Abstract

There is currently no standardised complication grading classification routinely used for paediatric orthopaedic surgical procedures. The Clavien-Dindo classification used in general surgery was modified and validated in 2011 by Sink et al. and has been used regularly to classify complications following hip preservation surgery. The aim of this study was to adapt and validate Sink et al.'s modification of the Clavien-Dindo classification system for grading complications following surgical interventions of the upper and lower extremities and spine in paediatric orthopaedic patients.

Sink et al.'s modification of the Clavien-Dindo classification system was further modified for paediatric orthopaedic procedures. The modified grading scheme was based on the treatment required to treat the complication and the long term morbidity of the complication. Grade I complications do not require deviation from standard treatment. Grade II complications deviate from the normal post-operative course and require outpatient treatment. Grade III complications require investigations, re-admission or re-operation. Grade IV complications are limb or life threatening or have a potential for permanent disability (IVa: with no long term disability and IVb: with long-term disability). Grade V complications result in death. Forty-five complication scenarios were developed. Seven paediatric orthopaedic surgeons were trained to use the modified system and they each graded the scenarios on two occasions. The scenarios were presented in a different random order each time they were graded. Fleiss' and Cohen's k statistics were performed to test for inter-rater and intra-rater reliabilities, respectively.

The overall Fleiss' k value for inter-rater reliability was 0.772 (95% CI, 0.744–0.799). The weighted k was 0.765 (95% CI, 0.703–0.826) for Grade I, 0.692 (95% CI, 0.630–0.753) for Grade II, 0.733 (95% CI, 0.671–0.795) for Grade III, 0.657(95% CI, 0.595–0.719) for Grade IVa, 0.769 (95% CI, 0.707–0.83) for Grade IVb and 1.000 for Grade V (p value <0.001). The Cohen's k value for intra-rater reliability was 0.918 (95% CI, 0.887–0.947). These tests show that the adapted classification system has high inter- and intra-rater reliabilities for grading complications following paediatric orthopaedic surgery.

Given the high intra- and inter-rater reliability and simplicity of this system, adoption of this grading scheme as a standard of reporting complications in paediatric orthopaedic surgery could be considered. Since the evaluation of surgical outcomes should include the ability to reliably grade surgical complications, this reproducible, reliable system to assess paediatric surgical complications will be a valuable tool for improving surgical practices and patient outcomes.

Gentamicin sulphate is a potent antibiotic, widely used by clinicians to treat Staphylococcus aureus bacterial complications in orthopaedic surgery and osteomyelitis. Antibiotics as administered are poorly localised and can accumulate with toxic effects. Achieving a better targeted release and controlled dosage has been an ongoing unmet microengineering challenge.

In this study we evaluated the antibiotic release potential of beta tricalcium phosphate (β-TCP) micro and macrospheres to eradicate Staphylococcus aureus and maintain osteoblast biocompatibility. Gentamicin was absorbed onto and within the spheres at an average amount of 4.2 mg per sample. Human osteoblast cell studies at five days incubation showed attachment and growth on the spheres surface with no detrimental effect on the cell viability. A time delayed antibacterial efficacy test was designed with the bacteria introduced at predetermined time intervals from 0–60 minutes.

We demonstrated that hydroxyapatite covered Foraminifera nano-, micro- macrospheres facilitated the slow release of the encapsulated pharmaceutical agent. Principally, this arises owing to their unique architecture of pores, struts and channels, which amplifies physiological degradation and calcium phosphate dissolution to release attached pharmaceuticals in a controlled manner. The Staphylococcus aureus growth response following exposure to the gentamicin incorporated microspheres at various time intervals showed the complete elimination of the bacteria within 30 minutes. Gentamicin release continued with no re-emergence of bacteria.

β-TCP nano to macro size spheres show promise as potential bone void filler particles with, in this case, supplementary delivery of antibiotic agent. Owing to their unique structure, excellent drug retention and slow release properties, they could be used in reconstructive orthopaedics to treat osteomyelitis caused by Staphylococcus aureus and possibly other sensitive organisms.

Objective

There is mounting evidence to suggest a vascular insult is responsible for Perthes' disease, and it is suggested that this may have long-term implications for the vascular health of affected individuals. This study sought to use ultrasound measures to investigate vascular structure and function in children affected by Perthes' disease.

