The Wnt5a Receptor, Receptor Tyrosine Kinase-Like Orphan Receptor 2, Is a Predictive Cell Surface Marker of Human Mesenchymal Stem Cells with an Enhanced Capacity for Chondrogenic Differentiation.

Multipotent mesenchymal stem cells (MSCs) have enormous potential in tissue engineering and regenerative medicine. However, until now, their development for clinical use has been severely limited as they are a mixed population of cells with varying capacities for lineage differentiation and tissue formation. Here, we identify receptor tyrosine kinase-like orphan receptor 2 (ROR2) as a cell surface marker expressed by those MSCs with an enhanced capacity for cartilage formation. We generated clonal human MSC populations with varying capacities for chondrogenesis. ROR2 was identified through screening for upregulated genes in the most chondrogenic clones. When isolated from uncloned populations, ROR2+ve MSCs were significantly more chondrogenic than either ROR2-ve or unfractionated MSCs. In a sheep cartilage-repair model, they produced significantly more defect filling with no loss of cartilage quality compared with controls. ROR2+ve MSCs/perivascular cells were present in developing human cartilage, adult bone marrow, and adipose tissue. Their frequency in bone marrow was significantly lower in patients with osteoarthritis (OA) than in controls. However, after isolation of these cells and their initial expansion in vitro, there was greater ROR2 expression in the population derived from OA patients compared with controls. Furthermore, osteoarthritis-derived MSCs were better able to form cartilage than MSCs from control patients in a tissue engineering assay. We conclude that MSCs expressing high levels of ROR2 provide a defined population capable of predictably enhanced cartilage production. Stem Cells 2017;35:2280-2291.

Augmentation and repair of tendons using demineralised cortical bone.

BACKGROUND: In severe injuries with loss of tendon substance a tendon graft or a synthetic substitute is usually used to restore functional length. This is usually associated with donor site morbidity, host tissue reactions and lack of remodelling of the synthetic substitutes, which may result in suboptimal outcome. A biocompatible graft with mechanical and structural properties that replicate those of normal tendon and ligament has so far not been identified. The use of demineralised bone for tendon reattachment onto bone has been shown to be effective in promoting the regeneration of a normal enthesis. Because of its properties, we proposed that Demineralised Cortical Bone (DCB) could be used in repair of a large tendon defect. METHODS: Allogenic DCB grafts in strip form were prepared from sheep cortical bone by acid decalcification and used to replace the enthesis and distal 1 cm of the ovine patellar tendon adjacent to the tibial tuberosity. In 6 animals the DCB strip was used to bridge the gap between the resected end of the tendon and was attached with bone anchors. Force plate analysis was done for each animal preoperatively and at weeks 3, 9, and 12 post operatively. At week 12, after euthanasia x-rays were taken and range of movements were recorded for hind limbs of each animal. Patella, patellar tendon - DCB and proximal tibia were harvested as a block and pQCT scan was done prior to histological analysis. RESULTS: Over time functional weight bearing significantly increased from 44% at 3 weeks post surgery to 79% at week 12. On retrieval none of the specimens showed any evidence of ossification of the DCB. Histological analysis proved formation of neo-enthesis with presence of fibrocartilage and mineralised fibrocartilage in all the specimens. DCB grafts contained host cells and showed evidence of vascularisation. Remodelling of the collagen leading to ligamentisation of the DCB was proved by the presence of crimp in the DCB graft on polarized microscopy. CONCLUSION: Combined with the appropriate surgical techniques, DCB can be used to achieve early mobilization and regeneration of a tendon defect which may be applicable to the repair of chronic rotator cuff injury in humans.

Silicate-substituted calcium phosphate with enhanced strut porosity stimulates osteogenic differentiation of human mesenchymal stem cells.

