Influence of phytochemicals on initial protein adsorption/adhesion over calcium silicate biomaterials: a pilot study.
Importance of biomaterials and protein adhesion has been proved by various investigators. According to them, protein adhesion gains substantial importance with regard to the surface and biocompatibility. This is more relevant in case of bone biomaterials that stay for longer time in the clinical service. It is also known that that protein adhesion is the first phenomenon to happen when a material is implanted in bone. While this happens, proteins show the change in conformation due to the influence of surface physics and chemistry (1).
It is well known that collagen constitutes almost one quarter of the total protein content in animals, being the major component of several connective tissues such as skin, tendons, ligaments, cartilage, bone, teeth, basement membranes, blood vessels etc. Hence it is felt logical that protein adhesion studies should be carried out using soluble fraction of collagen so that the interaction would be more similar to that happening in the physiological system.
Calcium silicate (CS) based ceramics are regarded as a potential bioactive material for bone tissue regeneration due to their osseointegration properties (2). In order to improve the bioactivity and as a substitute to expensive growth factors, phytochemicals are being used (3). There always remains a question about the effect of phytochemicals on the initial protein adhesion on the scaffolds". In order to investigate further, 5 osteogenic herbs viz. Cissus quadrangularis (CQ), Butea monosperma (BM), Ficus benghalensis (FB), Ficus religiosa (FRel) and Ficus Racemosa (FR) were selected to be incorporated to CS scaffolds in various concentrations and protein adhesion was studied using depletion study.
Material and Methods
Synthesis of Calcium Silicate Powder
Sodium silicate and 33% hydrochloric acid were purchased from Himedia (India). Calcium hydroxide was from Sd finechem., Mumbai, India. All the other chemicals were of analytical grade. The wet chemical process of synthesis was from McFarlane et al (4).
Isolation of collagen from rat skin (5, 6, 7)
Skins harvested from sacrificed and discarded wistar rats' carcasses were obtained from approved laboratory animal facility. Rat skins were cut into small sizes (linch approx.) and soaked in 0.5% aqueous acetic acid solution for 3-5 days, till swelling was incipient. The hair was removed from the soaked skin and subsequently washed with distilled water for 3-4 times. The skin was soaked in Chloroform: Methanol (3:1) solution for 2 hours (defatting). Then, the skins were immersed in 10% w/v sodium chloride solution for 4-5 days (at 4[degrees]C) followed by grinding in mixer to homogenize it and stirred in magnetic stirrer at 4[degrees]C for 1-2 days. The mixture was centrifuged at 8000 rpm for 15 minutes at 4 [degrees]C. Supernatant was dialyzed against cold distilled water for 3-4 days. The solution was stored at 4[degrees]C till further use. Collagen was confirmed using circular dichroism spectroscopy using Jasco J815 CD Spectrometer from 190 to 260 nm at 0.5 nm resolution and a protein concentration of ~0.5 mg/ml.
Phytochemical Extraction (3)
10 gm dry twig powder of Butea monosperma, Ficus benghalensis(FB), Ficus religiosa (FRel) and Ficus Racemosa (FR) & fresh aerial parts of Cissus quandrangularis are soaked respectively in 100 ml ethanol at 37[degrees]C for 2 days. Then filtered, diluted to contain 10 mg/ml solids and refrigerated at 4oC for further use.
Each of CS pellets were made of 200mg of powder and prepared using stainless steel dyes of 1cm diameter in a hydraulic press, at pressure 50 MPa for 5secs. (PCI Analytics Pvt. Ltd, Mumbai, India). The phytochemical containing pellets were made as follows: 200mg of CS was added with 200[micro]l of respective extract, dried and pelletized and labeled as "20". Similarly added 400p.l, 600p.l, 800p! of respective extract, labeled as "40", "60" and "80" respectively preceded with the abbreviation of the herb. Eg: BM40.
Depletion Studies (8)
The samples were placed in ~0.5 mg/ml solution of salt soluble collagen. At definite time intervals, i.e. (0, 2, 10, 20, 45 minutes), absorbance in UV-Vis spectrophotometry was recorded at 280 nm to estimate residual protein in the solution. The absorbance was plot against time to obtain the rate of protein adhesion.
Results and Discussion
Circular Dichroism was used in confirming the structure of isolated collagen. The salt soluble collagen is basically uncross-linked tropocollagen random coils, which is testified by the CD spectra that clearly shows the random coil orientation of the isolated collagen (Fig. 1) (8,9).
