PROJECT TOPIC – EVALUATION OF THE OSTEOGENIC POTENTIAL AND ANTIBACTERIAL ACTIVITY OF AUTOLOGOUS PLATELET-RICH PLASMA IN NIGERIAN LOCAL DOGS

 

ABSTRACT

This study was conducted to assess the osteogenic potentials of platelet rich plasma (PRP) in Nigerian local dogs and to determine antibacterial activity of the PRP. Forty-five Nigerian local dogs, aged 8-12 months, used for this study were acclimatized for two weeks. The study was conducted in four  experiments. In experiment one, 8.5 ml of blood was collected in duplicates from each of 20 dogs. The blood was collected into test tubes preloaded with
anticoagulant. PlateltexR PRP (PPRP) and CaCl2 PRP (CPRP) were prepared using two standard methods. The platelet concentrations of the whole blood and the prepared PRPs were determined following standard procedures.

The platelet derived growth factor (PDGF) and transforming growth factor (TGF) contents in the whole blood and the two PRPs were quantified using  enzyme-linked immunosorbent assay. The results of the platelet counts and  the growth factor (GF) contents of the whole blood and the two PRPs were analyzed using one-way analysis of variance (ANOVA) at 5% level of probability. In experiment 2, the osteogenic effects of the PPRP and autologous cancellous bone grafts (ACBG) on caudolateral ulna osteotomies were evaluated in 12 dogs. Partial ostectomy of the caudal cortical surface of the right ulna was performed under anaesthesia.

The 12 dogs used for the experiment were randomly assigned into four groups namely; platelet rich plasma group (PRPG), cancellous bone graft group (CBG), platelet rich plasma/cancellous bone graft group (PRP/CBG) and control group (CG).The bone defects of the PRPG were treated by applying autologously prepared PRP onto the defects before closure of the soft tissues. The dogs in the CBG were treated by applying ACBGs from the proximal tibia. The defects in the PRP/CBG were treated by applying both autologous PRP and cancellous bone graft. Normal saline was applied on the defects of the CG before closure. Radiographs of the defects were taken at weeks 1, 4, 6 and 8 post-operation (PO). Callus proliferation, mineralization and the radiographic optical densities (ROD) of the osteoid were recorded.

Histological sections of the formed callus on the healing bone defects were also prepared at week 10 (PO) and the results evaluated semi quantitatively using standard procedures. In experiment 3, the effects of the two different PRPs on tibia fractures were evaluated in 9 dogs. The rate of healing of the fractures was subjectively evaluated without bias following a standard procedure. In experiment 4, an in vitro antibacterial activity of the PPRPs was  evaluated using the disc diffusion method. The inhibition zone diameter (IZD) around each PRP containing disc was measured to the nearest whole millimeter. Variations in the susceptibilities of the test isolates to the PRPs were analyzed by analysis of variance (ANOVA). Variant means were separated
using Duncan’s multiple range test (DMRT).

All analyses were done at 5% probability level. The mean platelet concentration (PC), PDGF and TGF concentrations of the PPRP were 20.47±92(x105/μl), 14.73±0.31(ng/ml) and 90.55±1.97(ng/ml), respectively while the values for the CPRP were 12.46±0.59(x105/μl), 8.30±35(ng/ml) and 43.60±1.05(ng/ml),
respectively. The mean PC, PDGF and TGF of the PPRP and CPRP were significantly (p<0.05) higher than those of the whole blood. PPRP had a significantly (p<0.05) higher mean concentrations of PDGF and TGF than CPRP. Bone defects in dogs in the PRP/CBG showed superior osteogenic indices in terms of mineralization, ROD, and maturation of the osteoids. Those in PRP group however, performed better than the CBG group. The osteogenic indices in the control group were least. Periosteal reaction was evident in 100%of the fractures in PPRP group and 66.7% in both CPRP and the CG at week 4 post operation.
At week 8 PO, the PPRP group exhibited extensive periosteal incorporation into the cortex, better mineralization of osteoid (sclerosis) and total loss of  fracture lines compared to the fractures in the CPRP and CG. The mean IZD produced by PRP against Staphylococcus sciuri, Pseudomonas aeruginosa and Escherichia coli were 19.50±0.50mm, 16.63±0.47mm and 13.88±0.31mm respectively. However, the variation in the mean IZD for the three isolates was not significant (p>0.05). With the better osteogenic indices and antibacterial activity demonstrated by the PRP-treated defects compared to the control, it was established that PRP has both osteogenic and antibacterial activity in Nigerian local dogs.

 

CHAPTER ONE

 

INTRODUCTION

1.1 Background of the study

Bone is defined as a biological tissue composed of dynamically active cells which are integrated into a rigid framework (Canalis et al., 1991). Bone cells  consist of osteoblasts, osteoclasts, osteocytes, osteoprogenator cells and hematopoietic components including platelets (Canalis et al., 1991). Bone  healing, whether in fracture repair or in any given fusion model, is a delicate balance between bone depositon, resorption, and remodeling which are
influenced by numerous biochemical, biomechanical, cellular, and pathological mechanisms (Everts et al., 2007). Researches into the biology of bone, ligament and tendon healing have led to the development of variety of products designed to help stimulate biologic factors and promote healing (Foster et al., 2009).

Applications of mechanical vibration along the axis of the fractures, ionic resonance electromagnetic field stimulation, and static magnetic force with
samarian cobalt magnets have all been employed. In our rural setting, many herbs have been investigated with varying degrees of successes and failures (Akpulu et al., 1994; Asgar, 1999). The use of exogenous recombinant proteins including bone morphogenic proteins is being investigated in our department. The use of autologous recombinant products in tissue engineering is rapidly growing, focusing on manipulation of growth factors and secretory proteins to maximize healing of bones and soft tissues (Everts et al., 2007).

In spite of these, surgeons still continue to face challenges relating to the healing of bone. There still abound many cases of delayed and non-union fractures in many orthopedic clinics all over the world. This has resulted in increased morbidity coupled with added financial burden on the patients or the clients (Anon, 2007).Platelet-rich plasma (PRP) represents an emerging biotechnology in current tissue engineering and cellular therapy. Platelet rich plasma is defined as a portion of the plasma fraction of autologous blood having a platelet concentration above baseline (Mehta and Watson, 2008; Marx, 2001). It has been postulated that platelet gel will be used widely in future for soft and bony tissue repair (Jameson, 2007). Platelet-rich plasma has been used to treat soft tissue wounds since 1985 (Driver et al., 2006).

It is a growth factor agonist (Petrova and Edmonds, 2006) with both mitogenic and chemotactic properties (Marx, 2001; Millington and Norris, 2000). The secretory proteins contained in the -granules of platelets are:

1. Platelet-derived growth factor (PDGF-AA, BB, and AB isomers) (Nikolidakis and Jansen, 2008; Knighton et al., 1986)

.2. Transforming growth factor- (TGF-) (Weibrich et al., 2001). 3. Platelet factor 4 (PF4) (Nikolidakis and Jansen, 2008)
4. Interleukin-1 (IL-1).
5. Platelet-derived angiogenesis factor (PDAF)
6. Vascular endothelial growth factor (VEGF) (El-Sharkawy et al., 2007). The PRP is also a buffy coat product which, apart from being riched in platelets, also  contains a high concentration of viable neutrophilic polymorphonuclear leucocytes or neutrophils.These cells ordinarily play an important role in the innate immune defense against infections. As an autologous preparation, PRP is safer to use than allogenic or homologous preparations and is free from concerns over transmissible diseases (Mehta and Watson, 2008) such as HIV, hepatitis, West Nile fever, and Creutzfeldt-Jakob disease. Platelet rich plasma requires no special considerations regarding antibody formation (Edmonds et al., 2000), effectively preventing the risk of graft versus host disease (McAleer et al., 2006) and leading to better acceptance by patients (Mehta and Watson, 2008)

EVALUATION OF THE OSTEOGENIC POTENTIAL AND ANTIBACTERIAL ACTIVITY OF AUTOLOGOUS PLATELET-RICH PLASMA IN NIGERIAN LOCAL DOGS



1.2 Statement of the problem

Enhancing bone healing has been a long standing challenge to man. The following have been tried:
1. Application of mechanical vibration along the axis of fracture.
2. Ionic resonance electromagnetic field stimulation.
3. Autologous bone grafts.
4. Use of demineralized bone matrix.
5. Use of exogenous recombinant proteins.
6. Herbal remedies among others.

In spite of these, there are still many cases of delayed union, non-union and limb amputations. Infection is a great threat in management of bone defects. Many antibiotics are costly and problem of resistance abound. So there is the need to develop products which can promote osteosynthesis and prevent bacterial infections.

1.3 Research questions

i. Do different PRP preparation methods affect its platelet and growth factor content?
ii. What is the outcome of treatment of osteotomies of Nigerian local dogs using two differently prepared PRP?
iii. Does PRP have any antibacterial potential?

 

EVALUATION OF THE OSTEOGENIC POTENTIAL AND ANTIBACTERIAL ACTIVITY OF AUTOLOGOUS PLATELET-RICH PLASMA IN NIGERIAN LOCAL DOGS

 

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