MOLECULAR BIOLOGY

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CHAPTER 3: BONE DUST RESULTS

Systematic Review – Spine Deformity

 Introduction

The first bone graft material we evaluated was human posterior spinal bone dust. The reason for starting with bone dust was due to its autologous nature and the potential for it to demonstrate therapeutic potential. As part of this PhD, we conducted a systematic review to provide an up-to-date summary of in vitro, preclinical in vivo and clinical evidence on the efficacy of bone dust, created using high speed burrs, as a simple, safe and free local autologous bone graft. This systematic review has been published in the journal Spine Deformity (Chapter 8.3).
The total cost of low back pain in the United States alone exceeds $100 billion per year (Katz, 2006). Treatment of low back pain includes analgesia, activity modification (e.g. avoiding heavy lifting and high impact sport) and physiotherapy. In patients who are unresponsive to non-operative management and in patients with sinister pathology, surgical treatment is indicated. Spinal fusion, is a common surgical procedure performed to alleviate low back pain in a significant proportion of patients.
Despite advances in modern medicine and surgical techniques, non-union remains a significant problem for spinal fusion surgery, with up to 50% non-union reported for cervical and lumbar fusion (Buchowski et al., 2008; Lee et al., 2004). Bone graft and synthetic grafts are used to encourage bony fusion through osteoconduction, osteoinduction and osteogenesis. Currently, autologous iliac crest bone grafting remains the gold standard, but has disadvantages of prolonged operating time, increased blood loss and graft site complications occurring in up to 49% of patients (Arrington et al., 1996; Banwart et al., 1995; Dhawan et al., 2006; Robertson & Wray, 2001). Biological augmentation is being thoroughly investigated, however, products such as bone morphogenetic proteins (BMPs) are expensive and can be associated with significant adverse effects (Carragee et al., 2011; Garrison et al., 2007). Therefore, there is a need to provide simple, safe and cost-effective alternatives.
The use of locally harvested bone, such as morselised spinous processes, has been well described and is in clinical use (Betz et al., 2010). Bone dust generated via high speed burr is an alternate source of autologous bone graft that has received much less attention. Bone dust generated during the burring process is usually lost through suction along with blood. However, bone dust can be collected using simple suction traps and used to augment fusion (Ekanayake & Shad, 2010; Heidari et al., 2007; Malhotra et al., 2009; Nichter et al., 1988).
In this section, an up-to-date summary of in vitro, preclinical in vivo and clinical evidence on the efficacy of posterior human spinal bone dust will be discussed.

 Material and methods

A comprehensive review of the literature was performed with reference to the methods outlined in the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. Medline, PubMed, OVID, Scopus and Cochrane library were searched. Two independent reviewers performed separate searches and combined articles found. Once duplicates had been excluded, the remaining studies were then screened by title and abstract in order to determine relevance to the systematic review. Once the articles had been screened, a hand search of all remaining articles’ references was performed.
Key words: bone dust, bone burring, bone pate, bone paste
Inclusion criteria: All pre-clinical and clinical studies, published in any language, assessing the efficacy of bone dust, created using a high-speed burr, in osseous fusion.
Exclusion criteria: Studies not assessing the use of bone dust created via high-speed burr.
Assessing the quality of clinical studies: All clinical articles were assigned a level of evidence according to The Oxford Centre for Evidence-Based Medicine (OCEBM) Levels of Evidence Working Group. “The Oxford 2011 Levels of Evidence”.

