Development of Tri-Layered Biomimetic Atelocollagen Scaffolds with Interfaces for Osteochondral Tissue Engineering.

The development of biomimetic scaffolds containing cartilage, calcified cartilage, and bone regeneration for precise osteochondral repair remains a challenge. Herein, a novel tri-layered scaffold-with a top layer containing type II atelocollagen and chondroitin sulphate for cartilage regeneration, an intermediate layer with type II atelocollagen and hydroxyapatite for calcified cartilage formation, and a bottom layer with type I atelocollagen and hydroxyapatite for bone growth-that can be built using liquid-phase cosynthesis, is described. The tri-layered scaffolds are mechanically demonstrably superior and have a lower risk of delamination than monolayer scaffolds. This is due to higher cohesion arising from the interfaces between each layer. In vitro results show that although monolayer scaffolds can stimulate bone marrow stem cells to differentiate and form cartilage, calcified cartilage, and bone separately (detected using quantitative polymerase chain reaction analysis and staining with safranin-O and Alizarin Red S), the tri-layered scaffolds support the regeneration of cartilage, calcified cartilage, and bone simultaneously after 2 and 4 months of implantation (detected using gross and micro-computed tomography images, histological staining, and Avizo, a software used to detect microlevel defects in metals). This work presents data on a promising approach in devising strategies for the precise repair of osteochondral defects.

Effect of tear size, corticosteroids and subacromial decompression surgery on the hierarchical structural properties of torn supraspinatus tendons.

OBJECTIVES: The effects of disease progression and common tendinopathy treatments on the tissue characteristics of human rotator cuff tendons have not previously been evaluated in detail owing to a lack of suitable sampling techniques. This study evaluated the structural characteristics of torn human supraspinatus tendons across the full disease spectrum, and the short-term effects of subacromial corticosteroid injections (SCIs) and subacromial decompression (SAD) surgery on these structural characteristics. METHODS: Samples were collected inter-operatively from supraspinatus tendons containing small, medium, large and massive full thickness tears (n = 33). Using a novel minimally invasive biopsy technique, paired samples were also collected from supraspinatus tendons containing partial thickness tears either before and seven weeks after subacromial SCI (n = 11), or before and seven weeks after SAD surgery (n = 14). Macroscopically normal subscapularis tendons of older patients (n = 5, mean age = 74.6 years) and supraspinatus tendons of younger patients (n = 16, mean age = 23.3) served as controls. Ultra- and micro-structural characteristics were assessed using atomic force microscopy and polarised light microscopy respectively. RESULTS: Significant structural differences existed between torn and control groups. Differences were identifiable early in the disease spectrum, and increased with increasing tear size. Neither SCI nor SAD surgery altered the structural properties of partially torn tendons seven weeks after treatment. CONCLUSIONS: These findings may suggest the need for early clinical intervention strategies for torn rotator cuff tendons in order to prevent further degeneration of the tissue as tear size increases. Further work is required to establish the long-term abilities of SCI and SAD to prevent, and even reverse, such degeneration. Cite this article: Bone Joint Res 2014;3:252-61.

Characterisation of freeze-dried type II collagen and chondroitin sulfate scaffolds.

Collagen type-II is the dominant type of collagen in articular cartilage and chondroitin sulfate is one of the main components of cartilage extracellular matrix. Afibrillar and fibrillar type-II atelocollagen scaffolds with and without chondroitin sulfate were prepared using casting and freeze-drying methods. The scaffolds were characterised to highlight the effects of fibrillogenesis and chondroitin sulfate addition on viscosity, pore structure, porosity and mechanical properties. Microstructure analysis showed that fibrillogenesis increased the circularity of pores significantly in collagen-only scaffolds, whereas with it, no significant change was observed in chondroitin sulfate-containing scaffolds. Addition of chondroitin sulfate to afibrillar scaffolds increased the circularity of the pores and the proportion of pores between 50 and 300 µm suitable for chondrocytes growth. Fourier transform infrared spectroscopy explained the bonding between chondroitin sulfate and afibrillar collagen- confirmed with rheology results- which increased the compressive modulus 10-fold to 0.28 kPa. No bonding was observed in other scaffolds and consequently no significant changes in compressive modulus were detected.

Tenocyte proliferation on collagen scaffolds protects against degradation and improves scaffold properties.

Tissue engineering scaffolds encourage cell proliferation whilst degrading to facilitate tissue regeneration. Their mechanical properties therefore change, decreasing due to scaffold degradation and increasing due to extracellular matrix deposition. This work compares the changing properties of collagen scaffolds incubated in culture medium, with and without human tenocytes, in order to investigate the relationship between degradation and tenocyte proliferation. The material properties of scaffolds are compared over 26 days using mechanical testing, differential scanning calorimetry, infra-red spectroscopy, and histology and biochemical assays. For medium-only scaffolds, the mechanical properties decrease rapidly, while culture medium sulfhydryl content increases significantly, with no significant changes in the denaturation temperature of scaffold collagen content. Conversely, the mechanical properties and collagen content of tenocyte-seeded scaffolds increase significantly while culture medium sulfhydryl content decreases and denaturation temperature remains the same. These results indicate that tenocytes proliferation both reduces the degradation of collagen scaffolds incubated in culture medium and produces scaffolds with improved properties.

Atomic Force Microscopy of bulk tendon samples: affect of location and fixation on tissue ultrastructure.

Atomic Force Microscopy (AFM) is a surface characterisation technique which analyses topology. To date, AFM studies of tissue ultrastructure have focussed on single collagen fibrils extracted from different tissues prior to analysis. Using sample preparation techniques used in electron microscopy studies, this work uses AFM to analyse the collagen ultrastructure of bulk samples from bovine deep digital flexor tendons (DDFTs). DDFT ultrastructure in regions of the tendon which experience different loading conditions are compared. Samples are analysed post-freezing and post-aldehyde fixation with either 10% formalin or 4% glutaraldehyde in order to investigate the affect of tissue preservation on tissue ultrastructure. The results demonstrate that both fibril diameter and repeat unit of the tendon vary between different regions in the dorsoventral plane, with regions subjected to both tensile and compressive forces exhibiting smaller fibril diameter and repeat unit compared to regions subjected to tensile forces alone. These differences are detectable regardless of the tissue preservation technique used. However these measured differences do vary with preservation techniques with aldehyde-fixed samples exhibiting smaller fibril diameters and larger repeat units compared to frozen samples. These results demonstrate that AFM is a highly suitable technique for the characterisation of different ultrastructures in bulk samples but that it is important to be consistent in the choice of preservation technique.

Identification of growth increments in the shell of the bivalve mollusc Arctica islandica using backscattered electron imaging.

Annually resolved growth increments in the shell of the bivalve mollusc Arctica islandica have previously been used in combination with geochemical measurements to successfully construct high-resolution proxy records of past marine environmental conditions. However, to ensure the accuracy of these paleoenvironmental reconstructions it is essential that the annual growth series of increments within the examined shells are reliably identified, and can be distinguished from spurious lines caused by nonannual perturbations such as those resulting from storm disturbance. The current methods used for identifying the growth increment series are sometimes compromised because of ambiguity that results from the employed preparation methods. Here it is shown that backscattered electron imaging of polished shell cross sections may be used to clearly discriminate between the two compositionally and structurally distinct increments that comprise 1 year of outer shell growth. This method, involving minimal specimen preparation, is likely to be primarily useful as a validation technique of particular value in cases where increment identification using existing methods is difficult or ambiguous.

Macrophage-mediated degradation of crosslinked collagen scaffolds.

Biological scaffolds used in tissue engineering are incorporated in vivo by a process of cellular in-growth, followed by host-mediated degradation and replacement of these scaffolds, in which phagocytic cells from the monocyte/macrophage cell lineage play a key role. The chemical degradation of scaffolds with collagenases is well established, but to date this has not been correlated with an in vitro model of cell mediated scaffold degradation. RAW264.7, a murine monocyte/macrophage cell line, was cultured on collagen scaffolds crosslinked either by dehydrothermal treatment (DHT) or by carbodiimide (EDC). These cells attached to collagen scaffolds, proliferated and exhibited macrophage aggregation to form giant cells. Crosslinking the scaffolds by either DHT or EDC increased the resistance of the scaffold to degradation by macrophages. Increasing the amount of crosslinking in the scaffold made them more resistant to degradation by collagenase. However, while EDC increased the scaffolds' thermal and mechanical properties and decreased the swelling ratio, DHT increased the mechanical properties, but decreased the denaturation temperature and swelling ratio. Altering the scaffold properties by crosslinking affects the rate of degradation by macrophages, and this is correlated with chemical degradation (r=0.658, p<0.01). This will help in the design of scaffolds with task-specific profiles for use in tissue engineering.

