On the Role of Reverse Micelles Other Aggregates in the System

70 A. Gadomski, I. Santamaria-Holek, N. Kruszewska et al. [212]. A leading argument regarding the biofluids involved in the biolubrication of AC is that the glycoprotein lubricin on hydrophilic surface active PLs, supported by the hyaluro- nan that are held together by protoglycans is responsible for the ultra-low friction in the joint in certain pH and surface tension [204, 208, 212, 209, 213, 214]. The pH value of SF can distinguish normal from osteoarthritic condition. In previous studies of samples of aspirated SF, the pH of normal SF was found to be between 7 .3 and 7 .43 [209]. In contrast the pH values of SF in various inflammatory conditions from joints with osteoarthritis OA and rheumatoid arthritis RA were 7 .4 − 8.1 mean of 7.9 for 16 joints with OA, and 7 .4 − 7.6 7.5 for the six joints with RA [213]. It is known that multi- layer film prepared by sequential electrostatic adsorption of polyL-lysine and hyaluronic acid, PLLHA onto charged silicon surfaces can provide an insight into the understanding of surface friction and wettability. In particular, studies have shown that surface friction can be altered by a factor of 10 and the degree of swelling by a factor of 8 for films com- posed of the two polyelectrolytes, by simply varying the pH [215]. Synovial fluid surface tension ST, free energy measure in surface layer was measured for inflammatory joint diseases. The ST values of synovial fluid in various inflammatory conditions from joints with seronegative spandylarthropathies Spa and rheumatoid arthritis RA were mean of 42 .42 mNm for 6 joints with Spa, and mean of 47.99 mNm for the 19 joints with RA. It was connected with significantly higher concentration of total proteins 5 .0 gdL for Spa and 3 .9 gdL for ST [214].

3.3. On the Role of Reverse Micelles Other Aggregates in the System

Reverse micelles RMs are aggregates formed by certain surfactant molecules in apolar solvents in vitro [216] and aqueous solutions by PLs as liposomes in living organisms [217]. The RMs have the polar or charged head-groups, with counter ions being localized in the polar core of the aggregate. The external part of RMs is a shell made up of hydrocarbon chains. Water, ions and several other polar macromolecules, are readily solubilized in the polar core, forming in the case of water, a so-called “water pool”. The ratio of molarity of water [H 2 O ] to the molarity of surfactant [S] was termed as a parameter R; R = [H 2 O ][S]. The most studied surfactant in organic solvents is sodium bis-2-ethylhexyl sulfosuccinate AOT forming RMs in aprotic solvents, e.g., isooctane, heptane, benzene and chloroform [216, 24]. Figure 26. Normal micelle NM and reverse micelles RMs. Water is solubilized by anionic, cationic and zwitterionic micelles, RMs, exhibit two ab- sorption bands in the near infrared spectral region 5200 − 4700 cm −1 [216]: this finding Can Modern Statistical Mechanics Unravel Some Practical Problems . . . 71 has been interpreted as due to two populations, one bound to the surfactant polar head and the other dispersed in the bulk phase. In the core of RMs of PL on the basis of NMR spectra described various type of water: tightly bound to the polar head to PL, inner hydration shell and weakly bound water together with trapped and bulk water [218]. The presence of guest chemical species in aqueous pool of RMs can considerably modify the physico-chemical characteristics of the water. The solubilized ions, peptides, proteins, and glycoproteins will create own water environment resulting in dramatically changed properties and the shape of RMs is modified. Water in core of RMs represents a kind of novel solvent with new phys- ical parameters, e.g., activity, microviscosity, and dielectric constant [216]. The optimum pH activity in the RMs was higher by about one pH unit than that determined in bulk water. The higher order structure of proteins solubilized in an AOT reverse micellar system was observed [219]. Factors affecting solubilization are largely determined by the condition in aqueous solution, namely pH, ionic strength, surfactant concentration and charge distribu- tion. Solubilization occurs when charged macromolecules and the surfactant has opposite charges. Increasing ionic strength of the aqueous phase will reduce electrostatic interactions as a result of the Debye screening effect. Thus, at a higher ionic strength, the interactions between hydrophilic biomolecules and the surfactant polar group are reduced, by decreas- ing concentration of surfactant when dealing with large charged macromolecules. Higher solubilization corresponds with higher positive surface charge, so process