Cartilage and bone degeneration are major healthcare problems affecting millions of individuals worldwide. Elucidation of the processes modulating the cell-matrix interactions involved in cartilage or bone formation offer tremendous potential in the development of clinically relevant strategies for cartilage and bone regeneration. We have therefore adopted an ex vivo tissue engineering approach to investigate chondrogenesis and osteogenesis using a mix human mesenchymal progenitor populations encapsulated in biomineralised polysac-charide templates with or without the addition of type-I collagen.

Alginate/chitosan polysaccharide capsules containing 2.5mg/ml type-I collagen and TGF-beta-3 were encapsulated with human bone marrow cells (HBMC), articular chondrocytes or a co-culture at a ratio of 2:1 respectively and placed in a rotating (Synthecon) biore-actor or held in static 2D culture conditions for 28 days, to determine whether the presence of type-I collagen within the alginate could promote the synthesis of an extracellular matrix.

Constructs were stained with alcian blue, sirius red and von Kossa. In bioreactor samples encapsulated with HBMC and type-I collagen, viable cells were present within lacunae, surrounded by a matrix of proteo-glycans and fibrous collagen, which was mineralized. Immunohistochemistry and polarised light microscopy indicated an organised collagenous matrix with extensive expression of type I collagen and bone sialoprotein with small regions of type II collagen. Type X collagen was also expressed indicating the presence of hypertrophic chondrocytes. Within the static HBMC groups, smaller areas of matrix were generated with decreased expression of type-I and type-II collagen. Co-culture bioreactor samples also demonstrated regions of new mineralised bone matrix; however these were less prominent than in the HBMC only groups. No matrix formation was observed in chondrocyte cultures although the cells remained viable as assessed by live/dead staining. Biochemical analysis indicated significantly increased (p< 0.05) DNA in all bioreactor samples in comparison with static constructs and significantly increased protein in HBMC bioreactor constructs in comparison with other cell types.

These studies outline a unique tissue engineering approach, utilizing individual and mixed human mesen-chymal progenitor populations coupled with innovative polysaccharide templates containing type I collagen and bioreactor systems to promote chondrogenic and osteo-genic differentiation.

The ability to generate replacement human tissues on demand is a major clinical need. Indeed the paucity of techniques in reconstructive surgery and trauma emphasize the urgent requirement for alternative strategies for the formation of new tissues and organs. The idea of biomimesis is to abstract good design principles and optimizations from nature and incorporate them in the construction of synthetic materials and structures. Direct appropriation of natural inorganic skeletons is also biomimetic since their unique properties inform us on ways to generate functional, optimized scaffolds.

A number of well characterized natural skeletons were investigated as potential scaffolds for tissue regeneration using mesenchymal stem cell populations. Marine sponges, sea urchin skeletons and nacre were found to possess unique functional properties that supported human cell attachment, growth and proliferation and provided organic/ inorganic extracellular matrix analogues for guided tissue regeneration.

A good understanding of the processses involved in biomineralisation and the emergence of complex inorganic forms has inspired synthetic strategies for the formation of biological analogues (organised inorganic materials with biological form). We have developed two functional examples of biological structures generated using biomimetic materials chemistry with applications for human tissue regeneration. Mineralised biopoly-saccharide microcapsules provided enclosed micro-environments with an appropriate physical structure and physiological milieu, for the support of the initial stages of tissue regeneration combined with a capacity to deliver human cells, plasmid DNA and controlled release of biological factors such as cytokines. Calcium carbonate porous microspheres analogous to microscopic coccolithophore shells provided a template for tissue formation and a mechanism for the delivery of DNA and functional biological factors. These biomi-metic structures have considerable potential as scaffolds for skeletal repair and regeneration, particularly when combined with inductive and stimulatory biological factors (cytokines, morphogens, signal molecules) and plasmid DNA carrying with them chemical cues that modulate and direct permanent tissue formation complimentary with the host.

Introduction: The ability to generate bone for skeletal repair, replacement or restoration is a major clinical need. Indeed the paucity of techniques in reconstructive surgery and trauma emphasise the need for alternative bone formation strategies. Natural biological ceramic structures possess arrangements of structural elements that govern and optimise tissue function, nutrition and organisation. The aim of this study was to fabricate biomineral microporous shells with highly complex forms and to examine their ability to interact with human osteoprogenitor cells as cell and growth factor delivery vehicles.

