Platelet Adhesion Properties of PCL-TCP Composites

Transcription

1 Platelet Adhesion Properties of PCL-TCP Composites L.H Yong a, E.Y.L Teo a, SH Teoh a,b a Centre for Biomedical Materials Applications and Technology (BIOMAT), Dept of Mechanical Engineering, National University of Singapore b Osteopore International Pte Ltd, 10 Science Park Road, The Alpha, #02-28, Singapore ABSTRACT It was shown from Teo et al that Fused Deposition Modeled Polycaprolactone- Tricalcium Phosphate (FDM PCL-TCP) scaffolds have an outstanding capacity to attract and trap platelets in comparison with commercially available wound dressings [1]. This project aims to explore the platelet adhesion properties of heat pressed PCL and PCL- TCP composite sheets so as to establish whether the percentage of TCP have a significant effect on the platelet adhesion properties of PCL. Scanning Electron Microscopy and manual platelet counting will be used to evaluate platelet adhesion properties INTRODUCTION Platelets are important players in initiating clotting formation in blood, a process commonly known as haemostasis. The three primary functions of platelets include maintainance of vascular integrity to provide growth of the blood vessel, formation of a platelet plug to fill the breached vessel in an injury and stabilization of the plug with its adhesion property with the exposed subendothelium [2]. Platelets would first adhere in seconds upon the injury site (adhesion) and will activate via several feedback pathways of cofactors, one of which is calcium ions, commonly known as factor IV, stimulates the

2 release of adenosine diphosphate (ADP) and serotonin, causing shape changes to occur [3, 4]. Commercial wound dressings aim to be more effective in the healing of wounds and extensive research has been carried to improve on current wound dressing technologies. Haemostasis begins immediately after wounding and so it is important for a material to induce haemostasis rapidly to allow for other processes like epithelialisation to occur [5]. In addition, calcium ions can be found in TCP as a phosphate [6]. We therefore hypothesise that TCP, as a source of calcium ions, has the ability to attract platelets and that increased percentages of TCP will increases the amount of platelet adhesion and also its activation state, enhancing the material s haemostatic capability. MATERIALS AND METHODS PCL produced by Sigma Aldrich and medical grade TCP was used. Heat pressed sheets of PCL-TCP is made by mixing relevant contents of materials, into a roll mill with temperatures set at 95 C and rolled mixed into a uniform composition. The material is scraped off from the rollers and placed into a heat press machine with temperatures set at 95 C and hydraulic pressure first at 100MPa for the first 10 minutes and at 280MPa for the subsequent hour. After 1 hour, the heating element of the machine is switched off and the sheet allowed to cool in room temperature at pressure maintained at 280 MPa. A total of 4 different compositions of PCL TCP sheets were generated (0%, 10%, 20%, 25%) by repeating the above process. Squares of sizes 8mm sides were cut from the prepared PCL- TCP sheets and sodium hydroxide treatment was performed on these squares by

3 soaking in 5M NaOH for 3 hours, washed with phosphate buffer solution (PBS) before immersing in 70% ethanol for 12 hours. The ethanol was then removed and the specimen dried prior to PRP loading. LOADING OF PRP PRP was prepared from whole blood collected from one rat source. 8 ml heparinised blood was centrifuged at 140g for 10 min to separate PRP and platelet-poor plasma (PPP) portions from the red blood cell fraction. 80µl of PRP was extracted and dripped onto each individual squares of 0%, 10%, 20% and 25%. Similarly, 80µl of 1 PBS was loaded onto separate samples to serve as controls. The loaded samples were then incubated at 37 C for 15 minutes. SCANNING ELECTRON MICROSCOPY (SEM) ANALYSIS After incubation, the samples were removed from the 24-well plate and placed in 4% formaldehyde at room temperature for 30 minutes. The samples were then rinsed 3 times in deionised water and then dehydrated in a graded ethanol series of 25%, 50%, 75%, 95% and 100% for 10 minutes, vacuum-dried and gold-sputtered with JFC-1200 finecoater for 40s at 10 ma under high vacuum. The specimens were examined with a JEOL JSM 5600LV SEM operating at 15 kv under high vacuum mode at a magnification of pictographs were taken from every percentage and at various areas around the samples.

