Title

Longitudinal Intravital Imaging to Quantify the “Race for the Surface” Between Host Immune Cell and Bacteria for Orthopaedic Implants with S. aureus Colonization in a Murine Model

Abstract

Serial section electron microscopy (SSEM) was initially developed to map the neural connections in the brain. SSEM eventually led to the term ‘Connectomics’ to be coined to describe process of following a cell or structure through a volume of tissue. This permits the true three-dimensionality to be appreciated and relationships between cells and structures. The purpose of this study was to utilize this methodology to interrogate S. aureus infected bone.

Bone samples were harvested from mice tibia infected with S. aureus and were fixed, decalcified, and osmicated. The samples were paraffin embedded and 5-micron sections were cut to identify regions of bacterial invasion into the osteocyte-lacuna-canalicular-network (OLCN). This area was cut from the paraffin block, deparaffinized, post-fixed and reprocessed into epoxy resin. Serial sections were cut at 60nm and collected onto Kapton tape utilizing the Automated Tape-collecting Ultramicrotome (ATUMtome) system. Samples were mounted onto 4” silicon wafers and post-stained with 2% uranyl acetate followed by 0.3% lead citrate and carbon coated. A ZEISS GeminiSEM 450 scanning electron microscope fitted with an electron backscatter diffusion detector was used to image the sections. The image stack was aligned and segmented using the open-source software, VASTlite.

264 serial sections were imaged, representing approximately 40 × 45 × 15-micron (x, y, z) volume of tissue. 70% of the canaliculi demonstrated infiltration by S. aureus.

This study demonstrates that SSEM can be applied to the skeletal system and provide a new solution to investigate the OLCN system. It is feasible that this methodology could be implemented to investigate why some canaliculi are resistant to colonization and potentially opens up a new direction for the prevention of chronic osteomyelitis. In order to make this a realistic target, automated segmentation methodologies utilizing machine learning must be developed and applied to the bone tissue datasets.

INTRODUCTION

Staphylococci species account for ∼80 % of osteomyelitis cases. While the most severe infections are caused by Staphylococcus aureus (S. aureus), the clinical significance of coagulase negative Staphylococcus epidermidis (S. epidermidis) infections remain controversial. In general, S. epidermidis was known to be a protective commensal bacterium. However, recent studies have shown that intra-operative low-grade S. epidermidis contamination prevents bone healing. Thus, the purpose of this study is to compare the pathogenic features of S. aureus and S. epidermidis in an established murine model of implant-associated osteomyelitis.

Background

Glenoid component aseptic loosening is the most common source of total shoulder arthroplasty (TSA) revision. In an attempt to strengthen cemented glenoid component fixation, divergent pegged glenoids were designed. Divergent peg creation was intended to increase cement purchase and provide resistance to component rocking.

Controlling postoperative pain and nausea after total joint arthroplasty remains an important challenge. We conducted a prospective, randomized controlled trial with 120 patients to determine if the addition of perioperative dexamethasone to a multimodal regimen improves antiemetic and analgesic control, enhances mobility, and shortens hospital length of stay after total hip and knee arthroplasty. Patients administered 10 mg of intravenous dexamethasone intraoperatively consumed less daily rescue anti-emetic and analgesic medication, reported superior VAS nausea and pain scores, ambulated further distances, and had a significantly shorter length of stay compared to the control group (p < 0.05). A second, 24-hour postoperative dose of 10 mg intravenous dexamethasone provided significant additional pain and nausea control and further reduced length of stay (p < 0.05). No adverse events were detected with the administration of the intraoperative and/or postoperative dexamethasone.

The Royal National Orthopaedic Hospital has completed an extensive trial of ACI versus MACI in the treatment of symptomatic osteochondral defects of the knee. A new technique has now been proposed which is quicker and easier to perform. This is the Gel-Type Autologous Chondrocyte Transplantation, CHONDRONTM. At Stanmore CHONDRON has been used for the past 17 months. Our aim was to assess the short term functional outcome of patients who have undergone CHONDRONTM using validated outcome scoring questionnaires. We retrospectively reviewed the notes of 43 patients that had undergone CHONDRONTM over one year ago and scored them using the Modified Cincinnati Score, the Visual Analogue Score and the Benltey Stanmore Functional Rating Score.

