Biotechnol Lett (2014) 36:403–415 DOI 10.1007/s10529-013-1374-4

REVIEW

Imaging the hard/soft tissue interface Alistair Bannerman • Jennifer Z. Paxton Liam M. Grover



Received: 12 March 2013 / Accepted: 25 September 2013 / Published online: 16 October 2013 Ó Springer Science+Business Media Dordrecht 2013

Abstract Interfaces between different tissues play an essential role in the biomechanics of native tissues and their recapitulation is now recognized as critical to function. As a consequence, imaging the hard/soft tissue interface has become increasingly important in the area of tissue engineering. Particularly as several biotechnology based products have made it onto the market or are close to human trials and an understanding of their function and development is essential. A range of imaging modalities have been developed that allow a wealth of information on the morphological and physical properties of samples to be obtained non-destructively in vivo or via destructive means. This review summarizes the use of a selection of imaging modalities on interfaces to date considering the strengths and weaknesses of each. We will also consider techniques which have not yet been utilized to their full potential or are likely to play a role in future work in the area.

A. Bannerman  J. Z. Paxton  L. M. Grover (&) School of Chemical Engineering, University of Birmingham, Birmingham, UK e-mail: [email protected] A. Bannerman e-mail: [email protected] J. Z. Paxton e-mail: [email protected]

Keywords Biomechanics  Enthesis  Interface  Imaging  Native tissues  Osteochondral junction  Osteotendinous junction

Introduction In a multicomponent system such as the musculoskeletal system, the interface between different parts or materials is fundamental to maintaining function and integrity whilst the system is in motion or under a load. The anatomy and physiology of connective tissues has been well studied for the osteotendious junction (boneligament/tendon), osteochondral junction (bone-cartilage) and cementum periodontal complex (Fig. 1). One of the most frequently injured tissues that comprises a hard/soft tissue interface is the ligament/tendon bone interface, which may be graduated in terms of mineralization and matrix organization. Such graduated interfaces are termed entheses. Recent developments in biotechnology have led to the development of therapies that enable the reconstruction of these graduated interfaces and the characterization of these therapies has required the application of a range of novel imaging methodologies. The enthesis is an interfacial region and may have either two or four distinct zones. If the enthesis has two zones, it is known as an indirect or fibrous enthesis and, if it has four zones, it is known as a direct or fibrocatilagenous enthesis (Benjamin and McGonagle 2009). The fibrous enthesis is typically found in

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tendons and some ligaments, and it is an interface where the soft tissue connects directly to the bone. Fibrous entheseses are also found in the teeth as the periodontal ligament attaches to the cementum and alveolar bone, anchoring the teeth in place. The fibrocartilaginous enthesis has a more gradually graded interface going from bone, through mineralized fibrocartilage, unmineralized fibrocartilage, to the sinew and features a steady increase in collagen alignment and decrease in mineralization (Fig. 1). The biomechanical significance of this is that it acts as a means of smoothly transmitting forces between sinew and bone, reducing the chance of impedance mismatch and injury. The development, anatomy and function of both enthesis types has been well documented (Benjamin et al. 2004, 2006; Benjamin and McGonagle 2009). Ligaments such as the commonly damaged anterior cruciate ligament (ACL) have poor native healing ability and, in cases where a torn ligament has been surgically reattached by a graft, the

Fig. 1 The constituent zones of the hard/soft tissue interface in the osteotendinous junction, osteochondral junction and cementum periodontal complex

