INTRODUCTION

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.

Introduction

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.

Re-rupture rates after rotator cuff repair remain high because of inadequate biological healing at the tendon-bone interface. Single-growth factor therapies to augment healing at the enthesis have so far yielded inconsistent results. An emerging approach is to combine multiple growth factors over a spatiotemporal distribution that mimics normal healing. We propose a novel combination treatment of insulin-like growth factor 1 (IGF-1), transforming growth factor β1 (TGF-β1) and parathyroid hormone (PTH) incorporated into a controlled-release tyraminated poly-vinyl-alcohol hydrogel to improve healing after rotator cuff repair. We aimed to evaluate this growth factor treatment in a rat chronic rotator cuff tear model.

A total of 30 male Sprague-Dawley rats underwent unilateral supraspinatus tenotomy. Delayed rotator cuff repairs were then performed after 3 weeks, to allow tendon degeneration that resembles the human clinical scenario. Animals were randomly assigned to: [1] a control group with repair alone; or [2] a treatment group in which the hydrogel was applied at the repair site. All animals were euthanized 12 weeks after rotator cuff surgery and the explanted shoulders were analyzed for biomechanical strength and histological quality of healing at the repair site.

In the treatment group had significantly higher stress at failure (73% improvement, P=0.003) and Young's modulus (56% improvement, P=0.028) compared to the control group. Histological assessment revealed improved healing with significantly higher overall histological scores (10.1 of 15 vs 6.55 of 15, P=0.032), and lower inflammation and vascularity.

This novel combination growth factor treatment improved the quality of healing and strength of the repaired enthesis in a chronic rotator cuff tear model. Further optimization and tailoring of the growth factors hydrogel is required prior to consideration for clinical use in the treatment of rotator cuff tears. This novel treatment approach holds promise for improving biological healing of this clinically challenging problem.

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 Staphylococcus aureus and Escherichia coli.

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.

Abstract

Method.

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.

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.

The aim was to assess the wear rate of highly Cross Linked (X3) polyethylene with the use of 36mm femoral heads in total hip arthroplasty (THA). We have previously reported our early results and raised some concern regarding the potential excessive femoral head penetration rates. These results give the 2 year wear rates following this initial bedding-in phase.

There were 100 consecutive patients who had a THA with the same femoral and acetabular components using a 36mm femoral head and X3 polyethylene that were assessed prospectively. Validated computer software (Polyware) was used to assess linear and 3 dimensional wear using standardised x-rays. Examinations were performed at 2,12,18 and 24 months.

There were 40 hips that had completed the 2 year x-ray examination (average 2.4 years). The mean 2-dimensional linear wear rate was 0.17 mm/yr and the mean volumetric wear rate was 113.73 mm3/yr. Steady state wear was achieved after the 2 month and before the 1 year examination. The steady state wear rate was 0.001mm/yr. There was no difference in wear rate with the different sized cups used and wear rate was independent of liner thickness.

The early high wear rates reported have now settled into a more expected pattern of steady state wear similar to other results presented in literature with the use of smaller femoral heads. Using a 36 mm femoral head has not adversely increased the wear rates compared to smaller head sizes when used in conjunction with X3 polyethylene in the short term for THA. These results suggest that the wear rate of X3 is not compromised even with thinner liners and raise the possibility of safely using even larger head sizes with this polyethylene.

Replacement of damaged or diseased tissues with permanent metal implants based on stainless steel, cobalt chrome and titanium alloys has been at the forefront of classical biomaterials research and the orthopaedic medical device industry for decades. Biodegradable polymers have also reached the market but often have limited capacity in load bearing orthopaedic applications due to their low stiffness and poor mechanical properties. The development of biodegradable metals based on magnesium (Mg) could be heralded as a major breakthrough in the field of orthopaedic surgery. Degradable implants eliminate the time and cost associated with a secondary surgery to remove hardware, and reduces the period the implant is exposed to instability, fibrous encapsulation, stress shielding and inflammation. The metabolism of Mg and its excretion via the kidneys is a natural physiological process that is well understood, however, controlling the rapid degradation of Mg biomaterials in vivo is a major challenge yet to be resolved for the safe and effective use of Mg in orthopaedic implants.

In this study, we describe a novel manufacturing method for fabricating Mg/Mg alloy implants, as well as the development of an in vitro method for screening Mg/Mg alloy degradation rate by considering both their electrochemical corrosion behaviour and biological characteristics.

