Autologous fat grafting has favourable potential as a regenerative strategy and is the current gold-standard to repair large contour defects, as needed in breast reconstruction after mastectomy and traumatic soft tissue reconstruction. Clinically, there is a limit on the volume of lipoaspirate which can be utilised to repair a soft-tissue defect. Surgical complications are the result of poor structural fidelity of lipoaspirate and graft resorption as a filling material and are hindered further by poor graft vascularisation. This study aims to develop injectable lipoaspirate-derived adipose tissue grafts with enhanced biologically and clinically-admissible structural and functional properties adopting light photocrosslinking of unmodified lipoaspirate. Patient-derived lipoaspirate was harvested and crosslinked using novel photoinitiator and exposure to visible light (wavelength 450nm) in surgery, establishing bonds between extracellular matrix (ECM) proteins within the material. The degree of crosslinking was tuned (photoinitiator concentration, light exposure, light intensity) and covalent bond formation measured using mass spectrometry. To predict patient response, SWATH-MS was used to identify differences in patient ECM and crosslinked grafts were implanted in vivo using a subcutaneous mouse model. Functional vessel formation and resorption were quantified using micro-CT and tissue-remodelling was assessed via histology.Introduction
Methods
Stimulation of angiogenesis via the delivery of growth factors (GFs) like vascular endothelial growth factor (VEGF) is a promising strategy for the treatment of avascular necrosis (AVN). Tyraminated poly-vinyl-alcohol hydrogels (PVA-Tyr), which have the ability to covalently incorporate GFs, were proposed as a platform for the controlled delivery of therapeutic levels VEGF to the necrotic areas[1]. Nevertheless, PVA hydrophilicity and bioinertness limits its integration with the host tissues. The aim of this study was to investigated the effectiveness of incorporating gelatin, an FDA-approved, non-immunogeneic biomaterial with biological recognition sites, as a strategy to facilitate blood vessels invasion of PVA-Tyr hydrogels and to restore the vascular supply to necrotic tissues. Progressively higher gelatin concentrations (0.01–5wt%) were incorporated in the PVA-Tyr network. Hydrogel physico-chemical properties and endothelial cell attachment were evaluated. Afterwards, the capability of the released VEGF and gelatin to promote vascularization was evaluated via chorioallantoic membrane (CAM) assay. VEGF-loaded PVA-Tyr hydrogels with or without gelatin (n=7) were implanted in a subcutaneous mouse model for 3 weeks. Vascularization (CD31+ cells) and cell infiltration (H&E) were evaluated. Finally, AVN was induced in 6 weeks old male piglets as previously described [2]. A transphyseal hole (3mm) was drilled and PVA-Tyr hydrogels with 1% gelatin were delivered in the defects. Piglets were euthanized after 4 weeks and microCT analysis was performed.INTRODUCTION
METHODS
Introduction: The mechanobiology and response of bone formation to strain under physiological loading is well established, however investigation into exceedingly soft scaffolds relative to cancellous bone is limited. In this study we designed and 3D printed mechanically-optimised low-stiffness implants, targeting specific strain ranges inducing bone formation and assessed their biological performance in a pre-clinical in vivo load-bearing tibial tuberosity advancement (TTA) model. The TTA model provides an attractive pre-clinical framework to investigate implant osseointegration within an uneven loading environment due to the dominating patellar tendon force. A knee finite element model from ovine CT data was developed to determine physiological target strains from simulated TTA surgery. We 3D printed low-stiffness Ti wedge osteotomy implants with homogeneous stiffness of 0.8 GPa (Ti1), 0.6 GPa (Ti2) and a locally-optimised design with a 0.3 GPa cortex and soft 0.1 GPa core (Ti3), for implantation in a 12-week ovine tibial advancement osteotomy (9mm). We quantitatively assessed bone fusion, bone area, mineral apposition rate and bone formation rate. Optimised Ti3 implants exhibited evenly high strains throughout, despite uneven wedge osteotomy loading. We demonstrated that higher strains above 3.75%, led to greater bone formation. Histomorphometry showed uniform bone ingrowthin optimised Ti3 compared to homogeneous designs (Ti1 and Ti2), and greater bone-implant contact. The greatest bone formation scores were seen in Ti3, followed by Ti2 and Ti1. Results from our study indicate lower stiffness and higher strain ranges than normally achieved in Ti scaffolds stimulate early bone formation. By accounting for loading environments through rational design, implants can be optimised to improve uniform osseointegration. Design and 3D printing of exceedingly soft titanium orthopaedic implants enhance strain induced bone formation and have significant importance in future implant design for knee, hip arthroplasty and treatment of large load-bearing bone defects.