Synthesis and properties of a novel anisotropic self-inflating hydrogel tissue expander.

The advent of self-inflating hydrogel tissue expanders heralded a significant advance in the reconstructive techniques available for the surgical restoration of a wide variety of soft tissue defects. However, their use in specific applications such as cleft palate surgery is limited on account of their isotropic expansion. An anisotropic self-inflating hydrogel tissue expander has been developed which markedly increases the potential indications for which this restorative tool may be employed. These include complex pediatric soft tissue reconstructions of the palate, nose, ear and digits. Anisotropic expansion in a hydrogel polymer network composed of methyl methacrylate and vinylpyrrolidone has been achieved by annealing the xerogel under a compressive load for a specified time period. By controlling the anisotropic processing conditions and composition we have been able to accurately tailor the ultimate expansion ratio up to 1500%. The expansion rate of the xerogel has also been significantly reduced by encapsulating the polymer within a semi-permeable silicone membrane. The structure and properties of the novel anisotropic hydrogel were characterized by attenuated total reflectance infrared spectroscopy, differential scanning calorimetry, thermogravimetric analysis and small-angle neutron scattering.

Gradient collagen/nanohydroxyapatite composite scaffold: development and characterization.

This paper reports an in situ diffusion method for the fabrication of compositionally graded collagen/nanohydroxyapatite (HA) composite scaffold. The method is diffusion based and causes the precipitation of nano-HA crystallites in situ. A collagen matrix acts as a template through which calcium ions (Ca(2+)) and phosphate ions (PO4(3-)) diffuse and precipitate a non-stoichiometric HA. It was observed that needle-like prismatic nano-HA crystallites (about 2 x 2 x 20 nm) precipitated in the interior of the collagen template onto the collagen fibrils. Chemical and microstructural analysis revealed a gradient of the Ca to P ratio across the width of the scaffold template, resulting in the formation of a Ca-rich side and a Ca-depleted side of scaffold. The Ca-rich side featured low porosity and agglomerates of the nano-HA crystallites, while the Ca-depleted side featured higher porosity and nano-HA crystallites integrated with collagen fibrils to form a porous network structure.

On the process capability of the solid free-form fabrication: a case study of scaffold moulds for tissue engineering.

This study applies the methodology and procedure of process capability to investigate a solid free-form fabrication technique as a manufacturing method to produce scaffold moulds for tissue engineering. The process capability Cpk and process performance Ppk of scaffold mould manufacture using a solid free-form fabrication technique has been analysed with respect to the dimension deviations. A solid free-form fabrication machine T66 was used to fabricate scaffold moulds in this study and is able to create features that ranged from 200 microm to 1000 microm. The analysis showed that the printing process under the normal cooling conditions of the printing chamber was in statistical control but gave low process capability indices, indicating that the process was 'inadequate' for production of 'dimension-consistent' scaffold moulds. The study demonstrates that, by lowering the temperature of the cooling conditions, the capability Cpk of the printing process can be improved (about threefold) sufficiently to ensure the consistent production of scaffold moulds with dimension characteristics within their specification limits.

Novel 3D collagen scaffolds fabricated by indirect printing technique for tissue engineering.

This article reports the mechanical properties and in vitro evaluation of a collagen scaffold fabricated using an indirect 3D printing technique. Collagen scaffolds, featuring predefined internal channels and capillary networks, were manufactured using phase change printing. It was observed that the collagen scaffolds featured internal channels and a hierarchical structure that varied over length scales of 10-400 microm. In vitro evaluation using hMSCs demonstrated that the resultant collagen based scaffolds have the ability to support hMSC cell attachment and proliferation; cells can migrate and survive deep within the structure of the scaffold. The cell numbers increased 2.4 times over 28 days in culture for the lysine treated scaffolds. The cells were spread along the collagen fibers to form a 3D structure and extracellular matrix was detected on the surface of the scaffolds after 4 weeks in culture. The crosslinking treatment enhanced the biostability and dynamic properties of the collagen scaffolds significantly.

Micro- and nano-injectable composite biomaterials containing calcium phosphate coated with poly(DL-lactide-co-glycolide).