While many synthetic ceramic bone graft substitutes (BGSs) have osteoconductive properties (e.g. provide a physical scaffold for osteointegration of surrounding bone tissue), certain BGSs are osteostimulative in that they actively upregulate mesenchymal stem cell proliferation and stimulate differentiation into osteoblast-like cells. The osteostimulative properties of silicate-substituted calcium phosphate with enhanced porosity (SiCaP EP) were evaluated in vitro with STRO-1+ immunoselected human bone marrow derived mesenchymal stem cells (HBMSCs). Osteostimulative materials (SiCaP) and Bioglass 45S5 (Bioglass) were also assessed as positive controls along with non-silicate substituted hydroxyapatite as a negative control. HBMSCs were also assessed on Thermanox discs cultured in basal and osteogenic media to determine when osteogenic differentiation could be significantly detected with this in vitro cell system. HBMSC viability and necrosis, total DNA content, alkaline phosphatase (ALP) expression, and osteocalcin expression were evaluated after 7, 14, 21, and 28 days. It was demonstrated that SiCaP EP is osteostimulative based on its propensity to support STRO-1+ HBMSC proliferation and ability to promote the differentiation of HBMSCs down the osteoblastic lineage from ALP-expressing, matrix-producing osteoblasts to Osteocalcin-producing pre-osteocytes without the presence of external osteogenic factors. SiCaP EP permitted greater HBMSC attachment as well as ALP and Osteocalcin expression than Bioglass which may be attributed to its microstructure and chemistry.

A study using a canine hydrotherapy treadmill at five different conditions to kinematically assess range of motion of the thoracolumbar spine in dogs.

BACKGROUND: Incline treadmill and underwater treadmill (UWTM) exercises are common canine rehabilitation modalities , which are often used in isolation in dogs recovering from spinal surgery. Early use of an incline during UWTM exercise may have the potential to improve rehabilitation outcomes in dogs, but, it is hypothesised that dorsoventral movement of the spine may be excessive meaning it is unsuitable in some circumstances. OBJECTIVES: The purpose of this study was to identify changes in canine spinal kinematics in dogs when using a dry treadmill at different angles of incline compared to an underwater treadmill using the same inclines. METHODS: Eight dogs were encouraged to walk on a dry, horizontal, underwater treadmill as well as under the same conditions with both a 10% and 20% incline. This was then repeated at a 10% and 20% incline with the addition of water to hock level. Data were collected using reflective anatomical markers placed at the occipital protuberance, T1, T13, L3, L7 and sacral apex, captured by a high-speed camera facing the lateral aspect of the treadmill. Dorsoventral motion of the spine as well as flexion, extension and range of motion (ROM) of T1, T13, L3 and L7 were recorded. RESULTS: We found significant differences in dorsoventral spinal ROM at T1, L3 and L7, but no significant differences in T13 ROM. No significant differences were found in flexion and extension of any of the joints assessed when comparing dry conditions to the use of water (P>0.05). CONCLUSIONS: The lack of significant differences in joint flexion and extension at T1, T13, L3 and L7 indicates the potential safe use of combining underwater treadmill and incline exercise in canine rehabilitation. However, a lack of uniformity in results makes distinguishing any patterns of significance difficult. More research is needed to establish the effects of these exercises in additional planes of motion before a treatment protocol can be established.

Limbs kinematics of dogs exercising at different water levels on the underwater treadmill.

BACKGROUND: With hydrotherapy rising in the United Kingdom, before understanding the effect of hydrotherapy in animals with pathologies, kinematics data for healthy animals is required. OBJECTIVES: To assess how different water levels on an underwater treadmill (UWTM) can affect joint kinematics. METHODS: Zinc oxide markers were placed on bony landmarks on the limbs of 10 healthy dogs, randomly split into five groups. An UWTM was used with water levels to the digits, tarsus, stifle and hip. The maximum flexion, extension and ROM were determined and a repeated measures ANOVA or Friedman's was used to determine significant differences. RESULTS: We have detected various changes in kinematics following exercise at different water levels, in comparison with a dry treadmill, including consistent increases in flexion of the elbow, stifle and tarsal joints, which were observed for all water levels. The carpal joint had increases in flexion all water levels apart from digit level. An increase in shoulder flexion was seen only with water on or above stifle level, while hip kinematics had the fewest changes with only ROM increasing at high water level (hip level). Extension of the limbs joints was not markedly affected, with only a few data being significant. The carpal joint had an overall decrease in extension with water at all levels, and the stifle joint had a decreased extension when water was at stifle height. CONCLUSIONS: Water level can significantly affect joint kinematics, and knowledge of how each water level affects the joints is relevant to design tailored hydrotherapy protocols.

Optimising soft tissue in-growth in vivo in additive layer manufactured osseointegrated transcutaneous implants.