4 The adhesion properties in this context are defined as the number of adhered platelets in the SEM pictographs and its activation level achieved upon adhesion. Marta Carretero et al had defined various stages of platelet activation on a polymeric material under static conditions and are summarized in Fig 2 [6]. This classification will be used to qualitatively analyse the activation stages present in the SEM pictographs in the next section. Type 0 Type 1 Type 2 Type 3 Fig 1 Schematic of platelet activation in 4 different stages of activations. Taken from Kuwahara M,Sugimoto M,Tsuji S,et al, Platelet shape changes and adhesion under high shear flow. Arterioscler Thromb Vasc Biol. 2002;22: Type Description 1 Round platelets 2 Early dentritic, pseudopods extending radially 3 Late dendritic, cytoplasm fill spaces between pseudopods 4 Fully spread, cytoplasm completely filled pseudopods Fig 2 Summary of morphologies of platelets undergoing increasing types of activations. Taken from Marta Carretero,et al, Platelet adhesion onto a polystyrene surface under static conditions: role of specific platelet receptors and effect of divalent cations, Platelets, vol 13, 2002, p The entire experiment was repeated in order to confirm our findings as stated below.

5 RESULTS A B Manual counting was employed in the calculation of the platelet adhesion number on all SEM pictographs. An average platelet count, general platelet morphology and presence of network formations were taken and tabulated in Fig 4. A representative picture from each percentage of TCP in all experiments was taken and complied in Fig 3 for comparison. The number of adhered platelets as seen in Fig 3A is minimum amongst all C D percentages of TCP. It has a mostly Type I activation level with visible pseudopods spreading from the platelets and some aggregation among platelets. At 10% TCP (Fig 3B), there is an increase in number of adhered platelets from 0% TCP. A mixture of Type I and II platelets is also noticed, indicating a higher platelet activation level. At 20% TCP (Fig 3C), the number of platelets are also now seen to be greater than that of those at 10% TCP. Moreover, the platelets appear bigger with a flatter appearence and a larger proportion of platelets undergoing aggregation. This can be classified as a late Type II with equal number of Type III activation. The number of adhered platelets and activation level on 20% TCP (Fig 3D) is observed to be the highest of all, with major aggregations taking place among platelets. Type III activation of platelets can be seen as the majority in all.

6 A B C D Fig 3 SEM pictographs of platelet adhesion at different percentages of TCP in PCL, incubated at 15 minutes. A 0% TCP, B 10%TCP, C 20% TCP, D 25% TCP. Magnification X 4500 Specimen Average platelet number Morphology Network formation 0% 15 Type 1 No formation 10% 30 Type 1 Slight 20% 50 Type 2 Moderate 25% 75 Type 3 Large Fig 2 Summary of results for average platelet number, morphology and presence of network formations

7 CONCLUSION From preliminary data, it can be seen that increasing percentages of TCP in PCL can enhance its platelet adhesion properties, defined as the number of adhered and the activation level of platelets found. It is also inferred that TCP can be a source of calcium ions for platelets to activate. This haemostatic phenomenon can be potentially applied to wound dressing applications using PCL-TCP composites as compared to other more expensive alternatives. FUTURE WORKS Lena Kikuchi et al had reported that surface morphology on materials play a bigger part in platelet activations instead of calcium and phosphate ions [7]. A suggestion to the extension of this work could be to study other forms of manufacturing methods in producing PCL TCP composites with varying surface microtopographies, for example biaxially-stretched sheets or chemically-etched surfaces. A comparison between the various surface energies of different manufacturing methods and percentages of TCP in the composite can then be achieved and discussed.

8 REFERENCES 1 E.Y.L Teo et al, Development of 3D Bioactive scaffolds for Haemostatic applications 2 Platelet information from MediaLab, Inc., 3 Shapiro SS, Treating thrombosis in the 21st century, N. Engl J. Med, 2003, 349(18): Peter G Hayward, Wayne A Morrison, Current concepts in wound dressings, Australian Prescriber, 1996, 19: S.H Teoh, Engineering Materials for Biomedical Applications Vol.1, World Scientific, Marta Carretero, Maribel D õaz-ricart, Berta Fust e, Eva Estebanell, Gin es Escolar, Antonio Ordinas, Platelet adhesion onto a polystyrene surface under static conditions: role of specific platelet receptors and effect of divalent cations, Platelets, 2002, 13: Lena Kikuchi, Jun Y. Park, Charles Victor, John E. Davies, Platelet interactions with calcium-phosphate-coated surfaces, Biomaterials 2005, 26: