligned matrices with axially directed pore architectures were obtained where single unidirectional temperature gradients were induced. Altering the freezing conditions by the introduction of multiple temperature gradients allowed collagen scaffolds to be produced with complex pore orientations, and anisotropy in pore size and alignment.

Osteoinduction by combining bone morphogenetic protein (BMP)-2 with a bioactive novel nanocomposite.

OBJECTIVES: There is increasing application of bone morphogenetic proteins (BMPs) owing to their role in promoting fracture healing and bone fusion. However, an optimal delivery system has yet to be identified. The aims of this study were to synthesise bioactive BMP-2, combine it with a novel α-tricalcium phosphate/poly(D,L-lactide-co-glycolide) (α-TCP/PLGA) nanocomposite and study its release from the composite. METHODS: BMP-2 was synthesised using an Escherichia coli expression system and purified. In vitro bioactivity was confirmed using C2C12 cells and an alkaline phosphatase assay. The modified solution-evaporation method was used to fabricate α-TCP/PLGA nanocomposite and this was characterised using X-ray diffraction and scanning electron microscopy. Functionalisation of α-TCP/PLGA nanocomposite by adsorption of BMP-2 was performed and release of BMP-2 was characterised using an enzyme-linked immunosorbent assay (ELISA). RESULTS: Alkaline phosphatase activity of C2C12 cells was increased by the presence of all BMP-2/nanocomposite discs compared with the presence of a blank disc (p = 0.0022), and increased with increasing incubation concentrations of BMP-2, showing successful adsorption and bioactivity of BMP-2. A burst release profile was observed for BMP-2 from the nanocomposite. CONCLUSIONS: Functionalisation of α-TCP/PLGA with BMP-2 produced osteoinduction and was dose-dependent. This material therefore has potential application as an osteoinductive agent in regenerative medicine.

Regeneration and repair of tendon and ligament tissue using collagen fibre biomaterials.

Collagen fibres are ubiquitous macromolecular assemblies in nature, providing the structures that support tensile mechanical loads within the human body. Aligned type I collagen fibres are the primary structural motif for tendon and ligament, and therefore biomaterials based on these structures are considered promising candidates for mediating regeneration of these tissues. However, despite considerable investigation, there remains no collagen-fibre-based biomaterial that has undergone clinical evaluation for this application. Recent research in this area has significantly enhanced our understanding of these complex and challenging biomaterials, and is reinvigorating interest in the development of such structures to recapitulate mechanical function. In this review we describe the progress to date towards a ligament or tendon regeneration template based on collagen fibre scaffolds. We highlight reports of particular relevance to the development of the underlying biomaterials science in this area. In addition, the potential for tailoring and manipulating the interactions between collagen fibres and biological systems, as hybrid biomaterial-biological ensembles, is discussed in the context of developing novel tissue engineering strategies for tendon and ligament.

Collagen-hyaluronic acid scaffolds for adipose tissue engineering.

Three-dimensional (3-D) in vitro models of the mammary gland require a scaffold matrix that supports the development of adipose stroma within a robust freely permeable matrix. 3-D porous collagen-hyaluronic acid (HA: 7.5% and 15%) scaffolds were produced by controlled freeze-drying technique and crosslinking with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride. All scaffolds displayed uniform, interconnected pore structure (total porosity approximately 85%). Physical and chemical analysis showed no signs of collagen denaturation during the formation process. The values of thermal characteristics indicated that crosslinking occurred and that its efficiency was enhanced by the presence of HA. Although the crosslinking reduced the swelling of the strut material in water, the collagen-HA matrix as a whole tended to swell more and show higher dissolution resistance than pure collagen samples. The compressive modulus and elastic collapse stress were higher for collagen-HA composites. All the scaffolds were shown to support the proliferation and differentiation 3T3-L1 preadipocytes while collagen-HA samples maintained a significantly increased proportion of cycling cells (Ki-67+). Furthermore, collagen-HA composites displayed significantly raised Adipsin gene expression with adipogenic culture supplementation for 8 days vs. control conditions. These results indicate that collagen-HA scaffolds may offer robust, freely permeable 3-D matrices that enhance mammary stromal tissue development in vitro.

Entrainment of lactose inhalation powders: a study using laser diffraction.

We have investigated the mechanism of entrainment of lactose inhalation blends released from a dry powder inhaler using a diffraction particle size analyser (Malvern Spraytec). Whether a powder blend entrains as a constant stream of powder (the "erosion" mechanism) or as a few coarse plugs (the "fracture" mechanism) was found by comparing transmission data with particle size information. This technique was then applied to a lactose grade with 0, 5 and 10wt% added fine particles. As the wt% fines increased, the entrainment mechanism was found to change from a mild fracture, consisting of multiple small plugs, to more severe fracture with fewer plugs. The most severe fracture mechanism consisted of either the powder reservoir emptying as a single plug, or of the reservoir emptying after a delay of the order of 0.1s due to the powder sticking to its surroundings. Further to this, three different inhalation grades were compared, and the severity of the fracture was found to be inversely proportional to the flowability of the powder (measured using an annular ring shear tester). By considering the volume of aerosolised fine particles in different blends it was determined that the greater the volume of fines added to a powder, the smaller the fraction of fines that were aerosolised. This was attributed to different behaviour when fines disperse from carrier particles compared with when they disperse from agglomerates of fines. In summary, this paper demonstrates how laser diffraction can provide a more detailed analysis of an inhalation powder than just its size distribution.