Calcium phosphate/poly(dl-lactide-co-glycolide) (CP/DLPLG) composite biomaterial, in which each CP particle was coated with DLPLG, was synthesized. Two kinds of composites were prepared: microcomposite, with particles 150-200mum in size, and nanocomposite, with the particles 40+/-5nm in size. Using nanoparticles, a new class of injectible composite biomaterials was produced. Based on scanning electron microscopy, atomic force microscopy, differential thermal analysis, thermogravimetric analysis, differential scanning calorimetry and Fourier transform infrared analyses, the structure and phase organization in both biomaterials was identified and in both studied cases CP particles were coated with DLPLG polymer. An injectable composite biomaterial, the characteristics of which depend on the ratio of the phases, was prepared by mixing physiological solution with the nano-CP/DLPLG composite. Rheological studies indicated a possible agglomeration of particles of the injectable nano-CP/DLPLG composite biomaterial with a CP content of 65%.

Collagen-hydroxyapatite composites for hard tissue repair.

Bone is the most implanted tissue after blood. The major solid components of human bone are collagen (a natural polymer, also found in skin and tendons) and a substituted hydroxyapatite (a natural ceramic, also found in teeth). Although these two components when used separately provide a relatively successful mean of augmenting bone growth, the composite of the two natural materials exceeds this success. This paper provides a review of the most common routes to the fabrication of collagen (Col) and hydroxyapatite (HA) composites for bone analogues. The regeneration of diseased or fractured bones is the challenge faced by current technologies in tissue engineering. Hydroxyapatite and collagen composites (Col-HA) have the potential in mimicking and replacing skeletal bones. Both in vivo and in vitro studies show the importance of collagen type, mineralisation conditions, porosity, manufacturing conditions and crosslinking. The results outlined on mechanical properties, cell culturing and de-novo bone growth of these devices relate to the efficiency of these to be used as future bone implants. Solid free form fabrication where a mould can be built up layer by layer, providing shape and internal vascularisation may provide an improved method of creating composite structures.

Making tissue engineering scaffolds work. Review: the application of solid freeform fabrication technology to the production of tissue engineering scaffolds.

Tissue engineering is a new and exciting technique which has the potential to create tissues and organs de novo. It involves the in vitro seeding and attachment of human cells onto a scaffold. These cells then proliferate, migrate and differentiate into the specific tissue while secreting the extracellular matrix components required to create the tissue. It is evident, therefore, that the choice of scaffold is crucial to enable the cells to behave in the required manner to produce tissues and organs of the desired shape and size. Current scaffolds, made by conventional scaffold fabrication techniques, are generally foams of synthetic polymers. The cells do not necessarily recognise such surfaces, and most importantly cells cannot migrate more than 500 microm from the surface. The lack of oxygen and nutrient supply governs this depth. Solid freeform fabrication (SFF) uses layer-manufacturing strategies to create physical objects directly from computer-generated models. It can improve current scaffold design by controlling scaffold parameters such as pore size, porosity and pore distribution, as well as incorporating an artificial vascular system, thereby increasing the mass transport of oxygen and nutrients into the interior of the scaffold and supporting cellular growth in that region. Several SFF systems have produced tissue engineering scaffolds with this concept in mind which will be the main focus of this review. We are developing scaffolds from collagen and with an internal vascular architecture using SFF. Collagen has major advantages as it provides a favourable surface for cellular attachment. The vascular system allows for the supply of nutrients and oxygen throughout the scaffold. The future of tissue engineering scaffolds is intertwined with SFF technologies.

Novel collagen scaffolds with predefined internal morphology made by solid freeform fabrication.

Novel collagen scaffolds possessing predefined and reproducible internal channels with widths of 135 microm and greater have been produced. The process employed to make the collagen scaffold utilises a sacrificial mould, manufactured using solid freeform fabrication technology, and critical point drying technique. A computer aided design (CAD) file of the mould to be produced is created. This mould is manufactured using a phase change ink-jet printer. A dispersion of collagen is then cast into the mould and frozen. The mould is dissolved away with ethanol and the collagen scaffold is then critical point dried with liquid carbon dioxide. The effect of processing on the tertiary structure of collagen is assessed by monitoring the wavenumber of the N-H stretching vibration peak using Fourier transform infra-red spectroscopy and it is found that processing does not denature the collagen. Ultraviolet-visual spectroscopy was used to detect the presence of any contamination from the sacrificial mould on the collagen. The ability to use computer aided design and manufacture (CAD/CAM) provides a route to optimise scaffold designs using collagen in tissue engineering applications.

A simple method for orienting silk and other flexible fibres in transmission electron microscopy specimens.

When microstructures are characterized by transmission electron microscopy (TEM), the interpretation of results is facilitated if the material can be sectioned in defined orientations. In the case of fibres, it is especially useful if transverse and longitudinal sections can be obtained reliably. Here we describe a procedure for orienting spider silk and other flexible fibres for TEM investigation. Prior to embedding in epoxy resin, the silk is wound around a notched support made from polyester film. No glue is required. After the silk and its supporting film have been embedded and the resin has been cured the film can be peeled away to reveal nearly perfectly orientated silk threads. Both transverse and longitudinal sections can then be cut with a microtome. The method can be extended to obtain sections at any intermediate orientation.

Material removal during the sliding of hydroxyapatite against UHMWPE.

Hydroxyapatite has been rubbed against ultra-high-molecular-weight-polyethylene (UHMWPE) under calcium-containg aqueous solutions. Further, hardness tests were carried out in air and in calcium-containing solutions whose pH ranged from pH 5 to pH 9. Hardness was found to vary with pH with a peak at around pH 7, i.e. - a chemomechanical effect was observed. Wear tests consisted in sliding hydroxyapatite samples against a UHMWPE disk for eight hours when lubricated by the same solutions as those used for the hardness tests. Volume loss, pH and calcium concentration were measured for up to 8 hours of sliding. Linking wear tests results with hardness results and supersaturation levels, it was concluded that two wear mechanisms occurred. A chemical mechanism depending on supersaturation occurred at the early stages of sliding. The wear rate was essentially independent of hardness during this stage. After a few hours, depending on the supersaturation of the lubricant, the chemical mechanism turned into a chemomechanical mechanism dependant on hardness.

Compositional and structural control in bone regenerative coatings.

The development of a low-temperature method of producing bioactive coatings for medical implants has been shown to bypass the problems associated with high temperature processing routes, in particular the appearance of amorphous phases and non-stoichiometric hydroxyapatite (HA), and delamination of the coating from the substrate. An electric field/aqueous solution technique for producing adherent, crack-free calcium phosphate coatings on titanium and stainless steel substrates is described. The characteristics of the coating are a function of electrode spacing, supersaturation, temperature and current and voltage conditions. Scanning electron microscopy (SEM) characterized the surface morphology of the coatings, which were shown to be HA. The possibility of producing a coating of carbonate-substituted HA having the same chemical composition as bone apatite, and forming at physiological temperatures, has also been demonstrated. The size of the microstructure decreased and the morphology changed as the carbonate ion concentration in the calcium and phosphate ion solution increased.

Collagen--calcium phosphate composites.

There is a widespread clinical need for bone augmentation and replacement. The major solid phases of bone are collagen and calcium phosphate and a bone analogue based on these two constituents should have some useful properties. In this review this theme is developed and the properties of natural and naturally based composites are compared. Composites have been produced by the precipitation of calcium phosphates on to collagen and a summary of the methods and results from mechanical testing and scanning electron microscopy are presented. Composites with mechanical properties intermediate between cancellous and cortical bone have been produced. The review concludes by explaining some of the mechanical properties of the composites, using knowledge of the hierarchical architecture of bone and results from microscopical examination of the fractured composites.

The effect of two types of cross-linking on some mechanical properties of collagen.

Samples of collagen were cross-linked by two different methods: (a) glutaraldehyde and (b) a combination of dehydrothermal treatment and cyanamide. The elastic modulus, the ultimate tensile strength (fracture stress), strain to failure, work of fracture, and fracture toughness were measured before and after cross-linking in ambient laboratory conditions, and during immersion in water. These tests were all performed over a range of strain rates. For collagen tested in the wet condition, it was found that cross-linking increased the elastic modulus from approximately 25-30 MPa, to between 55 and 60 MPa, but there was little effect on fracture stress, and strain to failure was reduced. The work of fracture of the collagen decreased on cross-linking. Cross-linking had the same effect on the elastic modulus, fracture stress, and strain to failure of dry collagen, but the work of fracture was unaffected. In conclusion, cross-linking increased the elastic modulus, reduced the strain to failure, and had little effect on the fracture stress of collagen under the present experimental conditions.