Orthopaedic surgeons are astounded with the strength of bone found in Polynesians. Furthermore the rate at which new Polynesian bone over-grows metal fixation of a recent fracture is impressive. Studies demonstrate that Polynesians have a higher Bone Mineral Density (BMD) than age and weight matched Europeans in NZ (1, 2). In addition, Polynesians have a lower incidence of hip fractures when compared to other ethnic groups (3). This suggests that the higher BMD or other inherent differences must account for the lower incidence of hip fractures in Polynesians.

The aim of this study was to identify (if any) a difference in osteoblast mitosis between European and Polynesian bone. Samples were collected from 13 patients that had joint replacements in accordance with the MCNZ ethics approval. The bone is processed and osteoblasts cultured in the lab to 50% confluence. The cells are then tagged with Propidium Iodide. Using Fluorescence-Activated Cell Sorting (or FACS) the number of osteoblasts in the different phases of the cell cycle are counted. The percentage of cells in G0/G1, S and G2/M phase can be determined by entering the FACS data into a program called mod-fit. This study shows that Polynesians have a greater proportion of cells undergoing replication (i.e S-phase) than their European counterparts.

Incidentally we have also shown that the proportion of cells undergoing mitosis lowers with age irrespective of ethnicity.

Chondral injuries of the knee are extremely common and present a unique therapeutic challenge due to the poor intrinsic healing of articular cartilage. These injuries can lead to significant functional impairment. There are several treatment modalities for articular osteochondral defects, one of which is autologous chondrocyte implantation. Our study evaluates the mid to long term functional outcomes in a cohort of 828 patients who have undergone an autologous chondrocyte implantation procedure (either ACI or MACI), identifying retrospectively factors that may influence their outcome.

The influence of factors including age, sex, presence of osteoarthritis and size and site of lesion have been assessed individually and with multivariate analysis. All patients were assessed using the Bentley Functional Score, Visual Analogue Score and the Cincinnati Functional Score. Assessment were performed pre-operatively and of their status in 2010.

The longest follow-up was 12 years (range 24 to 153 months) with a mean age of 34 years at time of procedure. The mean defect size was 409 mm² (range 64 to 2075 mm²). The distribution of lesions was 51% Medial Femoral Condyle, 12.5% Lateral Femoral Condyle, 18% Patella (single facet), 5% Patella (Multifacet) and 6% Trochlea. 4% had cartilage transplant to multiple sites.

High failure rates were noted in those with previous cartilage regenerative procedures or evidence of early osteoarthritis and those with transplantation to multiple sites.

Autologous chondrocyte implantation is an effective method of decreasing pain and increasing function, however patient selection plays clear role in the success of such procedure.

Chondral injuries of the knee are extremely common and present a unique therapeutic challenge due to the poor intrinsic healing of articular cartilage. These injuries can lead to significant functional impairment. There are several treatment modalities for articular osteochondral defects, one of which is autologous chondrocyte implantation. Our study evaluates the mid to long term functional outcomes in a cohort of 828 patients who have undergone an autologous chondrocyte implantation procedure (either ACI or MACI), identifying retrospectively factors that may influence their outcome.

The influence of factors including age, sex, presence of osteoarthritis and size and site of lesion have been assessed individually and with multivariate analysis. All patients were assessed using the Bentley Functional Score, Visual Analogue Score and the Cincinnati Functional Score. Assessment were performed pre-operatively and of their status in 2010. The majority of patients had several interim scores performed at varying intervals.

The longest follow-up was 12 years (range 24 to 153 months) with a mean age of 34 years at time of procedure. The mean defect size was 486 mm2 (range 64 to 2075 mm2). The distribution of lesions was 51% Medial Femoral Condyle, 12.5% Lateral Femoral Condyle, 18% Patella (single facet), 5% Patella (Multifacet) and 6% Trochlea. 4% had cartilage transplant to multiple sites. 30% failed following this procedure at a mean time of 72 months. 52% patients stated a marked improvement in their functional outcomes within the first two years. 49% stated an excellent result following their procedure.

High failure rate was noted in those with previous cartilage regenerative procedures, transplants occurring on the patella, particularly if involving multifacets. Multiple site cartilage transplantation was also associated with a high failure rate.

Autologous chondrocyte implantation is an effective method of decreasing pain and increasing function, however patient selection plays clear role in the success of such procedure.