 In vitro studies

In vitro studies are limited in number and have predominantly focused on the ability of bone dust to provide an alternate source of osteogenic cells, with one study determining the tissue composition of the collected bone dust (Table 3-1) (Ichiyanagi et al., 2010; Ye et al., 2013). Histological analysis of bone dust created during a laminectomy demonstrated that it was not purely bone tissue. In their sample, the authors quantified the product of high speed burring as being 65% bone, combined with other tissue types including blood products, fibrous tissue, cartilage and marrow (Patel et al., 2009). Furthermore, the authors did not identify any evidence of microscopic damage to the cellular components of the bone dust harvested using high-speed burrs.
There have been three studies that have cultured osteoblast-like cells from human bone dust and assessed their ability to proliferate and differentiate (Eder et al., 2011; Ichiyanagi et al., 2010). One study identified a cell population with mesenchymal stem cell characteristics in bone dust harvested during a transforaminal lumbar interbody fusion (TLIF) procedure and collected using a suction trap. These cells were able to proliferate when seeded within an osteoconductive serum glue and were capable of differentiating towards an osteoblastic lineage (Ichiyanagi et al., 2010).
When comparing the rate of proliferation and viability of osteoblasts cultured from bone dust versus bone chips collected from human laminae and spinous processes of patients undergoing lumbar fusion, osteoblast-like cells grew out of 57% of bone dust samples, compared to 100% of bone chips (Eder et al., 2011). Cell growth was also significantly slower, taking 14.8 days to reach confluence for bone dust compared to 5.6 days for bone chips. Furthermore, there were fewer cells in bone dust samples compared to bone chips (1.25×106 cells and 1.73×105 cells, respectively). Once grown, however, there was no difference in cell viability. The authors postulated that the lower osteoblast yield may be due to thermal or mechanical damage to cells caused by the high-speed burr, however, histological assessment suggested that this was not the case and may have been a reflection of the mixed tissue population present in bone dust (Eder et al., 2011).
A similar study compared the osteogenic potential of cells harvested from bone dust collected from human laminae and spinous processes versus cells harvested from iliac crest bone chips (Ye et al., 2013). Bone dust were collected using 3mm, 4mm and 5mm burrs from 10 patients (age 28-49 years) undergoing posterior spinal fusion for burst fracture or fracture dislocation. The authors concluded that while cells cultured from bone dust demonstrated osteogenic potential, it was inferior to iliac bone chips in terms of cell viability, alkaline phosphatase (ALP) activity and calcium deposition. It was interesting to note, however, that there were no differences between cells harvested from bone dust created from different size burrs. The authors also analysed similar samples in an animal model, which will be discussed in the preclinical in vivo section (Ye et al., 2013).
In addition to studies utilising human cells, several in vitro studies have also used animal tissue to investigate the efficacy of bone dust. Gupta et al. collected samples of bone dust, bone fragments and periosteum from five New Zealand white rabbits and cultured cells from these tissues (Gupta et al., 2009). The cells obtained from each of these samples were cultured for five weeks then assessed for collagen, calcium and ALP release. Collagen production appeared to be the same in each cell population. However, calcium and ALP concentrations were significantly higher in cells cultured from bone dust and periosteum, compared to those from bone chips. Contrary to these findings, Hassanein et al. found particulate bone graft (harvested from New Zealand white rabbits via a hand-driven 16mm bit and brace) contained significantly more viable cells, and had higher ALP activity (0.13 vs. 0.06 μU/μg), than bone dust (Hassanein et al., 2012).

CHAPTER 1: INTRODUCTION
1.1 THE STRUCTURE OF BONE
1.2 BONE MASS
1.3 BONE COMPOSITION
1.4 BONE CELLS
1.5 BONE FORMATION
1.6 FRACTURE
1.7 FRACTURE HEALING
1.8 FRACTURE NON-UNION
1.9 SPINAL FUSION
1.10 BONE LOSS
1.11 SURGERY
1.12 MESENCHYMAL STEM CELLS
1.13 SCAFFOLDS
1.14 GROWTH FACTORS
1.15 PROMISING NOVEL BONE GRAFT SUBSTITUTES
CHAPTER 2: MATERIALS AND METHODS
2.1 MATERIALS AND REAGENTS
2.2 ETHICS APPROVAL
2.3 PRIMARY HUMAN OSTEOBLASTS
2.4 CELL CULTURE
2.5 MOLECULAR BIOLOGY
2.6 PROTEIN DETECTION
2.7 ETHICS APPROVAL
2.8 RAT CRITICAL-SIZED CALVARIAL DEFECT MODEL
2.9 MICRO-CT
2.10 HISTOLOGY
CHAPTER 3: BONE DUST RESULTS 
3.1 SYSTEMATIC REVIEW – SPINE DEFORMITY
3.2 BONE DUST IN VITRO RESULTS – SPINE (IN PRESS)
CHAPTER 4: MCH-CAL™ RESULTS 
4.1 INTRODUCTION
4.2 MATERIAL AND METHODS
4.3 MCH-CAL™ IN VITRO RESULTS
4.4 MCH-CAL™ IN VIVO RESULTS
CHAPTER 5: LACTOFERRIN RESULTS 
5.1 INTRODUCTION
5.2 MATERIAL AND METHODS
5.3 LACTOFERIN IN VIVO RESULTS
5.4 DISCUSSION
5.5 CONCLUSION:H
CHAPTER 6: PHB-V RESULTS 
6.1 INTRODUCTION
6.2 MATERIAL AND METHODS
6.3 PHB-HV IN VITRO
6.4 PHB-HV IN VIVO RESULTS
6.5 DISCUSSION
6.6 CONCLUSION
CHAPTER 7: GELLAN GUM/HA RESULTS
7.1 INTRODUCTION
7.2 MATERIAL AND METHODS
7.3 GELLAN GUM/HA IN VITRO RESULTS
7.4 GELLAN GUM/HA IN VIVO RESULTS
7.5 DISCUSSION
7.6 CONCLUSION
CHAPTER 8: CONCLUDING DISCUSSION 
8.1 DISCUSSION AND FUTURE DIRECTIONS
8.2 CONCLUSION
8.3 SPINE DEFORMITY
8.4 SPINE
8.5 JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE
REFERENCES
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