Polyetheretherketone (PEEK) interbody fusion cages combined with autologous bone graft is the current clinical gold standard treatment for spinal fusion, however, bone graft harvest increases surgical time, risk of infection and chronic pain. We describe novel low-stiffness 3D Printed titanium interbody cages without autologous bone graft and assessed their biological performance in a pre-clinical in vivo interbody fusion model in comparison to the gold standard, PEEK with graft. Titanium interbody spacers were 3D Printed with a microporous (Ti1: <1000μm) and macroporous (Ti2: >1000μm) design. Both Ti1 and Ti2 had an identical elastic modulus (stiffness), and were similar to the elastic modulus of PEEK. Interbody fusion was performed on L2-L3 and L4-L5 vertebral levels in 24 skeletally mature sheep using Ti1 or Ti2 spacers, or a PEEK spacer filled with iliac crest autograft, and assessed at 8 and 16 weeks. We quantitatively assessed bone fusion, bone area, mineral apposition rate and bone formation rate. Functional spinal units were biomechanically tested to analyse range of motion, neutral zone, and stiffness. Results: Bone formation in macroporous Ti2 was significantly greater than microporous Ti1 treatments (p=.006). Fusion scores for Ti2 and PEEK demonstrated greater rates of bone formation from 8 to 16 weeks, with bridging rates of 100% for Ti2 at 16 weeks compared to just 88% for PEEK and 50% for Ti1. Biomechanical outcomes significantly improved at 16 versus 8 weeks, with no significant differences between Ti and PEEK with graft. This study demonstrated that macroporous 3D Printed Ti spacers are able to achieve fixation and arthrodesis with complete bone fusion by 16 weeks without the need for bone graft. These significant data indicate that low-modulus 3D Printed titanium interbody cages have similar performance to autograft-filled PEEK, and could be reliably used in spinal fusion avoiding the complications of bone graft harvesting.
Hypochlorous acid (HOCl) is a potent anti-bacterial agent which could reduce periprosthetic joint infection. Early infection complications in joint replacements are often considered to be due to local contamination at the time of surgery and result in a significant socioeconomic cost. Current theatre cleaning procedures produce “clean” operating theatres which still contain bacteria (colony forming units, CFU). Reducing this bacterial load may reduce local contamination at the time of surgery. HOCl is produced naturally in the human neutrophil and has been implicated as the primary agent involved in bacterial killing during this process. In vitro research confirms its efficacy against essentially all clinically relevant bacteria. The recent advent of commercial production of HOCl, delivered as a fog, has resulted in extensive use in the food industry. Reported lack of corrosion and high anti-bacterial potency are seen as two key factors for the use of HOCl in the orthopaedic environment. Prior work by the authors comparing human cell toxicity of HOCl, chlorhexidine and iodine solutions shows favourable results. This study evaluates use of neutral HOCl applied as a dry room fog to decrease bacteria in the operating theatre environment. Using an animal operating theatre as the test site, bacterial swabs were taken from ten 100cm2 sample areas before standard cleaning with detergent, after standard cleaning, and again after 60 minutes exposure to HOCl fog. After standard cleaning, 6 of 10 sample sites recorded significant bacterial growth (>10 CFU/100cm2). After exposure to HOCl fog, growth in all 10 sites was below detection limits (<10 CFU/100cm2). This was repeated with specific exposure to We can conclude that HOCl is effective when used as a fogging agent to reduce bacterial loading within an operating theatre environment and as such has significant potential to reduce intraoperative contamination and periprosthetic infection.
We prospectively investigated the radiological outcomes of the uncemented Oxford medial compartment arthroplasty in 231 consecutive patients performed in a single centre with a minimum two year follow up. The functional outcome using the Oxford knee score and the High Activity Arthroplasty Score were significantly improved on the pre-operative scores (p<0.001). There were 196 patients with a radiological examination that was acceptable for analysis of the bone-implant interface. Of the six tibial zones examined on the anteroposterior radiograph there were only three with a partial radiolucent line (3 out of 1176 zones). All were present in the medial aspect of the tibial base plate (Zone 1) and all were <1 mm in size. All of these patients were asymptomatic. There were no radiolucent lines seen around the femoral component or on the lateral tibial view. There was one revision for mal-seating of the tibial component.Method.
