Sensitivity of spatially offset Raman spectroscopy (SORS) to subcortical bone tissue

The development of spatially offset Raman spectroscopy (SORS) has enabled deep, non‐invasive chemical characterization of turbid media. Here, we use SORS to measure subcortical bone tissue and depth‐resolved biochemical variability in intact, exposed murine bones. We also apply the technique to study a mouse model of the genetic bone disorder osteogenesis imperfecta. The results suggest that SORS is more sensitive to disease‐related biochemical differences in subcortical trabecular bone and marrow than conventional Raman measurements.     

Inverse spatially‐offset Raman spectroscopy using optical fibers: An axicon lens‐free approach

Inverse spatially offset Raman spectroscopy (I‐SORS) seeks to interrogate deep inside a Raman‐active, layered, diffusely scattering sample. It makes a collimated laser beam incident onto the sample surface in the form of concentric illumination rings (of varying radii) from whose center the back‐scattered Raman signal is collected for detection. Since formation of illumination rings of different sizes requires an axicon to be moved along the axis of the collimated laser beam and axicons below a certain minimum size (~1 inch) are not readily available, this classical configuration incorporating an axicon cannot be used for designing a compact I‐SORS probe of narrower diameter. We report a novel scheme of implementing I‐SORS which overcomes this limitation by implementing ring illumination and point collection using two multi‐mode optical fibers. An important advantage of the proposed scheme is that unlike the previously reported inverse SORS configurations, it does not require physical movement of any of the optical components for generating spatial offsets needed for probing sub‐surface depths. Another advantage is its fiber‐optic configuration which is ideally suited for designing a compact and pencil‐sized I‐SORS probe, often desired in many practical situations for carrying out depth‐sensitive Raman measurements in situ from a layered turbid sample.      

Depth‐resolved multimodal imaging: Wavelength modulated spatially offset Raman spectroscopy with optical coherence tomography

A major challenge in biophotonics is multimodal imaging to obtain both morphological and molecular information at depth. We demonstrate a hybrid approach integrating optical coherence tomography (OCT) with wavelength modulated spatially offset Raman spectroscopy (WM‐SORS). With depth colocalization obtained from the OCT, we can penetrate 1.2‐mm deep into strong scattering media (lard) to acquire up to a 14‐fold enhancement of a Raman signal from a hidden target (polystyrene) with a spatial offset. Our approach is capable of detecting both Raman and OCT signals for pharmaceutical particles embedded in turbid media and revealing the white matter at depth within a 0.6‐mm thick brain tissue layer. This depth resolved label‐free multimodal approach is a powerful route to analyze complex biomedical samples.      

Evaluation of processed bovine cancellous bone matrix seeded with syngenic osteoblasts in a critical size calvarial defect rat model

INTRODUCTION: Biologic bone substitutes may offer alternatives to bone grafting procedures. The aim of this study was to evaluate a preformed bone substitute based on processed bovine cancellous bone (PBCB) with or without osteogenic cells in a critical size calvarial defect rat model. METHODS: Discs of PBCB (Tutobone) were seeded with second passage fibrin gel-immobilized syngenic osteoblasts (group A, n = 40). Cell-free matrices (group B, n = 28) and untreated defects (group C; n=28) served as controls. Specimens were explanted between day 0 and 4 months after implantation and were subjected to histological and morphometric evaluation. RESULTS: At 1 month, bone formation was limited to small peripheral areas. At 2 and 4 months, significant bone formation, matrix resorption as well as integration of the implants was evident in groups A and B. In group C no significant regeneration of the defects was observed. Morphometric analysis did not disclose differences in bone formation in matrices from groups A and B. Carboxyfluorescine-Diacetate-Succinimidylester (CFDA) labeling demonstrated low survival rates of transplanted cells. DISCUSSION: Osteoblasts seeded into PBCB matrix display a differentiated phenotype following a 14 days cell culture period. Lack of initial vascularization may explain the absence of added osteogenicity in constructs from group A in comparison to group B. PBCB is well integrated and represents even without osteogenic cells a promising biomaterial for reconstruction of critical size calvarial bone defects.     

Time‐lapsed imaging for in‐process evaluation of supercritical fluid processing of tissue engineering scaffolds

This article demonstrates the application of time‐lapsed imaging and image processing to inform the supercritical processing of tissue scaffolds that are integral to many regenerative therapies. The methodology presented provides online quantitative evaluation of the complex process of scaffold formation in supercritical environments. The capabilities of the developed system are demonstrated through comparison of scaffolds formed from polymers with different molecular weight and with different venting times. Visual monitoring of scaffold fabrication enabled key events in the supercritical processing of the scaffolds to be identified including the onset of polymer plasticization, supercritical points and foam formation. Image processing of images acquired during the foaming process enabled quantitative tracking of the growing scaffold boundary that provided new insight into the nature of scaffold foaming. Further, this quantitative approach assisted in the comparison of different scaffold fabrication protocols. Observed differences in scaffold formation were found to persist, post‐fabrication as evidenced by micro x‐ray computed tomography (μ x‐ray CT) images. It is concluded that time‐lapsed imaging in combination with image processing is a convenient and powerful tool to provide insight into the scaffold fabrication process. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Successful human long‐term application of in situ bone tissue engineering

Tissue Engineering (TE) and Regenerative Medicine (RM) have gained much popularity because of the tremendous prospects for the care of patients with tissue and organ defects. To overcome the common problem of donor‐site morbidity of standard autologous bone grafts, we successfully combined tissue engineering techniques for the first time with the arteriovenous loop model to generate vascularized large bone grafts. We present two cases of large bone defects after debridement of an osteomyelitis. One of the defects was localized in the radius and one in the tibia. For osseus reconstruction, arteriovenous loops were created as vascular axis, which were placed in the bony defects. In case 1, the bone generation was achieved using cancellous bone from the iliac crest and fibrin glue and in case 2 using a clinically approved β‐tricalciumphosphate/hydroxyapatite (HA), fibrin glue and directly auto‐transplanted bone marrow aspirate from the iliac crest. The following post‐operative courses were uneventful. The final examinations took place after 36 and 72 months after the initial operations. Computer tomogrphy (CT), membrane resonance imaging (MRI) and doppler ultrasound revealed patent arterio‐venous (AV) loops in the bone grafts as well as completely healed bone defects. The patients were pain‐free with normal ranges of motion. This is the first study demonstrating successfully axially vascularized in situ tissue engineered bone generation in large bone defects in a clinical scenario using the arteriovenous loop model without creation of a significant donor‐site defect utilizing TE and RM techniques in human patients with long‐term stability.     

Real‐time endoscopic Raman spectroscopy for in vivo early lung cancer detection

Currently the most sensitive method for localizing lung cancers in central airways is autofluorescence bronchoscopy (AFB) in combination with white light bronchoscopy (WLB). The diagnostic accuracy of WLB + AFB for high grade dysplasia (HGD) and carcinoma in situ is variable depending on physician's experience. When WLB + AFB are operated at high diagnostic sensitivity, the associated diagnostic specificity is low. Raman spectroscopy probes molecular vibrations and gives highly specific, fingerprint‐like spectral features and has high accuracy for tissue pathology classification. In this study we present the use of a real‐time endoscopy Raman spectroscopy system to improve the specificity. A spectrum is acquired within 1 second and clinical data are obtained from 280 tissue sites (72 HGDs/malignant lesions, 208 benign lesions/normal sites) in 80 patients. Using multivariate analyses and waveband selection methods on the Raman spectra, we have demonstrated that HGD and malignant lung lesions can be detected with high sensitivity (90%) and good specificity (65%).

     

Predicting the structural integrity of bone defects repaired using bone graft materials

Bone defects create stress concentrations which can cause fracture under impact or cyclic loading. Defects are often repaired by filling them with a bone graft material; this will reduce the stress concentration, but not completely, because these materials have lower stiffness than bone. The fracture risk decreases over time as the graft material is replaced by living bone. Many new bone graft materials are being developed, using tissue engineering and other techniques, but currently there is no rational way to compare these materials and predict their effectiveness in repairing a given defect. This paper describes, for the first time, a theoretical model which can be used to predict failure by brittle fracture or fatigue, initiating at the defect. Preliminary results are presented, concentrating on the prediction of stress fracture during the crucial post-operative period. It is shown that the likelihood of fracture is strongly influenced by the shape of the defect as well as its size, and also by the level of post-operative exercise. The most important finding is that bone graft materials can be successful in preventing fracture even when their mechanical properties are greatly inferior to those of bone. Future uses of this technique include pre-clinical assessment of bone replacement materials and pre-operative planning in orthopaedic surgery.     

Intra‐articular injection of autologous adipose‐derived mesenchymal stem cells in the treatment of knee osteoarthritis

Background

Osteoarthritis (OA) is a chronic degenerative joint disease and is considered to be the fourth leading cause of disability and the second cause of inability to work in men. Recently, adipose‐derived mesenchymal stem cells (AD‐MSCs) came into focus for regenerative medicine as a promising tool for the treatment of OA. The administration of stem cells into impaired joints results in pain relief and improves quality of life, accompanied by restoration of hyaline articular cartilage.

Methods

In the present study, nine patients (including two patients with bilateral symptoms) diagnosed with osteoarthritis (International Knee Documentation grade B in 5 and grade D in six knees) were treated using a single injection of AD‐MSCs at a concentration of 0.5–1.0 × 10⁷ cells and were followed up for 18 months. During follow‐up, all the cases were evaluated clinically by Knee Society score (KSS), Hospital for Special Surgery knee score (HSS‐KS), Tegner–Lysholm (T–L) score and visual analogue scale (VAS) of pain, as well as by plain radiography and by magnetic resonance imaging visualization with 2D Magnetic Resonance Observation of Cartilage Repair Tissue (MOCART) score assessment.

Results

Significant improvement of all four clinical scores was observed within the first 6 months (KSS for 41.4 points, HSS‐KS for 33.9 points, T–L score for 44.8 points, VAS of pain from 54.5 to 9.3) and improvement persisted throughout the rest of the follow‐up. MOCART score showed significant cartilage restoration (from 43 ± 7.2 to 63 ± 17.1), whereas radiography showed neither improvement, nor further joint degeneration.

Conclusions

The results obtained in the present study provide good basis for prospective randomized controlled clinical trials with respect to the use of AD‐MSCs in the treatment of osteoarthritis.     

Investigating marine bio‐calcification mechanisms in a changing ocean with in vivo and high‐resolution ex vivo Raman spectroscopy

Ocean acidification poses a serious threat to marine calcifying organisms, yet experimental and field studies have found highly diverse responses among species and environments. Our understanding of the underlying drivers of differential responses to ocean acidification is currently limited by difficulties in directly observing and quantifying the mechanisms of bio‐calcification. Here, we present Raman spectroscopy techniques for characterizing the skeletal mineralogy and calcifying fluid chemistry of marine calcifying organisms such as corals, coralline algae, foraminifera, and fish (carbonate otoliths). First, our in vivo Raman technique is the ideal tool for investigating non‐classical mineralization pathways. This includes calcification by amorphous particle attachment, which has recently been controversially suggested as a mechanism by which corals resist the negative effects of ocean acidification. Second, high‐resolution ex vivo Raman mapping reveals complex banding structures in the mineralogy of marine calcifiers, and provides a tool to quantify calcification responses to environmental variability on various timescales from days to years. We describe the new insights into marine bio‐calcification that our techniques have already uncovered, and we consider the wide range of questions regarding calcifier responses to global change that can now be proposed and addressed with these new Raman spectroscopy tools.     

Resonance Raman quantification of the redox state of cytochromes b and c in‐vivo and in‐vitro

We observe the redox state changes with respiration of cytochromes b and c in mitochondria in a living Saccharomyces cerevisiae cell as well as in isolated mitochondria with the very use of Raman microspectroscopy. The possibility of monitoring the respiration activity of mitochondria in vivo and in vitro by Raman microspectroscopic quantification of the cytochrome redox states is suggested. It will lead to a new means to assess mitochondrial respiration activity in vivo and in vitro without using any labelling or genetic manipulation. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Synthesis,development, characterization and effectiveness of bovine pure platelet gel‐collagen‐polydioxanone bioactive graft on tendon healing

Bovine platelet gel (BPG) is an accessible and cost‐effective source of growth factors which may have a value in tendon regenerative medicine. We produced a collagen implant (CI) as a tendon proper, covered it with polydioxanone (PDS) sheath to simulate paratenon and finally embedded the BPG as an active source of growth factor within the bioimplant to test whether BPG would be able to accelerate and enhance tendon regeneration and repair. After in vitro characterization of the bioactive grafts, the grafts were implanted in rabbit large tendon defect model. Untreated tendons and tendons treated with either CI or CI‐PDS were served as controls for the CI‐PDS‐BPG. The animals were investigated clinically, ultrasonographically and haematologically for 120 days. After euthanasia, dry matter content, water uptake and delivery characteristics and also gross morphological, histopathological and scanning electron microscopic features of the healing tendons were assessed. In vitro, the activated platelets in the scaffold, released their growth factors significantly more than the controls. BPG also increased cell viability, and enhanced cellular differentiation, maturation and proliferation inside the CI‐PDS compared with the controls. In vivo, the BPG modulated inflammation, increased quality and rate of fibroplasia and produced a remodelled tendon that had significantly higher collagen content and superior collagen fibril and fibre differentiation than controls. Treatment also significantly improved tendon water uptake and delivery characteristics, animals’ serum PDGF level, CI‐PDS biocompatibility and biodegradability and reduced peritendinous adhesions, muscle fibrosis and atrophy. BPG was effective on tendon healing and CI‐PDS‐BPG may be a valuable bioscaffold in tendon reconstructive surgery.     

An ex vivo model using human osteoarthritic cartilage demonstrates the release of bioactive insulin‐like growth factor‐1 from a collagen–glycosaminoglycan scaffold

Biomimetic scaffolds hold great promise for therapeutic repair of cartilage, but although most scaffolds are tested with cells in vitro, there are very few ex vivo models (EVMs) where adult cartilage and scaffolds are co‐cultured to optimize their interaction prior to in vivo studies. This study describes a simple, non‐compressive method that is applicable to mammalian or human cartilage and provides a reasonable throughput of samples. Rings of full‐depth articular cartilage slices were derived from human donors undergoing knee replacement for osteoarthritis and a 3 mm core of a collagen/glycosaminoglycan biomimetic scaffold (Tigenix, UK) inserted to create the EVM. Adult osteoarthritis chondrocytes were seeded into the scaffold and cultures maintained for up to 30 days. Ex vivo models were stable throughout experiments, and cells remained viable. Chondrocytes seeded into the EVM attached throughout the scaffold and in contact with the cartilage explants. Cell migration and deposition of extracellular matrix proteins in the scaffold was enhanced by growth factors particularly if the scaffold was preloaded with growth factors. This study demonstrates that the EVM represents a suitable model that has potential for testing a range of therapeutic parameters such as numbers/types of cell, growth factors or therapeutic drugs before progressing to costly pre‐clinical trials. © 2015 The Authors. Cell Biochemistry and Function Published by John Wiley & Sons Ltd.     

Raman spectroscopy and PCA‐SVM as a non‐invasive diagnostic tool to identify and classify qualitatively glycated hemoglobin levels in vivo

In this study we identify and classify high and low levels of glycated hemoglobin (HbA1c) in healthy volunteers (HV) and diabetic patients (DP). Overall, 86 subjects were evaluated. The Raman spectrum was measured in three anatomical regions of the body: index fingertip, right ear lobe, and forehead. The measurements were performed to compare the difference between the HV and DP (22 well controlled diabetic patients (WCDP) (HbA1c <6.5%), and 49 not controlled diabetic patients (NCDP) (HbA1c ≥6.5%)). Multivariable methods such as principal components analysis (PCA) combined with support vector machine (SVM) were used to develop effective diagnostic algorithms for classification among these groups. The forehead of HV versus WCDP showed the highest sensitivity (100%) and specificity (100%). Sensitivity (100%) and specificity (60%), were highest in the forehead of WCDP, versus NCDP. In HV versus NCDP, the fingertip had the highest sensitivity (100%) and specificity (80%). The efficacy of the diagnostic algorithm by receiver operating characteristic (ROC) curve was confirmed. Overall, our study demonstrated that the

combination of Raman spectroscopy and PCA‐SVM are feasible non‐invasive diagnostic tool in diabetes to classify qualitatively high and low levels of HbA1c in vivo.     

Raman spectroscopy for the non‐contact and non‐destructive monitoring of collagen damage within tissues

The non‐destructive and label‐free monitoring of extracellular matrix (ECM) remodeling and degradation processes is a great challenge. Raman spectroscopy is a non‐contact method that offers the possibility to analyze ECM in situ without the need for tissue processing. Here, we employed Raman spectroscopy for the detection of heart valve ECM, focusing on collagen fibers. We screened the leaflets of porcine aortic valves either directly after dissection or after treatment with collagenase. By comparing the fingerprint region of the Raman spectra of control and treated tissues (400–1800 cm^–1), we detected no significant differences based on Raman shifts; however, we found that increasing collagen degradation translated into decreasing Raman signal intensities. After these proof‐of‐principal experiments, we compared Raman spectra of native and cryopreserved valve tissues and revealed that the signal intensities of the frozen samples were significantly lower compared to those of native tissues, similar to the data seen in the enzymatically‐degraded tissues. In conclusion, our data demonstrate that Raman microscopy is a promising, non‐destructive and non‐contact tool to probe ECM state in situ. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Three‐dimensional imaging technologies: a priority for the advancement of tissue engineering and a challenge for the imaging community

Tissue engineering/regenerative medicine (TERM) is an interdisciplinary field that applies the principle of engineering and life sciences to restore/replace damaged tissues/organs with in vitro artificially‐created ones. Research on TERM quickly moves forward. Today newest technologies and discoveries, such as 3D‐/bio‐printing, allow in vitro fabrication of ex‐novo made tissues/organs, opening the door to wide and probably never‐ending application possibilities, from organ transplant to drug discovery, high content screening and replacement of laboratory animals. Imaging techniques are fundamental tools for the characterization of tissue engineering (TE) products at any stage, from biomaterial/scaffold to construct/organ analysis. Indeed, tissue engineers need versatile imaging methods capable of monitoring not only morphological but also functional and molecular features, allowing three‐dimensional (3D) and time‐lapse in vivo analysis, in a non‐destructive, quantitative, multidimensional analysis of TE constructs, to analyze their pre‐implantation quality assessment and their fate after implantation. This review focuses on the newest developments in imaging technologies and applications in the context of requirements of the different steps of the TERM field, describing strengths and weaknesses of the current imaging approaches.