The aim of this study was to determine whether the clinical outcome of autologous chondrocyte transplantation was dependent on the timing of a high tibial osteotomy in tibio-femoral mal-aligned knees. Between 2000 and 2005, forty-eight patients underwent autologous chondrocyte implantation with HTO performed at varying times relative to the second stage autologous chondrocyte implantation procedure. 24 patients had HTO performed simultaneously with their second stage cartilage transplantation, (the HTO Simultaneous Group). 5 patients had HTO prior to their cartilage procedure, (the HTO pre-ACI Group) and 19 had HTO performed between 1 to 4 years after their second stage cartilage implantation, (the HTO post-ACI Group). There were 29 men and 19 women with a mean age of 37 years (Range 28 to 50) at the time of their second stage procedure.

With average follow-up of 72 months we have demonstrated a significant functional benefit in performing the HTO either prior to or simultaneously with the ACI procedure in the mal-aligned knee. The failure rate in the Post-ACI group was 45% compared to the Pre-ACI and Simultaneous group, with failure rates of 20% and 25%, respectively.

An HTO performed prior to or simultaneously with an autologous chondrocyte implantation procedure in the mal-aligned knee, provides a significant protective effect by reducing the failure rate by approximately 50%.

Introduction

Autologous chondrocyte implantation (ACI) is contra-indicated in a joint rendered unstable by a ruptured anterior cruciate ligament (ACL). We present our experience of ACI repair with ACL reconstruction

Articular cartilage implantation (ACI) and associated procedures (MACI = Matrix-assisted cartilage implantation) are now established treatments for osteochondral defects in the knee. The quality of repair in terms of histological appearance is frequently not known, whilst the correlation of histology results with functional outcomes remains undefined. Histological data of the quality of the repair tissue is sparse and a precise classification proved difficult.

This was a single-centre, prospective study. Over 12 years (1998-2010) 406 patients that underwent articular cartilage implantation procedures at our institution (ACI = 170, MACI = 205) had biopsies taken at the 1-2 year interval, in order to assess whether these contained ‘hyaline-like’ cartilage, ‘mixed hyaline-like with fibrocartilage’, fibrocartilage or fibrous tissue alone.

Histological sections of the biopsies were prepared and stained with haematoxylin, eosin and proteoglycan stains and viewed under polarised light. All biopsies were studied by a single histopathologist in a specialist, dedicated musculoskeletal laboratory.

All patients were assessed by the Cincinnati, Bentley and Visual Analogue scores both pre-operatively and at the time of the review.

The findings revealed that 56 patients healed with ‘hyaline-like’ cartilage (14.9%), 103 with ‘mixed’ (27.5%), 179 with fibrocartilage (47.7%) and 37 with fibrous tissue (9.9%).

These findings showed that 42.4% of defects were filled with ‘hyaline-like’ or ‘mixed’ cartilage, with 70% of these achieving a ‘fair’ to ‘excellent’ functional outcome. This was also observed in the fibrocartilage group, where 72% achieved similar results. Predictably 89% of the patients that healed by fibrous tissue had a poor functional outcome.

This study shows that 71% of patients whose osteochondral defects healed by either ‘hyaline-like’, ‘mixed’ or fibrocartilage experienced an improvement in the function. In contrast, only 11% of the patients whose defects filled with fibrous tissue, showed some functional improvement. Additionally, this data indicates the advantage of biopsies in assessing the overall results of cartilage implantation procedures.

Patients with osteochondrodysplasia frequently require Total Hip Arthroplasty at a younger age, as a result of early degenerative disease of the hip joint. The outcome of Modular Total Hip Arthroplasty in this group of patients has been reported previously. In this retrospective study we evaluated the outcome of custom made (CADCAM) Total Hip Arthroplasty in patients with osteochondrodysplasia.

Between 1974 and 2009, twenty one CADCAM Total Hip Arthroplasty procedures were performed in fourteen patients in our institution. There were eight female and six male patients, with the mean age at time of surgery of 40.95 years (20 to 78). The patients were followed up clinically and with the Harris hip score for a mean of 7.12 years (0.5 to 17 years).

Four of the twenty-one hips (23.8%) required revision surgery at a mean of 11.54 years (6.5 to 17 years); one required it for aseptic loosening of the femoral component; one required complete revision of the acetabular component; one required exchange of acetabular liner; and one was for symptomatic non-union of a lesser trochanter avulsion.

This study shows encouraging clinical outcomes of custom made (CADCAM) Total Hip Arthroplasty in patients with osteochondrodysplasia,

Purpose

The rate of arthroplasty or osteotomy in patients who had undergone autologous chondrocyte implantation (ACI) for osteochondral defects in the knee was determined. Furthermore, we investigated whether any radiographic evidence of osteoarthritis (OA) prior to ACI was associated with poorer outcome following surgery.

Introduction

We report the initial 2 and 3 year follow-up results of this randomised controlled trial of autologous chondrocyte implantation (ACI) using porcine-derived collagen membrane as a cover (ACI-C) versus matrix-carried autologous chondrocyte implantation (MACI) for the treatment of osteochondral defects of the knee.

Aims

To investigate (1) The relationship between macroscopic grading and durability of cartilage repair following collagen-covered autologous chondrocyte implantation (ACI-C) in the knee; (2) The influence of histology on durability of cartilage repair; (3) The relationship between macroscopic appearance and histology of repair tissue.

The results for autologous chondrocyte implantation (ACI) in the treatment of osteochondral defects in the knee are encouraging. At present, two techniques have been described to retain the chondrocyte suspension within the defect. The first involves using a periosteal flap harvested from the distal femur and the second involves using a type I/III collagen membrane. To the authors' knowledge there are no comparative studies of these two techniques in the current literature.

A total of 68 patients with a mean age of 30.52 years (range 15 to 52 years) with symptomatic articular cartilage defects were randomised to have either ACI with a periosteal cover (33 patients) or ACI with a type I/III collagen cover (35 patients). The mean defect size was 4.54 cm² (range 1 to 12 cm²). All patients were followed up at 24 months.

A functional assessment using the Modified Cincinnati score showed that 74% of patients had a good or excellent result following the ACI with collagen cover compared with 67% after the ACI with periosteum cover at 2 years (p>0.05). Arthroscopy at 1 year also demonstrated similar results for both techniques. However, 36.4% of the periosteum covered grafts required shaving for hypertrophy compared with 1 patient for the collagen covered technique.

This prospective, randomised study has shown no statistical difference between the clinical outcome of ACI with a periosteal cover versus ACI with a collagen cover at 2 years. A significant number of patients who had the ACI with periosteum technique required shaving of a hypertrophied graft within the first year of surgery. We conclude that there is no advantage in using periosteum as a cover for retaining the chondrocytes within an osteochondral defect; as a result we advocate the use of an alternative cover such as a porcine-derived, type I/III collagen membrane.

Purpose

We report on minimum 2 year follow-up results of 71 patients randomised to autologous chondrocyte implantation (ACI) using porcine-derived collagen membrane as a cover (ACI-C) and matrix-induced autologous chondrocyte implantation (MACI) for the treatment of osteochondral defects of the knee.

Autologous chondrocyte implantation (ACII) has been shown to have favourable results in the treatment of symptomatic chondral and osteochondral lesions. However, there are few reports on the outcomes of this technique in adolescents.

The aim was to assess functional outcome and pain relief in adolescents undergoing autologous chondrocyte implantation (ACI).

Thirty-one adolescent patients undergoing ACI or Matrix-assisted chondrocyte implantation (MACI) were identified from a larger prospective study. Mean age was 16.3 years (range 14 – 18) with a mean follow-up of 66.3 months (12–126 months).

There were 22 males and nine females. All patients were symptomatic; 30 had isolated lesions and one had multiple lesions. Patients were assessed pre and postoperatively using the Visual Analogue Score (VAS), the Stanmore/Bentley Functional Rating Score and the Modified Cincinnati Rating System.

The mean VAS improved from 5.8 pre-operatively to 2 post-operatively. The Stanmore/Bentley Functional Rating Score improved from 2.9 to 0.9 whilst the Modified Cincinnati Rating System improved from 49.8 pre-operatively to 81.3 postoperatively with 87% of patients achieving excellent or good results. All postoperative scores exhibited statistically significant improvement from pre-operative scores.

The results show that, in this particular group of patients, this procedure produces reduction in pain and a statistically significant improvement in function postoperatively. We strongly recommend this procedure in the management of adolescents with symptomatic chondral defects.