Basis of the depletion study is the fact that as the protein adsorption proceeds, the concentration of residual protein in the solution decreases (10). Since the total amount protein is same, the amount of protein adsorbed is obviously taken from the solution. Hence, this study is a sensitive procedure to critically evaluate the protein adhesion in both qualitative and quantities terms. Adsorption kinetics allows one to understand protein adsorption and its relationship to cellular events. In a mixture of proteins, the protein at the highest concentration and with the highest diffusivity has the best chance of adsorbing to the surface. If this protein binds to the surface at a rate that depletes all available surface sites before any other protein approaches the surface, then the surface will be dominated by this protein. If however, this protein loosely binds to surface sites only, it can be easily replaced by other proteins that may be present at much lower concentrations but bind to the surface with much higher affinity. Adsorption is the accumulation and adhesion of molecules, atoms, ions, or larger particles to a surface, but without actually penetrating the surface.
[FIGURE 1 OMITTED]
The adsorption of larger biomolecules such as proteins is of high physiological relevance, and adsorb with different mechanisms than their molecular or atomic analogues. Some of the major driving forces behind protein adsorption include: surface energy, intermolecular forces, hydrophobicity, and ionic or electrostatic interaction. By knowing how these factors affect protein adsorption, they can then be manipulated by machining, alloying, and other engineering techniques to select for the most optimal performance in biomedical or physiological applications.
In case of Butea monosperma, the absorbance was gradually decreased due to the adsorption of protein into the pellet. The adsorption rate was less at lower concentration but when the concentration was increased, the adsorption rate was also increased till the optimum stage. On the other hand, in case of Cissus quandrangularis, the absorbance was decreased due to the increase of protein adsorption rate into the pellet. In this graph the rate of adsorption was slowly increased with respect to the concentration. In lower region of initial loading concentration, there was a marked increase in release but at higher concentration release did not increase in the same proportion (Fig. 2).
The Ficus species have shown drastic influence on the protein adhesion efficiency of calcium silicate scaffolds. The Ficus benghalensis has shown erratic protein adsorption character at all concentrations, in repetitive experiments. Hence was disregarded. This can probably be due to the discouraging nature of constituent phytochemicals. Probably the released phytochemicals may have denatured the approaching protein chain (Fig. 3). Hence, this might be the reason for absence of local osteogenic effects in F. benghalensis compared to other Ficus species. The F. racemosa and F religiosa have shown similar trait to Cissus and Butea. The protein loss from solution was dependent on time and final protein adhesion marginally increased with concentration.
This comparative pilot study has shed valuable light on the phenomenon of influence of phytochemicals on protein adsorption/adhesion. Few significant observations were made in this study. The protein adhesion was un-interfered in the control samples, which was verified repeatedly. Hence, the validity of the study methodology was proven. The adsorption of protein on surface was dependent on the phytochemical concentration/release. Though different herbs have dissimilar phytoconstituents, the interaction with adhering protein was strikingly similar. It should further be analyzed on what phytoconstituent is actually responsible for such a phenomenon. Nevertheless, this relation will be analyzed in future and reported.
[FIGURE 2 OMITTED]
[FIGURE 3 OMITTED]
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R Narasimha Raghavan  *, G Vignesh , S. Angelin Trinita , J. Pragathi , Gopi Krishna Giri , Arijit Chakraborty , T.P. Sastry 
 Centre for Laboratory Animal Technology and Research,  Department of Biomedical Engineering, Sathyabama University, Chennai 600119  Department of Biotechnology, Achariya Arts and Science College, Achariyapuram, Villianur, Puducherry 605110  Department of Pharmacology, School of Pharmaceutical Sciences, VELS University, Chennai 600117  Bioproducts Laboratory, CSIR-Central Leather Research Institute, Chennai
* Coresponding author: Dr. R. Narasimha Raghavan; E-mail: email@example.com
Received 18 April 2016; Accepted 7 June 2016; Published online 10 June 2016
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|Title Annotation:||Short Communication|
|Author:||Raghavan, R. Narasimha; Vignesh, G.; Trinita, S. Angelin; Pragathi, J.; Giri, Gopi Krishna; Chakrabo|
|Publication:||Trends in Biomaterials and Artificial Organs|
|Date:||Jan 1, 2016|
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