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fibrocartilaginous interface does not form properly leaving it exposed to further damage through sudden loads and thereby presenting a significant chance of reinjury (Spindler and Wright 2008; Deehan and Cawston 2005). The failure of clinical techniques or artificial grafts to regenerate damaged ACL replacements has led to a number of biotechnology-based tissue-engineered solutions being developed (Vunjak-Novakovic et al. 2004; Yang and Temenoff 2009; Yilgor et al. 2012). These replacement options use a large range of materials but are typically based around a hydrogel scaffold for the ligament tissue and calcium phosphate for the hard tissue if an interface is incorporated. Several studies have focused on the engineering of interfacial tissue, grown as either a single (Paxton et al. 2010a, b) or multiphase scaffold (Spalazzi et al. 2006, 2008). Further detail on interfacial tissue engineering can be found in a recent review by Paxton et al. (2012). Despite research into this area and successes shown in improving the interfacial strength of both engineered constructs and in vivo sinew repairs, the accurate reproduction of a graded native interface is proving hard to accomplish. Further to this understanding of the bioengineered products and their condition over time is limited, yet essential to their function. Imaging is a key technique for the use in both in vivo repair sites and interfacial tissue engineering. It allows the visualization of the interface and assists in the assessment of the development, composition and reaction to external factors of hard/soft tissue interfaces. A wide range of imaging methods have been developed and optimized toward biological imaging at the micro-scale and provide information on the morphological and chemical composition, and physical properties both in fixed and in vivo samples. A combination of imaging methods and information types allows the user to link microstructure with biomechanical and functional information. Despite the numerous imaging technologies available for biological samples, the highly contrasting properties of the materials present at the hard/soft interface create significant challenges for the preparation and imaging of samples. Techniques that provide suitable information and analysis on one constituent of the interface may have poor sensitivity or damage the other constituents during the preparation or imaging. Further to this, each imaging modality has its own particular advantages and limitations. Therefore for

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detailed investigations, a selection of two or more complementary techniques is required to provide complete information on the components of the interface. Imaging is aided by using stains and contrast agents to label specific components. The birefringent nature of biological tissues such as collagen, which is often the major component of hard/soft tissue interfaces allows polarized or other angle dependent methods to extract further information. This review will focus on currently applied methods used to image the hard/soft tissue interfaces both in vivo, ex vivo and in tissue engineered solutions. It will provide a brief summary of the literature on the use of optical, chemical, magnetic resonance, ultrasound, and X-ray imaging for the evaluation of hard/ soft tissue interfaces.

Modalities The imaging modalities used and ultimately the quantity and quality of data obtained are often dictated by the needs of the user. Table 1 shows an overview of the abilities and limitations of each modality. Table 2 lists specific applications of interface imaging for each technique. Example images for interfaces of a tissue engineered construct described by Paxton et al. (2010a) are shown in Fig. 2. Optical Optical microscopy using wavelengths in the visible spectrum is well established in the biological sciences. The resolution limit of optical microscopy defined by

Table 1 Basic overview of each imaging modality Optical

Raman

EM

MRI

Ultrasound

X-ray

Soft tissue

4

4

4

4

4

9

Hard tissue

94

4

4

4

4 3

4 3

Resolution range

Microscopy 250 nm OCT lm

Theoretical 250 nm

SEM-nm TEM50 pm

30 lm

25 lm

Micro-CT lm Beamline nm

Preparation

Histology (fixation, dehydration, embedding, sectioning) Brightfield/ confocal (staining) OCT (None)

None

Fixation and dehydration. Conductive coating. (TEM) thin sample required

None

None

None

Used in vivo

94

4

9

4

4

4

Destructive

94

9

4

9

9

9

Advantages

Reveals tissue architecture. Biological components can be stained. Birefringence of collagen

Non-invasive and nondestructive. Identification without chemical labels

High resolution images can be produced. Elemental mapping

Non-invasive and non-destructive. Good depth penetration. 3D reconstructions

Non-invasive and nondestructive. Good depth penetration. Mechanical information

3D reconstructions possible. Noninvasive and non-destructive. Good depth penetration

Disadvantages

Tissue must be fixed prior to processing (histology). Limited depth penetration

Large volumes of data. Limited depth penetration

Fixation and processing of tissue is laborious. Destructive. Identification of specific matrix compositions difficult

Low contrast between interface components. Identification of specific matrix compositions difficult

Identification of specific matrix compositions difficult

Radiation dose may be problematic in in vivo samples. Low attenuation of unstained soft tissues

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Table 2 Imaging applications of each technique in the literature Imaging technique

Interface structure

Form

Imaging mode

References

Optical

Bone-tendon\ligament

Ex vivo

Histological staining, light microscopy

Aydin et al. (2010), Clark and Stechschulte (1998), Milz et al. (2002), Moffat et al. (2008), Robson et al. (2004), Schwartz et al. (2012), Shaw et al. (2008), Spalazzi et al. (2006), Suzuki et al. (2002), Suzuki et al. (2003)

Bone-tendon

Ex vivo

Polarised light microscopy

Clark and Stechschulte (1998), Genin et al. (2009)

OCT

Adams Jr et al. (2003), Rashidifard et al. (2012)

Bone-tendon

Raman

EM

Bone-tendon (repair site)

Ex vivo

Histological staining, light microscopy

Nourissat et al. (2010)

Bone-tendon (repair site)

Ex vivo

Histological staining, light microscopy, polarised light microscopy

Hashimoto et al. (2007)

Bone ligament

In vitro

Fluorescence microscopy

Spalazzi et al. (2006)

Ligament/fibrocartilagebone

Ex vivo

Histological staining, light microscopy

Gao and Messner (1996)

Bone-fibrocartilageligament

In vitro

Histological staining, immunohistochemical staining, light microscopy

Spalazzi et al. (2008)

Bone-cartilage

Ex vivo

Histological staining, light microscopy

Bae et al. (2010), Im et al. (2010)

Bone-cartilage

In vitro

Histological staining

Khanarian et al. (2011)

Cementum-peridontal ligament

Ex vivo

Histological staining, immunohistochemical staining, light microscopy

Herber et al. (2012), Ho et al. (2010)

Cementum-peridontal ligament

Ex vivo

Polarised light microscopy

Lin et al. (2012), Ho et al. (2010)

Bone-tendon

Ex vivo

Raman spectroscopy

Schwartz et al. (2012), Wopenka et al. (2008)

Bone-tendon\ligament

In vitro

Confocal Raman spectroscopy

Paxton et al. (2010a), Genin et al. (2009)

Bone-tendon\ligament

Ex vivo

SEM

Clark and Stechschulte (1998), Oguma et al. (2001), Moffat et al. (2008), Paxton et al. (2010a), Spalazzi et al. (2006)

Bone-tendon

Ex vivo

TEM

Schwartz et al. (2012)

Cementum-peridontal ligament Cementum-peridontal ligament

Ex vivo

SEM, TEM

Lin et al. (2012)

Ex vivo

SEM

Ho et al. (2010), Herber et al. (2012)

In vitro

SEM

Khanarian et al. (2011)

Bone-cartilage

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Table 2 continued Imaging technique

Interface structure

Form

Imaging mode

References

MRI

Bone-tendon

In vivo

Ultrashort TE sequences

Du et al. (2005), Robson et al. (2004)

Bone-tendon

In vivo

T1-weighted spin echo sequences

Shaw et al. (2008)

Bone-tendon

In vivo

T1 weighted gradient echo and STIR sequences

Wiell et al. (2012)

Bone-cartilage

Ex vivo

Proton density weighted, T1 weighted, UTE sequences

Bae et al. (2010)

Ultrasound

X-ray

Bone-tendon

In vivo

Color Doppler

Ducher et al. (2010)

Bone-tendon

In vivo

Multiplaner scanning

Aydin et al. (2010)

Bone-tendon

In vivo

Power Doppler

Wiell et al. (2012)

Cementum-peridontal ligament

Ex vivo

Micro-CT

Lin et al. (2012), Herber et al. (2012), Ho et al. (2010)

Bone-tendon

Ex vivo

X-ray

Moriggl et al. (2003)

Dual energy CT

Johnson et al. (2007), Deng et al. (2009), Sun et al. (2008)

Bone-tendon

Others

Bone-cartilage

Ex vivo

Phase-contrast X-ray

Ismail et al. (2010)

Bone-cartilage Cementum-peridontal ligament

Ex vivo Ex vivo

AFM AFM

Campbell et al. (2012) Ho et al. (2010), Lin et al. (2012)

the fraction limit is dependent on the wavelength used but will be in the order of hundreds of nanometers, allowing imaging down to subcellular levels. Natural contrast in biological tissue is poor, but stains and labels allow an overall increase in the biological sharpness or specific biological components such as collagen or cell nuclei to be isolated. The majority of hard/soft tissue interface imaging in previous studies—particularly in ex vivo native tissue sections— has been performed using optical micrographs, typically of histologically stained specimens (Benjamin et al. 2006; Hems and Tillmann 2000; Herber et al. 2012; Suzuki et al. 2002, 2003). These techniques allow a visualization of the morphological and biological composition of the interface—with good contrast between the hard and soft components. Specific staining of individual biological components can be achieved through several histological (toluidine blue, Masson’s trichrome) or fluorescent stains (DAPI, phalloidin). The histological approach, however, is limited by the need for the destruction and possible distortion of the sample through a preparation process

involving; fixation, a series of alcohol baths and sectioning for soft tissue, and the decalcification of bone to allow sectioning—greatly reducing the practical application in investigating the development of tissue engineered samples. In addition to the information obtained from staining, more novel techniques such as polarization microscopy, which allows for the study of the alignment and directionality of collagen fibers at the insertion of into the bone can provide additional information (Clark and Stechschulte 1998; Longo et al. 2007). Episcopic reproduction of whole, histologically stained samples has been shown as effective means of producing three-dimensional stacks of mixed tissue samples (Weninger et al. 2006; Rosenthal et al. 2004). Other microscopy methods allow imaging of whole samples with the possibility of in vitro imaging and nondestructive three-dimensional reconstructions of tissue engineered samples. Fluorescence microscopy allows in vitro imaging through brightfield and confocal microscopy by using fluorescent stains that can be used on fixed or in vitro samples. Confocal microscopy

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Fig. 2 Imaging modalities applied to the hard/soft tissue interface of tissue engineered constructs as described by Paxton et al. 2010a a Confocal Raman mapping of the interface for the orthophosphate peak—scale bar = 200 lm b Confocal Raman mapping of the dataset from 2(a) for the Rayleigh peak showing soft tissue structure—scale bar = 200 lm c Light micrograph

of the interface—scale bar = 100 lm d Confocal microscopy showing anchor, sinew and DAPI stained cells—scale bar = 50 lm e SEM secondary backscattered electron image along the interface—scale bar = 100 lm f Micro-CT sagittal section of the interface—scale bar = 500 lm. H hard-tissue ceramic anchor, S sinew

produces images at a single focal plane allowing a sharper image and z-stacks to be produced to a depth of a few hundred microns depending on the sample properties (Pawley 2006). But it will provide limited information on hard tissues with only topological information being provided through reflectance imaging. Multispectral imaging, while not yet commonly available, has great potential with the use of multiple wavelengths at each point removing the need for stains and the associated sample preparation techniques, thus making it far less invasive and damaging than other microscopy techniques (Zipfel et al. 2003). An increasingly used technique, optical coherence tomography (OCT), covers the resolution and depth

gap between confocal microscopy and ultrasound for non/minimally invasive and in vivo imaging, able to produce images or videos with resolutions of 1–10 lm to depths of a few millimeters (Podoleanu 2005). Coherent infra-red light is split between the sample and a reference mirror with the interference pattern produced showing the structure via impedance boundaries. OCT has been well studied with respect to musculoskeletal disease (Rashidifard et al. 2013) with specific applications showing the practical success for examining tendon insertions in vivo (Rashidifard et al. 2012) and osteochondral junction (Rogowska and Brezinski 2002). Polarization sensitive OCT is able to detect collagen alignment and study the structure and

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find signs of damage at the interface (Adams Jr et al. 2003; Liu et al. 2012). Finally spectral data can be generated from OCT enabling the characterization of detected tissues and materials and increasing the quantitative information that can be obtained via the technique (Brezinski and Liu 2008). Raman spectroscopic imaging Raman spectroscopy is a chemical-imaging technique based on the anti-stokes Raman affect that can determine the chemical species present in a sample and semi-quantitatively determine their concentration. Imaging can be undertaken using microscopy on ex vivo or tissue engineered samples or by using fiberoptics in vivo. Information is provided by the vibrational properties of chemical bonds inside the sample, providing a spectrum at each data point where distinct peaks can be used to show the presence and quantity of a certain molecular bond. A distinct advantage of Raman spectroscopy is that stains and labels are not needed, reducing the preparation required and making live imaging more practical, particularly for tissue engineered samples. The location and shape of biological and organic peaks have been categorized for spectra (Movasaghi et al. 2007; Larkin 2011). The hyperspectral data generated means large file sizes are produced for even small numbers of data points requiring specialized data handling and storage methods to ensure practical use (Vidal and Amigo 2012). The choice of laser wavelength and exposure time may also cause damage to and disrupt the sample. In standard scattering mode the weak signal generated means that an integration time in the order of seconds may be required at each data point making imaging a slow process. Studies showing the effectiveness of Raman spectroscopy for detecting properties such as mineralization in hard and soft tissue components separately are wide-spread in the literature. These include detecting changes in bone and calcium phosphate-based materials (Maher et al. 2011; Penel et al. 2005) and for imaging and identification of single cells and tissues in soft tissue samples (Krafft et al. 2009; Navratil et al. 2006; Votteler et al. 2011). As a stand-alone imaging technique, the most practical application has been made by using confocal Raman microscopy. The method, however, is limited by low depth penetration due to the high scattering properties of tissue.

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Examples of research groups imaging the interface as a whole have been limited, although Paxton et al. (2010a) demonstrated recrystallization in a sinew by showing high local concentrations of phosphate bonds. Coherent anti-Stokes Raman spectroscopy (CARS) may provide an improvement to current Raman methods as it becomes increasingly available and commonly used. By taking advantage of the coherent Raman energy shift mechanism, the signal produced is far stronger allowing real time images to be produced, and vastly reducing the time required for data collection allowing video rate data to be collected (Day et al. 2011). The background is substantially higher than in scattered based Raman requiring more advanced processing techniques. Electron microscopy Electron microscopy takes advantage of the significantly short de Broglie wavelength of high energy electrons to produce topographic and elemental images with sub-nanometer resolution. For biological components, heavy element based stains can be applied to increase the contrast. Electron microscopy is limited to use on ex vivo and fixed samples due the destructive nature of the preparation and imaging environment. Scanning electron microscopy (SEM) produces high depth of field images via secondary backscattered electrons, or elemental information from X-rays or primary electrons. Environmental SEM can be used instead of conductive coating to minimize distortion to the sample but at the cost of reduced image quality (McGregor and Donald 2010). SEM of the interface for ex vivo and tissue engineering constructs has been widely reported, often as a complementary information source to histological samples; Villegas and Donahue (2010) and Oguma et al. (2001) reported on the use of SEM to study the morphology of collagen at the interface in ex vivo samples. Paxton et al. (2010a) reported use for interface topology and elemental mapping, with many other examples in the literature (Clark and Stechschulte 1998; Rufai et al. 1996). SEM is able to produce three-dimensional reconstructions by using of an ion beam to remove the outer layer of the sample after each image is acquired. Transmission electron microscopy (TEM) can resolve down to 50 pm by focusing electrons

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transmitted through a sample. TEM requires more complex preparation than SEM to enable transmission through up to hundred nanometers in thickness and millimeters in width. TEM use has been shown on ex vivo osteotendon by Schwartz et al. (2012) and cementum-peridontal ligament by Lin et al. (2012). Magnetic resonance imaging (MRI) MRI enables the production of a three-dimensional reconstruction of tissues to show morphology based on the intensity and distribution of water content via the alignment of magnetized nuclei. Contrast in magnetic resonance imaging is provided by the differences in relaxation time of nuclei in different tissues and environments. The sensitivity and resolution of MRI is determined by the strength of the magnetic field, radiofrequency coil, and imaging time (Driehuys et al. 2008). Magnetic resonance microscopy enables nondestructive, in vivo imaging of samples with a voxel size of 100 lm3 being common and down to 10 lm3 achievable (Driehuys et al. 2008). Widespread use of this technology, however, has been limited by a high cost and poor accessibility; this has limited practical use of the technology. Imaging of the enthesis with MRI is commonly performed in the clinical arena at the macro-scale to look for inflammation of the enthesis relating to osteoarthritis (Coates et al. 2012). Due to the short transverse relaxation times between the tissues in the enthesis, however, the minimum time-echo pulse sequence for differentiation between component tissues in the enthesis is beyond the capability of conventional clinical MRIs. In order to enable differentiation, it is necessary to use instruments capable of ultrashort time echo pulses (Gatehouse and Bydder 2003). The ‘magic angle’ effect has been exploited to image the interface where, due to the birefringent property of soft tissues, the angle between fibre orientation and the static magnetic field can improve contrast between materials like collagen fibres with different properties and orientations (Bydder et al. 2007). The use of ultra-short pulse echo sequences for MRI imaging of tendon and entheses has been shown in a range of studies (Robson et al. 2004). Du showed that ‘magic angle’ effect enabled imaging of an Achilles tendon in vivo (Du et al. 2005). Multiple studies by Benjamin and Bydder (2007); Benjamin et al. (2008a, b) have used a combination of ultra-short

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pulse echo sequences and ‘magic angle’ imaging to enable differentiation between the different components of the enthesis in vivo. Contrast agents are widely used in MRI for the tracking of particles, however, due to the generally static nature of the enthesis and size relative to the attainable resolution through MRI they will likely be redundant. For tissue engineered samples, hard tissue contrast might be improved through use of modified calcium phosphate based contrast agents which have been reported by Ventura et al. (2012) and Chesnick et al. (2011). Ultrasound Ultrasound uses electrically-induced pulses of high frequency sound to non-invasively map the subsurface of samples based on impedance changes between materials. These properties have also made it a popular choice for monitoring changes in scaffolds in tissue engineering. The data and resolution provided by ultrasonic imaging is dependent on the scan type and frequency used, respectively. In addition to greyscale spatial properties (B mode) ultrasound can also provide information on the composition (spectral) and mechanical properties (elastography) of samples. Contrast agents (bubbles, gas capsules) are used in ultrasound but are not applicable to the imaging of the hard/soft tissue interface. Resolution in ultrasound is proportional to the pulse frequency with a higher frequency producing a higher resolution at the cost of depth penetration and the requirement of specialized instrumentation, in high frequency (50 MHz) cases a resolution of 50 lm is common (Foster et al. 2000) and up to 25 lm is obtainable (Gudur et al. 2012). Like MRI, ultrasound is well established for the macroscopic imaging of the enthesis in the clinical setting, often used to examine changes related to arthritis due to its non-invasive nature (Aydin et al. 2010; Eder et al. 2012; Gandjbakhch et al. 2011). The most common application in ultrasound biomicroscopy is B mode imaging, producing a greyscale brightness image of the sample providing spatial, and if needed temporal information. Rice et al. (2009) have shown that the development and quantity of collagen can be monitored using ultrasound and Kreitz et al. (2011) reported a relationship between greyscale intensity and collagen content. Spectral ultrasound

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uses characteristic parameters obtained from the linear regression of calibrated power spectrum of raw radiofrequency data, by comparing these to databases of known samples the composition of the tissue can be determined (Lizzi et al. 2003). High resolution spectral ultrasound has been used by Gudar et al. in a study of mineralization in a hydrogel scaffold, and it was demonstrated that ultrasound imaging could be used to reconstruct images of the mineralized components in three-dimensions (Gudur et al. 2012). Ultrasound elastography (Zheng et al. 2004) has been undertaken by Spalazzi et al. (2006) who used the technique to determine the strain distribution across the interface in a native ACL. X-ray X-ray techniques covering radiographs and tomographic reconstructions based on the radio attenuation of the material are well known for their application in bone and similar hard materials and tissues. Modern bench top Micro-CT units can produce micron or submicron three-dimensional reconstructions of samples in relatively short time periods with little or no preparation. A comprehensive review of micro-CT imaging can be found by Ritman (2011). Micro-CT has been widely applied to calcium phosphate bone replacements materials to assess morphology, porosity, interconnectivity and bio-resorption under in vivo conditions (Fierz et al. 2008; Gauthier et al. 2005; Jones et al. 2007). The highest resolution and contrast in soft tissues is achieved through the use of synchrotron beamlines, which require the most preparation and are perhaps least practical means of acquiring data. Traditionally soft tissue has been poorly detected using micro-CT due to its very low attenuation, but the development of new techniques and contrast stains in recent years has created the potential for detailed imaging of soft tissue alongside the hard at the cost of more specialized equipment and/or preparation. The use of contrast agents, including iodine and barium, to enhance the contrast of soft tissues is well established in radiography. Studies by Metscher (2009a, b) have used iodine stains to produce hard and soft tissue reconstructions at histological resolution levels in developing fetal animals (Metscher 2009a, b). This will likely be more limited in the case of ligaments and tendons which are more homogenous in structure, however the staining and ability to resolve

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individual fibres in muscle has been shown (Jeffery et al. 2011) and may serve as a useful addition to the hard tissue information without the need for registration of complementary data from other modalities. Non-attenuation based methods such as phase-contrast X-ray detection have significantly increased the sensitivity for detection of soft tissue, Ismail et al. showed the contrast and detail that can be produced at the interface in the osteochondral interface of synovial joints through bench top and beamline phase contrast X-ray imaging techniques (Ismail et al. 2010). Dual-energy imaging uses a twin source/detector set-up to simultaneously capture data sets of the sample at two different energy levels optimized toward different material attenuations. This allows the data to be combined and fused images of high and low attenuation tissues to be produced. At the time of writing, no dual-energy studies have examined the microscopic hard/soft tissue interface but clinical level imaging of bone and tendon (Johnson et al. 2007; Sun et al. 2008; Deng et al. 2009) has shown a high level of detail in ligament/tendon structure. Emerging modalities Further imaging modalities are available and either have yet to be significantly applied to the area of interfacial tissue imaging or are still being developed. Atomic force microscopy has been applied ex vivo to the osteochondral junction, providing nanoscale mechanical measurements across the interface (Campbell et al. 2012). Non-invasive photo-accoustic imaging and tomography combining the contrast of optical methods with the depth of ultrasound has been applied to ex vivo samples, current studies have shown imaging about joints for arthritis based purposes (Chamberland et al. 2008). Optical projection tomography is a developing method that allows fluorescence tomographic imaging of samples 1–10 mm in size (Sharpe 2004) a much greater depth than can be achieved via confocal but is likely to be very limited by the hard tissue component. Due to the great increase in information that can be obtained through the use of multiple complimentary techniques in this and other applications a number of multi-modality imaging instruments have been developed and documented in the literature, these instruments are found either as in-house developments or commercially available systems. Raman spectroscopy

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has been combined with other imaging techniques, usually optical histology to provide more context and depth. The Thomopoulos group has combined optical images with Raman spectra at known points to provide information on the mineralization across the rotatorcuff tendon (Wopenka et al. 2008) and the changes in mineralization in developing tendons (Schwartz et al. 2012). Further examples of relevant studies or systems include fluorescence confocal microscopy-Raman spectroscopy (Caspers et al. 2003), Raman spectroscopy-OCT (Patil et al. 2008), OCT-fluorescence imaging (Yuan et al. 2009), and OCT-fluorescence spectroscopy (Barton et al. 2004).

Conclusion A review of the literature has found the majority of imaging of the hard/soft tissue interface has been done through light micrographs of plain or histologically stained samples. While this can provide a high level of morphological detail, the destructive nature of this method is problematic in tissue engineering where developing and inserted constructs will need to be monitored for process optimization with minimal disruption. The development of increasing numbers of advanced biological imaging modalities able to provide a range of information on samples has greatly increased in recent years enabling the hard/soft interface to be investigated through morphological, biological and chemical properties at a range of resolutions and in three-dimensional or planar views. Ultimately the imaging technique(s) chosen will depend on the user’s needs based on the required information and limitations from accessibility and sample preparation. Knowledge of the image production, capabilities and artifacts likely to be present in each technique is essential to producing the most effective and highest quality images. Further to the discussion given in this paper there are many digital processing techniques that can be applied during or after image acquisition to remove artifacts, improve image quality, allow automated handling of large data sets, and extract additional information. Separate hard and soft data may need to be acquired from different imaging techniques and recombined via an overlay or digital registration to build a comprehensive picture. An improved understanding of these complex interfaces, that are very difficult to image, will enable the

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development of new technologies and treatments for a range of skeletal pathologies. Acknowledgments The authors would like to the acknowledge the following for support and funding: Engineering and Physical Sciences Research Council, and Biotechnology and Biological Sciences Research Council Project number BB/G022356/1 and Orthopedic Research UK, Project number 472.

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soft tissue interface.

Interfaces between different tissues play an essential role in the biomechanics of native tissues and their recapitulation is now recognized as critic...
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