is governed by an electrostatic mechanism. In the next part of this paper we will focus on PLs as surfactants in aqueous solution, which are of the form of RMs, called liposomes and which form bilayers or membranes on surfaces Fig. 27. In particular, we will investigate the ability of bilayers to engage in solubilization, and the nature of the water in polar core “water pool”, or the interface of AC. Figure 27. The formation of bilayer on tissue and solid surfaces. Surface active Phospholipds, liposomes, bilayers and membranes - The PLs that are formed in aqueous solution of reverse micelles are referred to as liposomes which in turn form bilayers, with the major role of creating biological membranes that adsorb to solid sur- faces, including tissue surfaces. Because of its location in the membrane, the PL molecules possesses a high surface energy, while also possessing the ability to develop additional properties, such as, high electrical repulsive forces, adsorption of ions and charged macro- molecules, the adsorbed ions attract ions of opposite sign by forming a double layer of elec- tric charge at the interface. This double layers interface in cartilage giving rise to electrical repulsive forces which act to separate objects with like charges pushing them away from each other [216, 220, 221]. Liposomes are remarkably impermeable to cations, but per- 72 A. Gadomski, I. Santamaria-Holek, N. Kruszewska et al. meable to chloride ions and offer barrier to water penetration. These properties imply that liposomes would be a good osmometer [222]. The incorporation of lipophilic molecules in the matrix of the bilayer, e.g., cholesterol, proteins for transportation of hydrated ions, wa- ter and charged macromolecules are facilitating a cushioning structure of RMs. [221, 223]. The biological membrane comprising PLs sandwiched by two layers of side-chains and the folding of the protein molecules provides ion “channels” and has a strong hydrophilic na- ture [221, 2]. The content of PL and protein occupying surface sites in membrane and their function is controlled by the permeability of the structure [224]. The charged core of reverse micelle is well suited to organizing water molecules and ions in the synovial joint. In aqueous solutions, micelles play an essential role in the sta- bilization of charged particles and in the elimination of flocculation. Lubricin and another species electrostatically attached to hydrophilic SAPL-s allow them to maintain a double layer of electrostatic charges at the interface of contacting cartilages. The cartilages in a nor- mal joint are able to recharge themselves with exuded interstitial fluid. This phenomenon is connected with the charges on PLs on the articular surface membrane. The reverse mi- celles of hexagonal phases of PLs appear to be of fundamental relevance to many processes carried out by biomembranes [225]. Hence, we argue that the surface membrane of AC of the mammalian joint could operate in a similar manner to reverse micelles, thereby creating a the characteristic dynamic system in which the lipids play a central role. All mammalian biosurfaces are negatively charged, but AC with PLs has dipolar head groups. This char- acteristic charge situation is a powerful tool for manipulating the properties of the complex joint fluid system [220]. The new hydrophilic bilayer model H-philic B Model - The most common phase, formed by naturally occurring lipids i.e., phosphatidylcholines, PC, is the bilayer lamellar phase, which is adsorbed to the articular surfaces of AC Fig. 28. The adsorbed PLs create a structure similar to that of the core of the reverse micelle. The charged ends of PL molecules allow formation of water film between the two electrically charged surfaces Fig. 28. In our biolubrication hydrophilic bilayer model H-philic-B Model we do not propose a boundary or hydrodynamic lubrication process for normal articulating joints [207]. Figure 28. Schematic illustration of hydrophilic model A at equilibrium and hydrophobic B models in degeneration process of AC. Instead, we proposed a contrary opinion that a hydrophilic mechanism is responsible for the biolubrication of articulating mammalian joints. Biolubrication is provided by the action of surface-active PLs forming a hydrophilic multi-bilayer phase. This phase might function by binding to the articular surface of cartilage. Under our proposal, the articular surface will be characterized by hydrophilic properties and become electrically charged. This double layer or, multilayer consists of ions that are hydrated to form hydrofilm probably of a Can Modern Statistical Mechanics Unravel Some Practical Problems . . . 