Osseointegrated transcutaneous implants could provide an alternative and improved means of attaching artificial limbs for amputees, however epithelial down growth, inflammation, and infections are common failure modalities associated with their use. To overcome these problems, a tight seal associated with the epidermal and dermal adhesion to the implant is crucial. This could be achieved with specific biomaterials (that mimic the surrounding tissue), or a tissue-specific design to enhance the proliferation and attachment of dermal fibroblasts and keratinocytes. The intraosseous transcutaneous amputation prosthesis is a new device with a pylon and a flange, which is specifically designed for optimising soft tissue attachment. Previously the flange has been fabricated using traditional machining techniques, however, the advent of additive layer manufacturing (ALM) has enabled 3-dimensional porous flanges with specific pore sizes to be used to optimise soft tissue integration and reduce failure of osseointegrated transcutaneous implants. The study aimed to investigate the effect of ALM-manufactured porous flanges on soft tissue ingrowth and attachment in an in vivo ovine model that replicates an osseointegrated percutaneous implant. At 12 and 24 weeks, epithelial downgrowth, dermal attachment and revascularisation into ALM-manufactured flanges with three different pore sizes were compared with machined controls where the pores were made using conventional drilling. The pore sizes of the ALM flanges were 700, 1000 and 1250 µm. We hypothesised that ALM porous flanges would reduce downgrowth, improve soft tissue integration and revascularisation compared with machined controls. The results supported our hypothesis with significantly greater soft tissue integration and revascularisation in ALM porous flanges compared with machined controls.

Histological evaluation of cellular response to a multifilament electrospun suture for tendon repair.

BACKGROUND: Rotator cuff tendon repair in humans is a commonly performed procedure aimed at restoring the tendon-bone interface. Despite significant innovation of surgical techniques and suture anchor implants, only 60% of repairs heal successfully. One strategy to enhance repair is the use of bioactive sutures that provide the native tendon with biophysical cues for healing. We investigated the tissue response to a multifilament electrospun polydioxanone (PDO) suture in a sheep tendon injury model characterised by a natural history of failure of healing. METHODOLOGY AND RESULTS: Eight skeletally mature English Mule sheep underwent repair with electrospun sutures. Monofilament sutures were used as a control. Three months after surgery, all tendon repairs healed, without systemic features of inflammation, signs of tumour or infection at necropsy. A mild local inflammatory reaction was seen. On histology the electrospun sutures were densely infiltrated with predominantly tendon fibroblast-like cells. In comparison, no cellular infiltration was observed in the control suture. Neovascularisation was observed within the electrospun suture, whilst none was seen in the control. Foreign body giant cells were rarely seen with either sutures. CONCLUSION: This study demonstrates that a tissue response can be induced in tendon with a multifilament electrospun suture with no safety concerns.

Evaluation of the Effects of Synovial Multipotent Cells on Deep Digital Flexor Tendon Repair in a Large Animal Model of Intra-Synovial Tendinopathy.

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.

Histopathological and immunohistochemical evaluation of cellular response to a woven and electrospun polydioxanone (PDO) and polycaprolactone (PCL) patch for tendon repair.

We investigated endogenous tissue response to a woven and electrospun polydioxanone (PDO) and polycaprolactone (PCL) patch intended for tendon repair. A sheep tendon injury model characterised by a natural history of consistent failure of healing was chosen to assess the biological potential of woven and aligned electrospun fibres to induce a reparative response. Patches were implanted into 8 female adult English Mule sheep. Significant infiltration of tendon fibroblasts was observed within the electrospun component of the patch but not within the woven component. The cellular infiltrate into the electrospun fibres was accompanied by an extensive network of new blood vessel formation. Tendon fibroblasts were the most abundant scaffold-populating cell type. CD45+, CD4+ and CD14+ cells were also present, with few foreign body giant cells. There were no local or systemic signs of excessive inflammation with normal hematology and serology for inflammatory markers three months after scaffold implantation. In conclusion, we demonstrate that an endogenous healing response can be safely induced in tendon by means of biophysical cues using a woven and electrospun patch.

Treatment of a periarticular tibial fracture in a foal with a hybrid external fixator.

OBJECTIVE: To report repair of a right proximal tibial Salter-Harris type II fracture in a foal with a hybrid external fixator (HEF). STUDY DESIGN: Case report. ANIMALS: A 5-month-old male foal. METHODS: After open surgical reduction, an HEF built with wires, 2 half-rings, 3 half-threaded connecting rods, and Schanz pins was used to stabilize the fracture. RESULTS: Immediately after surgery, the foal had relatively good weight bearing. The HEF was removed at 60 days after radiographic confirmation of healing. CONCLUSIONS: HEF can be used to stabilize a proximal tibial Salter-Harris type II fracture in a 5-month-old foal. CLINICAL RELEVANCE: HEF should be considered as another option for repair of proximal tibial Salter-Harris type II fractures in foals.