Methods: Microporous vaterite shells were generated using a synthetic in-solution mineralisation technique in which mineral is spontaneously deposited around vesicular templates (Walsh and Mann 1999)* Porous and textured self-organising hollow microspheres (5–20 _m) were generated expressing controlled and uniform shapes. These micropores puncture the surface at high densities and are interconnected throughout the sphere. Primary human bone marrow cells labelled with Cell Tracker Green and ethidium homodimer-1 fluorescent labels and osteoprogenitors transfected with an adenoviral vector expressing Green Fluorescent Protein (AdGFP) were cultured with vaterite shells over three weeks.

Results: Cell biocompatibility of these biomimetic spheres was confirmed by confocal fluorescence and light microscopy in primary human bone marrow cultures labelled with CTG and bone marrow cultures transfected with AdGFP. At three weeks microspheres were encapsulated and integrated with osteoprogenitor cells. Histological analysis confirmed expression of alkaline phosphatase, extracellular matrix synthesis and the capacity for extensive mineralisation. Examination by SEM, fluorescent and light microscopy showed that the growth of osteoprogenitors transfected with AdGFP and microspheres in pellet culture showed vaterite spheres were encapsulated and integrated within the osteoprogenitor cell matrix indicating the potential of growth factor delivery. To determine the potential of the spheres to encapsulate selected proteins, microporous spheres were incubated with bovine haemoglobin. FITC microscopic evidence showed haemoglobin could be entrapped inside the spheres and between the biomineral crystal plates during self-assembly.

Discussion and Conclusion: These studies demonstrate the development of facile techniques for the generation of porous microsphere sponges that are biocompatible, possess the ability to aid mineralisation and for the delivery of cell and growth factors. These calcium carbonate structures provide a material with widespread application in a range of tissue engineering applications including skeletal tissue regeneration.

Introduction: The clinical need for a biodegradable material with broad application is evidenced by the fact that tissue loss as a result of injury or disease provides reduced quality of life for many at significant socio-economic cost. The development of simple biodegradable materials, with broad applicability and tissue/ cell specificity has to date proved elusive. Natural biopolymers such as alginate and chitosan are structural biomaterials of increasing significance to tissue repair and regeneration due to their potential for fabrication, design and efficient, environmentally benign synthesis. We describe the development of innovative microcapsule scaffolds based on chitosan and alginate that can be tailored to a range of cell types for a variety of tissues.

Methods: Semi-permeable polysaccharide microcapsules were produced by a one-step method, in which the deposition of a semi-permeable alginate/chitosan membrane around droplets of sodium alginate was coupled with in-situ precipitation of amorphous calcium phosphate as described by Leveque et al (2002)*. A variety of human cell types including mesenchymal stem cells, osteoprogenitors selected using the STRO-1 antibody by magnetically activated cell separation (MACS), osteoprogenitors transfected with adenovirus expressing Green Fluorescent Protein (GFP) and chondrocytes were mixed with sodium alginate and encapsulated within alginate/chitosan and calcium phosphate.

Results: Hybrid spheres (750–10,000um) were generated encapsulating primary human osteoprogenitor cells, STRO-1 selected osteoprogenitors and AdGFP transfected osteoprogenitors. Encapsulated cells remain viable inside the polysaccharide microcapsules for 2 weeks as shown by positive alkaline phosphatase staining of encapsulated cells. Cells expressing GFP were observed within microspheres indicating the e ability to deliver cells/factors as well as the potential for gene therapy. Encapsulation and delivery of active BMP-2 was confirmed using the promyoblast cell line C2C12 known to be exquisitely sensitive to BMP-2. Nucleation of calcium phosphate occurred within the polysaccharide membrane and could be controlled by the phosphate concentration in the alginate droplets to produce hybrid microcapsules with enhanced mechanical strength. Thin walled capsules were shown to split and degrade in culture within 2–4 days releasing viable osteoprogenitor cells indicating the ability to manipulate the mechanical integrity and to programme degradation of the microspheres. Finally we have shown that aggregation of the microspheres into extended frameworks can be achieved using a designed droplet/vapour aerosol system resulting in foams of aggregated beads.

Discussion and Conclusion: A variety of human skeletal cells have been encapsulated within polysaccharide/ calcium phosphate microspheres and extended frameworks with specifiable dimensions. These composite scaffolds offer stable mechanical and chemical biomimetic environments conducive to normal cell function. Natural polysaccharides are also highly amenable to complexation with a range of bioactive molecules and consequently offer tremendous potential in tissue engineering and regeneration of hard and soft tissues.