73 few hundred angstroms thick. If the two surfaces with multi-bilayers are oriented against each other, they give rise to electrical repulsive forces. Roberts [220] has suggested that electrical repulsive forces may be significant in biolubrication of surfaces by SF. The interface and nature of AC as a giant reverse micelle - The interface between AC with the adsorbed PLs bilayer highly charged on the surfaces, exuded interstitial fluid, synovial-fluid macromolecules and electrolyte creates the mechanism facilitating the lu- brication process. The synovial-fluid macromolecules such as glycoproteinlubricin and hyaluronan play important roles in hydrophilic lubrication [205]. Furthermore, lubricin is considered to play a critical role as a supplier of the PLs which are responsible for the struc- ture of the membrane overlaying the articular surface [204], might thus be a first example of biological polyelectrolytes supporting lubrication and wear-protection [2, 226]. Figure 29. Schematic representation of AC as a giant reverse micelle with various macro- molecular additives, compare with Fig. 32 and Fig. 33. The hydrophilic surface of AC and the space between the capsules are exaggerated in the figure for the sake of clarity. Lubricin is an active macro-ion in SF, which deposits the oligolamellar layer of PLs that possess the capabilities of high load bearing [204]. Hyaluronan is considered to play a criti- cal role in facilitating hydration by water in lubrication process [227]. It is also our opinion that the amount of water in the AC might be indicative of the high degree of hydration needed for effective lubrication. HA may also act to retain joint water in the articulation space outside the cartilage matrix. A friction coefficient coming from the CA value of .0007 was once recorded under high load using HA as carrier for PLs in recent works. Hyaluronan-phospholipid interaction has been shown in vitro to give rise to complexes which might exhibit peculiar lubricating and protective properties through the formation of a perforated membrane or thick cylinder roller system which is highly saturated by water [123]. Impact of the charge density of PL bilayer on lubrication of AC surfaces - Surface charge of phospholipid membrane can provide insights into its composition and demon- strate interaction between tissue and SF. The measured charge of AC for the human femoral head is 0 .037 Cm −2 . The addition of bovine SF 0 .0025 mmolml to the perfusion solution .015 M NaCl immediately reduced the streaming potential and reduced the surface charge 74 A. Gadomski, I. Santamaria-Holek, N. Kruszewska et al. by 25 [228]. In the following sections, we will introduce the results from our study of the microelectrophoretic mobility of liposomes as a function of hydrogen ion concentra- tion in sodium chloride solution within limit of 10 −5 to 0 .155 M or in dionized water [20]. We attempted to answer the question of how some changes in acid - base equilibrium have an impact on the charge density of a PL bilayer formed during lubrication occurring on the articular surfaces. Liposomes have been used to mimic biological PL membranes on AC surface where proteins are bounded, ions are transported, energy is transducted, and cellular processes take place. The charge density of the membrane was determined as a function of pH and electrolyte concentration from the microelectrophoretic method. Li- posome membrane has been prepared as an aqueous solution of NaCl under various pH conditions. Microelectrophoresis was used to examine the local acid-base equilibrium of the electrolytes with the membrane surface, which can be considered to be an interface of PLs in AC. The effects of the adsorption of ions H + , OH − , Na + , Cl − , which are present in solution upon electric charge of the liposome membrane assembly of phosphatidycholine PC, have also been found to exhibit pH-responsive behavior. Therefore, in this article, we will provide new data using micro-electrophoresis on the adsorption of ions H + , OH − , Na + , Cl − on the phosphatidycholine PC membrane which have also been found to ex- hibit pH-responsive behavior. Mathematical calculations of association constants for li- posome membrane surface in contact with ions in solution K AH , K ANa , K BOH , K BCl will also be carried out, leading to a model for adsorption of other ions, such as lubricin at the liposome membrane surface. Figure 30. The pH dependence of the surface charge density of liposomal membrane formed from PC. The experimental values are those obtained for dionized water and for .155 M NaCl solution [20]. The pH dependence of the surface charge of the liposomal membrane is plotted in Fig. 30. The experimental control curve, which was made in dionized water in the absence of sodium chloride. The other curve was obtained in the presence of 0 .155 M NaCl. It can be observed that in basic solution in the presence of the sodium chloride, a decrease of negative charge occurs. The −N + CH 3 groups of PC molecules are covered by OH − ions, whereas −PO − groups are uncovered. This fact indicates adsorption of Na + ions. A similar tendency can be observed in acidic solution: in the presence of sodium chloride, a decrease of positive charge occurs. The −PO − is A − groups are covered by H + ions, Can Modern Statistical Mechanics Unravel Some Practical Problems . . . 