A range of Mg alloys with varying amounts of calcium (0.8–28%) and zinc (3–10%) were cast and then machined into Ø4mm and 15mm discs for biocompatibility (HETCAM) and parallel in vitro testing. Alloys were placed in various simulated body fluid (SBF) solutions in vitro (7–28 days) to determine effect of alloy composition on degradation rate. These potentiostatic and potentiodynamic tests were designed to simulate, to varying degrees, the in vivo environment, with the crucial factors (e.g. temperature, pH, serum proteins, CO2 level) controlled to ensure consistency across the test methods. The mechanisms of corrosion on the Mg/Mg alloy microstructure and the effect of protein adsorption all played key roles in dictating the corrosion of alloys in vitro. Specifically the inclusion of physiological levels of serum proteins decreased the corrosion rate up to 600% over more standard SBF solutions described in literature.

This work provides an improved understanding of the effects of corrosion variables on Mg alloys, while making major steps towards deciding the most appropriate screening tests for new alloys for their use as a biomedical material prior to moving to in vivo animal studies.

The early results with highly cross-linked polyethylene have been encouraging and have increased the ability to use larger head diameters to improve the range of motion and decrease the dislocation rate, the commonest cause of early complications following total hip arthroplasty (THA). Wear rates with 32 mls heads have been satisfactory however there have been very few independent studies looking at early polyethylene wear in 36 mm heads. This study assessed the rate of polyethylene wear of a 36mm ceramic femoral head and a highly cross-linked polyethylene (X3 Stryker) liner in THA.

This prospective study reviewed 100 consecutive THAs in young patients (mean age 58 years) who had undergone THA with the same 36mm ceramic femoral head and highly cross linked polyethylene liner. All patients received the same femoral stem (ABG, Stryker) and acetabular cup (Trident, Stryker). Two surgeons performed all procedures. Patients were assessed radiologically immediately postoperatively, at 10 weeks and at one year. Validated computer software (Polyware) was used to assess both volumetric and linear wear.

At one year the mean two-dimensional linear wear rate was 0.51 mm/yr. Mean three-dimensional linear vector wear rate was 0.59 mls per year with a mean volumetric wear rate of 322.6 mms per three years. Cup size ranged from 52–62 mms and the correlation coefficient between cup size and three-dimensional linear wear rate was −0.100. The correlation coefficient between cup size and volumetric wear rate was −0.009 confirming no significant correlation between cup size and wear.

Larger size femoral heads are associated with a higher volumetric wear compared to linear wear rate when using conventional polyethylene. This study demonstrated much higher early linear wear rates compared to other studies using 28 and 32 mms heads. This higher rate may be associated with the creep phenomenon and early bedding-in in the early stages after a THA and although this is of concern these results should be interpreted with caution.

Deterioration in knee joint proprioception has been postulated to occur following injury, resulting in further instability due to disruption of receptors and feedback mechanisms. Surgical reconstruction techniques may also influence post-operative proprioceptive ability (PA). We hypothesised that anterior cruciate ligament (ACL) reconstruction techniques which disrupt the knee capsule would result in a decrease in PA.

Following ethical approval, a total of 48 subjects (mean age: 28.1 ± 10.5, 34 male, 14 female) undergoing ACL reconstruction surgery were included in the study. Fifteen subjects underwent “open” capsule ACL surgery and patellar tendon graft, whereas 33 subjects had “closed” capsule surgery with a hamstring tendon graft. Knee proprioception was measured on a custom-designed test apparatus incorporating electromagnetic position sensors (Polhemus Fastrack) located on femoral and tibial landmarks to accurately track knee angle during flexion-extension (no load). Leg flexion-extension under partial weight-bearing (5kg) was also evaluated. Pre-operative PA was assessed bilaterally, and then again on operated joints at three, six and twelve months post-op. Proprioceptive ability was measured as the cumulative absolute error in knee angle (°) between five repeat measurements and a target angle.

We observed no significant difference in PA between injured and contralateral knees prior to ACL reconstruction. Post-operatively, no significant difference in PA was observed between “open” versus “closed” ACL techniques, irrespective of loading conditions. While trends indicated that PA during knee extension (no load) and leg flexion (partial weight-bearing) improved over the 12 months compared to pre-operative values in closed ACL surgery, these were not significantly different to open ACL results.

The proportion of subjects whose PA improved in at least two out of the three post-op evaluations was also similar (approx 50%) across all groups, irrespective of joint loading. The only difference was PA during leg flexion under partial weight bearing, where 27% of open ACL surgery patients showed improvement in two or more follow-up tests, as opposed to 58% of closed ACL surgery patients.

We present a method to determine pre- and postoperative PA during knee flexion/extension under no load as well as under partial weight-bearing. We saw no significant difference in PA of the knee under no-load versus load. We also saw no significant difference in postoperative PA following open capsule, patellar tendon graft versus closed capsule, hamstring tendon graft ACL reconstruction technique after 1 year follow-up.