Results.
Articular cartilage has a limited regeneration capacity, and damage of cartilage often results in the onset of degenerative disease such as osteoarthritis (OA). MRI and CT imaging of cartilage and subchondral bone are becoming increasingly important in early detection and treatment of OA as well as for quantifying quality of tissue-engineered samples. Non-invasive CT scanners have been used to image cartilage tissue with the help of contrast agents. However, since only one energy source is available, imaging information of multiple soft and hard tissues is lost given that the overall x-ray attenuation is measured. Medipix All Resolution System (MARS) CT offers the possibility of applying more than one energy source. It is able to measure the energy of each photon individually and therefore determines the characteristics of attenuation. In this study, an ionic contrast agent (Hexabrix) was used to image the negatively charged extra-cellular matrix component, glycosaminoglycan (GAG), which is abundantly found in the middle and lower layers of healthy cartilage tissue. GAG distribution in the cartilage tissue could be imaged using an inverse relationship with Hexabrix signal (i.e. high signal represents low GAG content). Eight bovine cartilage-bone explants (3mm × 5mm) were incubated in 4 different Hexabrix concentrations ranging from 20% to 50% in PBS. Sections were imaged using the MARS scanner at high and low energies (13.32 keV and 30.84 keV). Images were pre-processed, reconstructed and colour-coded using different enhancement techniques and virtual experimental software. Histological (Safranin-O) staining and quantitative biochemical analysis of GAG content (DMMB dye assay) was performed to correlate GAG distribution and content with MARS-CT images. High resolution images of both cartilage and bone regions were obtained, with contrast enhanced CT of cartilage correlating well with histological staining. X-ray attenuation was high in regions poor in GAG content, whereas attenuation was low in GAG rich regions. Furthermore, there was a direct inverse correlation between Hexabrix signal and GAG content as measured in superficial (2.9 μg/mg) and middle/deep regions (10.6 μg/mg) in cartilage explants. It can be concluded that the MARS technique can be used to image GAG distribution and GAG content, and therefore could be used clinically to assess quality of healthy or osteoarthritic cartilage, as well as non-destructive imaging of GAG content in engineered tissues.
Cell-scaffold based cartilage tissue engineering strategies provide the potential to restore long-term function to damaged articular cartilage. A major hurdle in such strategies is the adequate (uniform and sufficient) population of porous 3D scaffolds with cells, but more importantly, the generation of engineered tissue of sufficient quality of clinically relevant size. We describe a novel approach to engineer cartilage grafts using pre-differentiated micro-mass cartilage pellets, integrated into specifically designed 3D plotted scaffolds. Expanded (P2) human nasal chondrocytes (HNCs) or bone marrow-derived mesenchymal stem cells (MSCs) from 3 donors (age 47–62 years) were micro-mass cell pellet cultivated at 5 × 105 cells/pellet for 4 days. Subsequently, pellets were integrated into degradable 3D Printed polymer (PEGT/PBT) scaffolds with 1mm fibre spacing. Constructs were cultured dynamically in spinner flasks for a total of 21 days. As a pellet-free control, expanded HNCs were spinner flask seeded into PEGT/PBT fibre plotted scaffolds. Constructs were assessed via histology (Safranin-O staining), biochemistry (glycosaminoglycan (GAG) and DNA content) and collagen type I and II mRNA expression. After 4 days, micro-mass cultured pellets could be successfully integrated into the fibre plotted scaffolds. DNA content of pellet integrated constructs was 4.0-fold±1.3 higher compared to single seeded constructs. At day 21, cartilage tissue was uniformly distributed throughout pellet integrated scaffolds, with minimal cell necrosis observed within the core. GAG/DNA and collagen type II mRNA expression were significantly higher (2.5-fold±0.5 and 3.1-fold±0.4 respectively) in pellet versus single cell seeded constructs. Furthermore, compared to single cell, the pellet seeded constructs contained significantly more total GAG and DNA (1.6-fold±0.1 and 3.1-fold±1.0 respectively). We developed a novel 3D tissue assembly approach for cartilage tissue engineering which significantly improved the seeding efficiency (∼100%), as well as tissue uniformity and integrity compared to more traditional seeding approaches using single cell suspensions. Furthermore, the integration of micro-mass cell pellets into 3D plotted PEGT/PBT scaffolds significantly improved the amount and quality of tissue engineered cartilage.