A novel ceramic coating for reduced metal ion release in metal-on-metal hip surgery.

An ovine total hip arthroplasty model was developed to evaluate metal ion release, wear, the biological response and adverse tissue reaction to metal-on-metal (MoM) bearing materials. The performance of an advanced superlattice ceramic coating (SLC) was evaluated as a bearing surface and experimental groups divided into; (1) MoM articulating surfaces coated with a SLC coating (SLC-MoM), (2) uncoated MoM surfaces (MoM), and (3) metal on polyethylene (MoP) surfaces. Implants remained in vivo for 13 months and blood chromium (Cr) and cobalt (Co) metal ion levels were measured pre and postoperatively. Synovial tissue was graded using an ALVAL scoring system. When compared with the MoM group, sheep with SLC-MoM implants showed significantly lower levels of chromium and cobalt metal ions within blood over the 13-month period. Evidence of gray tissue staining was observed in the synovium of implants in the MOM group. A significantly lower ALVAL score was measured in the SLC-MoM group (3.88) when compared with MoM components (6.67) (p = 0.010). ALVAL results showed no significant difference when SLC-MOM components were compared to MoP (5.25). This model was able to distinguish wear and the effect of released debris between different bearing combinations and demonstrated the effect of a SLC coating when applied onto the bearing surface. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1760-1771, 2019.

In vitro and in vivo study of commercial calcium phosphate cement HydroSet™.

The commercial calcium phosphate cement, HydroSet™, was investigated in vitro, studying phase formation, compressive strength and setting time, followed by an ovine in vivo study to measure osseointegration, bone apposition and bone-to-graft contact. The X-ray diffraction and 31 P Magic Angle Spinning Nuclear Magnetic Resonance (MAS NMR) results showed the initial formation of octacalcium phosphate and hydroxyapatite at one hour. Over 7 days the octacalcium phosphate transformed to apatite, which was the only crystalline phase of the cement at 28 days. This apatite phase is thought to be a calcium deficient apatite. In the scanning electron microscopy, histological images of 12-week ovine in vivo results showed a high degree of osseointegration, 92.5%. Compressive strength comparisons between in vitro and in vivo measurements showed a dramatic difference between the in vitro measurements (highest 25.4 MPa) and in vivo (95 MPa), attributed to bone ingrowth into the cement in vivo. To the best of our knowledge this is the first time phase evolution of HydroSet™ and the properties studied in vitro complement the in vivo evaluation of the cement in a publication. The significance of the new finding of initial formation of octacalcium phosphate in this cement is discussed. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 21-30, 2018.

Bone marrow mesenchymal stem cells do not enhance intra-synovial tendon healing despite engraftment and homing to niches within the synovium.

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.

In vivo biocompatibility and pacing function study of silver ion-based antimicrobial surface technology applied to cardiac pacemakers.

INTRODUCTION: Evidence suggests that the rate of cardiovascular implantable electronic device (CIED) infections is increasing more rapidly than the rates of CIED implantation and is associated with considerable mortality, morbidity and health economic impact. Antimicrobial surface treatments are being developed for CIEDs to reduce the risk of postimplantation infection within the subcutaneous implant pocket. METHODS AND ANALYSIS: The feasibility of processing cardiac pacemakers with the Agluna antimicrobial silver ion surface technology and in vivo biocompatibility were evaluated. Antimicrobially processed (n=6) and control pacemakers (n=6) were implanted into subcutaneous pockets and connected to a part of the sacrospinalis muscle using an ovine model for 12 weeks. Pacemaker function was monitored preimplantation and postimplantation. RESULTS: Neither local infection nor systemic toxicity were detected in antimicrobial or control devices, and surrounding tissues showed no abnormal pathology or over-reactivity. Semiquantitative scores of membrane formation, cellular orientation and vascularity were applied over five regions of the pacemaker capsule and average scores compared. Results showed no significant difference between antimicrobially processed and control pacemakers. Silver analysis of whole blood at 7 days found that levels were a maximum of 10 parts per billion (ppb) for one sample, more typically ≤2 ppb, compared with <<2 ppb for preimplantation levels, well below reported toxic levels. CONCLUSIONS: There was no evidence of adverse or abnormal pathology in tissue surrounding antimicrobially processed pacemakers, or deleterious effect on basic pacing capabilities and parameters at 12 weeks. This proof of concept study provides evidence of basic biocompatibility and feasibility of applying this silver ion-based antimicrobial surface to a titanium pacemaker surface.