Articular cartilage has a limited regenerative capacity. Tissue engineering strategies adopting seeding and differentiation of individual chondrocytes on porous 3D scaffolds of clinically relevant size remains a considerable challenge. A well documented method to produce small samples of differentiated cartilage tissue in vitro is via micro-mass (pellet) culture, whereby, high concentrations of chondrocytes coalesce to form. a spherical tissue pellet. However, pellet culture techniques are not applied clinically as it is only possible to produce small amounts of tissue (1–2mm). The aims of this study were to develop a method for mass-production of pellets, and investigate whether an alternative “pellet seeding” approach using smart 3D scaffold design would allow large numbers of spherical pellets to be fixed in place.

Chondrocytes were isolated from bovine articular cartilage via enzymatic digestion. Freshly isolated and expanded (passage 2) chondrocytes were placed in 96-well plates with round- or v-shaped wells at a range of densities from 0.1, 0.25 and 0.5 million cells per pellet, and centrifuged at 500g for 2 min. In order to assess pellet forming conditions, cells were treated with or without 300 mg/mL fibronectin (FN, Sigma) to improve cell-cell adhesion. Wells were also coated with or without silicone (Sigmacote) to prevent cell adhesion to wells. Pellets were cultured in vitro for up to 14 days and were assessed at various timepoints for size, shape, cell number (DNA assay) and cell differentiation capacity (histology). A robotic Bioplotter device was used to produce porous, biodegradable scaffolds by plotting −250μm polymer (PEGT/PBT) fibres in a layer-by-layer process. Scaffolds with specific 3D pore architecture were produced to allow spherical pellets to be press-fit in each pore thereby fixing them in place throughout the scaffold.

Primary and expanded chondrocytes plated at a density of 0.25 million cell/pellet in v-shaped 96-well plates without both FN and silicone treatment produced pellets with consistently better spherical shape and total cell number (as determined via DNA). Under these conditions, cell (re)differentiation and cartilage extracellular matrix formation was observed via positive staining for safranin-O. Mass production of pellets was achieved by culturing multiple 96-well plates in parallel. FN treatment promoted cell-cell adhesion, but also cell adhesion to well plates, irrespective of silicone treatment, resulting in irregular shaped pellets, as did the use of round-bottom shaped wells.

Smart scaffold design and layer-by-layer fabrication process allowed direct control over the fibre spacing and pore size (1.0–1.25mm). Multiple layers of spherical pellets (1.25–1.5mm) were press-fit in place, thereby limiting the need for direct cell adhesion to the scaffold. Continued culture of constructs containing pellets resulted in consistent tissue formation throughout the scaffold.

In this study, we describe an alternative approach to the design and seeding of scaffolds for cartilage tissue engineering. Current limitations involved with adherence and de-differentiation of single cell populations were avoided by taking advantage of smart 3D scaffold design and pellet-seeding and culture techniques. Further optimisation and automation of the process is necessary, however, such strategies could be beneficial for future scaffold-based cell therapies for repairing articular cartilage defects.

Tissue engineering techniques, combining autologous chondrocytes with biodegradable biomaterials, may offer significant advantages over current articular cartilage repair strategies. We present a series of experiments investigating the effect of 3D scaffold architecture and biomaterial composition on cartilage tissue formation in vitro and in vivo.

Porous polymer (PEGT/PBT) scaffolds with low (300/55/45) or high (1000/70/30) PEG molecular weight (MW) compositions were produced using novel solid free-form fabrication (3DF) techniques, allowing precise control over pore architecture, and conventional compression moulding (CM) foam techniques. Scaffolds were seeded with expanded human nasal chondrocytes, and cultured in vitro or implanted subcutaneously in vivo in nude mice for 4 weeks and cartilage tissue formation accessed.

3DF scaffolds contained highly accessible networks of large interconnecting pores (Ø525 μm) compared to CM scaffolds, containing complex networks of small interconnecting pores (Ø182 μm). 3DF scaffold architectures enhanced cell re-differentiation (GAG/DNA) and cartilaginous matrix accumulation compared to CM scaffolds, but only if 1000/70/30 compositions were used. Collagen type-II mRNA was significantly increased in 3DF architectures irrespective of scaffold composition. These effects were likely mediated by preferential protein adsorption to 1000/70/30 materials, promoting a spherical chondrocyte-like morphology, as well as efficient nutrient/waste exchange throughout interconnecting pores within 3DF architectures.

We observed synergistic effects of both composition and 3D scaffold architecture on human chondrocyte re-differentiation capacity, however, our data suggests that scaffold composition has a more significant influence than architecture alone. Such design criteria could be included in future scaffold architectures for repairing articular cartilage defects.