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Intra‐synovial tendon injuries are a common orthopedic problem with limited treatment options. The synovium is a specialized connective tissue forming the inner encapsulating lining of diarthrodial joints and intra‐synovial tendons. It contains multipotent mesenchymal stromal cells that render it a viable source of progenitors for tendon repair. This study evaluated the effects of autologous implantation of cells derived from normal synovium (synovial membrane cells [SMCs]) in augmenting repair in an ovine model of intra‐synovial tendon injury. For this purpose, synovial biopsies were taken from the right digital flexor tendon sheath following creation of a defect to the lateral deep digital flexor tendon. Mononuclear cells were isolated by partial enzymatic digestion and assessed for MSC characteristics. Cell tracking and tendon repair were assessed by implanting 5 × 106 cells into the digital flexor tendon sheath under ultrasound guidance with the effects evaluated using magnetic resonance imaging and histopathology. Synovial biopsies yielded an average 4.0 × 105 ± 2.7 × 105 SMCs that exhibited a fibroblastic morphology, variable osteogenic, and adipogenic responses but were ubiquitously strongly chondrogenic. SMCs displayed high expression of CD29 with CD271NEGATIVE and MHC‐IILOW cell‐surface marker profiles, and variable expression of CD73, CD90, CD105, CD166, and MHC‐I. Implanted SMCs demonstrated engraftment within the synovium, though a lack of repair of the tendon lesion over 24 weeks was observed. We conclude healthy synovium is a viable source of multipotent cells, but that the heterogeneity of synovium underlies the variability between different SMC populations, which while capable of engraftment and persistence within the synovium exhibit limited capacity of influencing tendon repair. © 2019 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society J Orthop Res 38:128–138, 2020

Background Intra-synovial tendon injuries display poor healing, which often results in reduced functionality and pain. A lack of effective therapeutic options has led to experimental approaches to augment natural tendon repair with autologous mesenchymal stem cells (MSCs) although the effects of the intra-synovial environment on the distribution, engraftment and functionality of implanted MSCs is not known. This study utilised a novel sheep model which, although in an anatomically different location, more accurately mimics the mechanical and synovial environment of the human rotator cuff, to determine the effects of intra-synovial implantation of MSCs. Methods A lesion was made in the lateral border of the lateral branch of the ovine deep digital flexor tendon within the digital sheath and 2 weeks later 5 million autologous bone marrow MSCs were injected under ultrasound guidance into the digital sheath. Tendons were recovered post mortem at 1 day, and 1–2, 4, 12 and 24 weeks after MSC injection. For the 1-day and 1–2-week groups, MSCs labelled with fluorescent-conjugated magnetic iron-oxide nanoparticles (MIONs) were tracked with MRI, histology and flow cytometry. The 4, 12 and 24-week groups were implanted with non-labelled cells and compared with saline-injected controls for healing. Results The MSCs displayed no reduced viability in vitro to an uptake of 20.0 ± 4.6 pg MIONs per cell, which was detectable by MRI at minimal density of ~ 3 × 104 cells. Treated limbs indicated cellular distribution throughout the tendon synovial sheath but restricted to the synovial tissues, with no MSCs detected in the tendon or surgical lesion. The lesion was associated with negligible morbidity with minimal inflammation post surgery. Evaluation of both treated and control lesions showed no evidence of healing of the lesion at 4, 12 and 24 weeks on gross and histological examination. Conclusions Unlike other laboratory animal models of tendon injury, this novel model mimics the failed tendon healing seen clinically intra-synovially. Importantly, however, implanted stem cells exhibited homing to synovium niches where they survived for at least 14 days. This phenomenon could be utilised in the development of novel physical or biological approaches to enhance localisation of cells in augmenting intra-synovial tendon repair.

Advancing age is a well-known risk factor for tendon disease. Energy-storing tendons [e.g., human Achilles, equine superficial digital flexor tendon (SDFT)] are particularly vulnerable and it is thought that injury occurs following an accumulation of micro-damage in the extracellular matrix (ECM). Several authors suggest that age-related micro-damage accumulates due to a failure of the aging cell population to maintain the ECM or an imbalance between anabolic and catabolic pathways. We hypothesized that ageing results in a decreased ability of tendon cells to synthesize matrix components and matrix-degrading enzymes, resulting in a reduced turnover of the ECM and a decreased ability to repair micro-damage. The SDFT was collected from horses aged 3-30 years with no signs of tendon injury. Cell synthetic and degradative ability was assessed at the mRNA and protein levels. Telomere length was measured as an additional marker of cell ageing. There was no decrease in cellularity or relative telomere length with increasing age, and no decline in mRNA or protein levels for matrix proteins or degradative enzymes. The results suggest that the mechanism for age-related tendon deterioration is not due to reduced cellularity or a loss of synthetic functionality and that alternative mechanisms should be considered.