75 whereas −N + CH 3 is B + groups are uncovered. This fact indicates adsorption of Cl − ions. Association constants of the surface groups with the solution ions were determined by the linear regression method [229]. The association constants determined in this way are equal to K BOH = 5.35 × 10 9 ± 1.56 × 10 8 , K BCl = 0.218 ± 0.011, K AH = 5.58 × 10 5 ± 2 .03 × 10 4 , K ANa = 0.051 ± 0.002 [m 3 mol]. From comparison of the association constants it appears that the H + ion is more strongly adsorbed than the Na + ion and the OH − ion is also more strongly adsorbed than the Cl − ion. The degree of coverage of the PC membrane surface by ions as function of pH of .155 M NaCl was also calculated. Beside the coverage with the H + and OH − ions, the coverage with other ions Na + and Cl − was considered to check if the coverage with these ions is as high as to affect of the PC membrane surface charge. The Na + ions adsorption starts when the amount of the H + ions becomes low at pH 6. In basic solution the degree of coverage of the membrane by the Na + ions is over 0 .8, e.g., in this pH range the membrane is covered by the Na + ions. A similar tendency can be observed for the Cl − ions: the adsorption of the Cl − ions begins when the amount of the OH − ions begins to decrease at pH 4. In a strongly acidic solution the degree of coverage of the membrane by the Cl − ions is almost one. The measurements of the surface charge of the PC membrane as a function of concen- tration of NaCl for physiological pH are presented in Fig. 31. The increase of the Na + ion concentration causes the decrease of the negative charge, and a similar action is seen in relation to the adsorption of the Na + ions. Figure 31. The surface charge density of the PC membrane as a function of the concentra- tion of sodium chloride within the range of 10 −5 to 0 .155 M in physiological pH condition [20]. In our experiment, the pH range 6 .4 to 8.4 7.4 is physiological condition of SF is of most interest. From these, we can conclude that sodium and hydrogen ions interaction with group −PO − or the degree of coverage of PLs membrane surface is high. Also, in the physiological pH condition, the degree of coverage of membrane by the OH ions is near one. The adsorption of the chloride ions, which is as a very weak base is not observed in pH range 6 .4 to 8.4 conditions. Our results do indeed indicate that the surface charge strongly influences the acid-base 76 A. Gadomski, I. Santamaria-Holek, N. Kruszewska et al. equilibrium of the adsorbing species. Surface-charge alterations, being closely related with pH changes modify transport conditions for the ions. We chose to alter the surface charge by changing the pH of the solution used to assemble the bilayer since the pH affects the degree of dissociation of both polyelectrolytes if present and the charge density on PLs bilayer. This liposome bilayer is a model for PL bilayers and will be applied for the investigation of lubricin and other macromolecules. If acid-base quasi-equilibra are keptrecovered by the system, it is more resistive to wear under static CA-type friction; hydration of PLs makes the coagulation ineffective, and the layers involving hydrated PLs, and being electrostatically adsorbed at the surfaces of AC, are more mechanically robust. The latter gives rise to weak-friction in the sense of CA [22] promoting sliding effect, due to electrostatic repulsion, and opposes possible peptization, which, however, depends upon keeping a balance of salts within the system. If the balance is not kept by the system, the coagulation effects may prevail, which leads to loosing one of the desired acid-base quasi-equilibra, thereby causing systemic disequilibrium. This may spoil the quasi-periodic character of the process, which would imply an imbalance in the ions involving a prone-to-friction viscoelastic membrane, while also causing the ions to flow [21]. This appear to be a good premise for studying the facilitated biolubrication of the AC.

3.4. aD3DS Description of Dynamic Friction-Lubrication FL Applied to