Fragility fractures, those fractures which result from low level trauma, have a large and growing socio-economic cost in countries with aging populations. Bone-density-based assessment techniques are vital for identifying populations that are at higher risk of fracture, but do not have high sensitivity when it comes to identifying individuals who will go on to have their first fragility fracture. We are developing Spatially Offset Raman Spectroscopy (SORS) as a tool for retrieving chemical information from bone non-invasively in vivo. Unlike X-ray-based techniques SORS can retrieve chemical information from both the mineral and protein phases of the bone. This may enable better discrimination between those who will or will not go on to have a fragility fracture because both phases contribute to bone's mechanical properties. In this study we analyse excised bone with Raman spectroscopy and multivariate analysis, and then attempt to look for similar Raman signals in vivo using SORS. We show in the excised work that on average, bone fragments from the necks of fractured femora are more mineralised (by 5-10%) than (cadaveric) non-fractured controls, but the mineralisation distributions of the two cohorts are largely overlapped. In our in vivo measurements, we observe similar, but as yet statistically underpowered, differences. After the SORS data (the first SORS measurements reported of healthy and diseased human cohorts), we identify methodological developments which will be used to improve the statistical significance of future experiments and may eventually lead to more sensitive prediction of fragility fractures. © 2015 The Authors. Journal of Raman Spectroscopy Published by John Wiley & Sons, Ltd.

The ability of Spatially Offset Raman Spectroscopy (SORS) to obtain chemically specific information from below the sample surface makes it a promising technique for non-invasive in vivo diagnosis of bone conditions by sampling bone through the skin. The depth below a surface interrogated by SORS depends on the system's optical properties and is difficult to estimate for complex bone material. This paper uses 830 nm laser excitation to investigate the influence of bone mineralization on photon migration properties in deer antler cortex, equine metacarpal cortex and whale tympanic bulla. Thin slices form each type of bone (thickness: 0.6-1.0 mm) were cut and put together on top of each other forming stacks with a total thickness of 4.1-4.7 mm. A 0.38 mm thin slice of polytetrafluoroethylene (PTFE) served as a test material for Raman signal recovery and was placed in between the individual bone slices within the stack. At SORS offsets of 8.0-9.5 mm Raman bands of materials not present in healthy bone (e.g. PTFE as an example) can be recovered through 4.4-4.7 mm of cortical bone tissue, depending on mineralization level and porosity. These findings significantly increase our understanding of SORS analysis through bones of different composition and provide information that is vital to determine the value of SORS as a medical diagnostic technique. The data serve to define which SORS offset is best deployed for the non-invasive detection of chemically specific markers associated with infection, degeneration and disease or cancer within bone.

Meniscal cartilage tears are common and predispose to osteoarthritis (OA). Most occur in the avascular portion of the meniscus where current repair techniques usually fail. We described previously the use of undifferentiated autologous mesenchymal stem cells (MSCs) seeded onto a collagen scaffold (MSC/collagen-scaffold) to integrate meniscal tissues in vitro. Our objective was to translate this method into a cell therapy for patients with torn meniscus, with the long-term goal of delaying or preventing the onset of OA. After in vitro optimization, we tested an ovine-MSC/collagen-scaffold in a sheep meniscal cartilage tear model with promising results after 13 weeks, although repair was not sustained over 6 months. We then conducted a single center, prospective, open-label first-in-human safety study of patients with an avascular meniscal tear. Autologous MSCs were isolated from an iliac crest bone marrow biopsy, expanded and seeded into the collagen scaffold. The resulting human-MSC/collagen-scaffold implant was placed into the meniscal tear prior to repair with vertical mattress sutures and the patients were followed for 2 years. Five patients were treated and there was significant clinical improvement on repeated measures analysis. Three were asymptomatic at 24 months with no magnetic resonance imaging evidence of recurrent tear and clinical improvement in knee function scores. Two required subsequent meniscectomy due to retear or nonhealing of the meniscal tear at approximately 15 months after implantation. No other adverse events occurred. We conclude that undifferentiated MSCs could provide a safe way to augment avascular meniscal repair in some patients. Registration: EU Clinical Trials Register, 2010-024162-22.

Summary: Loading increases bone mass and strength in a site-specific manner; however, possible effects of loading on bone matrix composition have not been evaluated. Site-specific structural and material properties of mouse bone were analyzed on the macro- and micro/molecular scale in the presence and absence of axial loading. The response of bone to load is heterogeneous, adapting at molecular, micro-, and macro-levels. Introduction: Osteoporosis is a degenerative disease resulting in reduced bone mineral density, structure, and strength. The overall aim was to explore the hypothesis that changes in loading environment result in site-specific adaptations at molecular/micro- and macro-scale in mouse bone. Methods: Right tibiae of adult mice were subjected to well-defined cyclic axial loading for 2 weeks; left tibiae were used as physiologically loaded controls. The bones were analyzed with μCT (structure), reference point indentation (material properties), Raman spectroscopy (chemical), and small-angle X-ray scattering (mineral crystallization and structure). Results: The cranial and caudal sites of tibiae are structurally and biochemically different within control bones. In response to loading, cranial and caudal sites increase in cortical thickness with reduced mineralization (−14 and −3%, p < 0.01, respectively) and crystallinity (−1.4 and −0.3%, p < 0.05, respectively). Along the length of the loaded bones, collagen content becomes more heterogeneous on the caudal site and the mineral/collagen increases distally at both sites. Conclusion: Bone structure and composition are heterogeneous, finely tuned, adaptive, and site-specifically responsive at the micro-scale to maintain optimal function. Manipulation of this heterogeneity may affect bone strength, relative to specific applied loads.

Bone diseases and disorders are a growing challenge in aging populations; so effective diagnostic and therapeutic solutions are now essential to manage the demands of healthcare sectors effectively. Spatially offset Raman spectroscopy (SORS) allows for chemically specific sub-surface probing and has a great potential to become an in vivo tool for early non-invasive detection of bone conditions. Bone is a complex hierarchical material and the volume probed by SORS is dependent on its optical properties. Understanding and taking into account the variations in diffuse scattering properties of light in various bone types is essential for the effective development and optimization of SORS as a diagnostic in vivo tool for characterizing bone disease. This study presents SORS investigations at 830 nm excitation on two specific types of bone with differing mineralization levels. Thin slices of bone from horse metacarpal cortex (0.6 mm thick) and whale bulla (1.0 mm thick) were cut and stacked on top of each other (4-7 layers with a total thickness of 4.1 mm). To investigate the depth origin of the detected Raman signal inside the bone a 0.38 mm thin Teflon slice was used as test sample and inserted in between the layers of stacked bone slices. For both types of bone it could be demonstrated that chemically specific Raman signatures different from those of normal bone can be retrieved through 3.8-4.0 mm of overlying bone material with a spatial offset of 7-8 mm. The determined penetration depths can be correlated with the mechanical and optical properties of the specimens. The findings of this study increase our understanding of SORS analysis of bone and thus have impact for medical diagnostic applications e.g. enabling the non-invasive detection of spectral changes caused by degeneration, infection or cancer deep inside the bone matrix.

Made available in DSpace on 2022-04-29T07:37:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-01-01 We explored the osseointegration potential of two macroporous titanium surfaces obtained using fast plasma sintering (FPS): Ti macroporous structures with 400-600 µmØ pores (TiMac400) and 850-1000 µmØ pores (TiMac850). They were compared against two surfaces currently in clinical use: Ti-Growth® and air plasma spray (Ti-Y367). Each surface was tested, once placed over a Ti-alloy and once onto a CoCr bulk substrate. Implants were placed in medial femoral condyles in 24 sheep. Samples were explanted at four and eight weeks after surgery. Push-out loads were measured using a material-testing system. Bone contact and ingrowth were assessed by histomorphometry and SEM and EDX analyses. Histology showed early osseointegration for all the surfaces tested. At 8 weeks, TiMac400, TiMac850 and Ti-Growths® showed deep bone ingrowth and extended colonisation with newly formed bone. The mechanical push-out force was equal in all tested surfaces. Plasma spray surfaces showed greater bone-implant contact and higher level of pores colonisation with new bone than FPS produced surfaces. However, the void pore area in FPS specimens was significantly higher, yet the FPS porous surfaces allowed a deeper osseointegration of bone to implant. FPS manufactured specimens showed similar osseointegration potential to the plasma spray surfaces for orthopaedic implants. FPS is a useful technology for manufacturing macroporous titanium surfaces. Furthermore, its capability to combine two implantable materials, using bulk CoCr with macroporous titanium surfaces, could be of interest as it enables designers to conceive and manufacture innovative components. FPS delivers functional graded materials components with macroporous structures optimised for osseointegration. Institute of Orthopaedics and Musculoskeletal Science University College London Royal National Orthopaedic Hospital Department of Veterinary Surgery and Anaesthesiology School of Veterinary Medicine and Animal Science São Paulo State University Research and Development Department Eurocoating Spa Department of Veterinary Surgery and Anaesthesiology School of Veterinary Medicine and Animal Science São Paulo State University

Summary Loading increases bone mass and strength in a site-specific manner; however, possible effects of loading on bone matrix composition have not been evaluated. Site-specific structural and material properties of mouse bone were analyzed on the macro- and micro/molecular scale in the presence and absence of axial loading. The response of bone to load is heterogeneous, adapting at molecular, micro-, and macro-levels. Introduction Osteoporosis is a degenerative disease resulting in reduced bone mineral density, structure, and strength. The overall aim was to explore the hypothesis that changes in loading environment result in site-specific adaptations at molecular/micro- and macro-scale in mouse bone. Methods Right tibiae of adult mice were subjected to well-defined cyclic axial loading for 2 weeks; left tibiae were used as physiologically loaded controls. The bones were analyzed with μCT (structure), reference point indentation (material properties), Raman spectroscopy (chemical), and small-angle X-ray scattering (mineral crystallization and structure). Results The cranial and caudal sites of tibiae are structurally and biochemically different within control bones. In response to loading, cranial and caudal sites increase in cortical thickness with reduced mineralization (−14 and −3%, p

The tendons in the turkey leg have specific well-defined areas which become mineralized as the animal ages and they are a thoroughly characterized model system for studying the mineralization process of bone. In this study, nondestructive Raman spectroscopic analysis was used to explore the hypothesis that regions of the turkey tendon that are associated with mineralization exhibit distinct and observable chemical modifications of the collagen prior to the onset of mineralization. The Raman spectroscopy features associated with mineralization were identified by probing (on the micrometer scale) the transition zone between mineralized and nonmineralized regions of turkey leg tendons. These features were then measured in whole tendons and identified in regions of tendon which are destined to become rapidly mineralized around 14 weeks of age. The data show there is a site-specific difference in collagen prior to the deposition of mineral, specifically the amide III band at 1270 cm(-1) increases as the collagen becomes more ordered (increased amide III:amide I ratio) in regions that become mineralized compared to collagen destined to remain nonmineralized. If this mechanism were present in materials of different mineral fraction (and thus material properties), it could provide a target for controlling mineralization in metabolic bone disease.

Raman Spectroscopy has become an important technique for assessing the composition of excised sections of bone, and is currently being developed as an in vivo tool for transcutaneous detection of bone disease using spatially offset Raman spectroscopy (SORS). The sampling volume of the Raman technique (and thus the amount of bone material interrogated by SORS) depends on the nature of the photon scattering in the probed tissue. Bone is a complex hierarchical material and to date little is known regarding its diffuse scattering properties which are important for the development and optimization of SORS as a diagnostic tool for characterizing bone disease in vivo. SORS measurements at 830 nm excitation wavelength are carried out on stratified samples to determine the depth from which the Raman signal originates within bone tissue. The measurements are made using a 0.38 mm thin Teflon slice, to give a pronounced and defined spectral signature, inserted in between layers of stacked 0.60 mm thin equine bone slices. Comparing the stack of bone slices with and without underlying bone section below the Teflon slice illustrated that thin sections of bone can lose appreciable number of photons through the unilluminated back surface. The results show that larger SORS offsets lead to progressively larger penetration depth into the sample; different Raman spectral signatures could be retrieved through up to 3.9 mm of overlying bone material with a 7 mm offset. These findings have direct impact on potential diagnostic medical applications; for instance on the detection of bone tumors or areas of infected bone.