Direct measurement of osmotic pressure of glycosaminoglycan solutions by membrane osmometry at room temperature

Articular cartilage is a hydrated soft tissue composed of negatively charged proteoglycans fixed within a collagen matrix. This charge gradient causes the tissue to imbibe water and swell, creating a net osmotic pressure that enhances the tissue's ability to bear load. In this study we designed and utilized an apparatus for directly measuring the osmotic pressure of chondroitin sulfate, the primary glycosaminoglycan found in articular cartilage, in solution with varying bathing ionic strength (0.015 M, 0.15 M, 0.5 M, 1 M, and 2 M NaCl) at room temperature. The osmotic pressure (pi) was found to increase nonlinearly with increasing chondroitin sulfate concentration and decreasing NaCl ionic bath environment. Above 1 M NaCl, pi changes negligibly with further increases in salt concentration, suggesting that Donnan osmotic pressure is negligible above this threshold, and the resulting pressure is attributed to configurational entropy. Results of the current study were also used to estimate the contribution of osmotic pressure to the stiffness of cartilage based on theoretical and experimental considerations. Our findings indicate that the osmotic pressure resulting from configurational entropy is much smaller in cartilage (based on an earlier study on bovine articular cartilage) than in free solution. The rate of change of osmotic pressure with compressive strain is found to contribute approximately one-third of the compressive modulus (H(A)(eff)) of cartilage (Pi approximately H(A)(eff)/3), with the balance contributed by the intrinsic structural modulus of the solid matrix (i.e., H(A) approximately 2H(A)(eff)/3). A strong dependence of this intrinsic modulus on salt concentration was found; therefore, it appears that proteoglycans contribute structurally to the magnitude of H(A), in a manner independent of osmotic pressure.     

Hydraulic conductivity of polymer matrices

We demonstrate that the chondroitin sulfate proteoglycan exhibits enhanced sensitivity to the flow of water compared to other macromolecules which is in accord with their functional role in conferring compressive resistance to cartilage. In order to understand factors that may contribute to its low hydraulic conductivity, a comparative study of hydraulic conductivity, as measured by the sedimentation velocity technique is made of various macromolecules representing variations in charge density, chemical composition, thermodynamic nonideality, size and flexibility. The polymers examined were dextran, poly(ethylene glycol), poly(vinyl alcohol), albumin, and dextran sulfate. The differences in hydraulic conductivity between the various macromolecules could not be explained by conventional theories which included prediction of hydraulic conductivity related to the radius of the molecule regarded as a uniform cylinder, nor the absolute charge density of the molecule and nor to the steric hindrance offered by the macromolecule to the diffusion of tritiated water. A qualitative relationship is established, however, between the noncounterion polymer contribution to osmotic activity and the resistance to water flow for polymers with high osmotic activity.     

A holographic sensor based on a rationally designed synthetic polymer

A new silver halide-containing holographic recording material has been designed and developed specifically for holographic chemical sensors. The hologram enables very small volume changes to be measured in a polymer layer throughout which the hologram is located. The holographic film is based on a fine-grain silver bromide emulsion suspended in a poly(vinyl alcohol) matrix crosslinked with Cr(III) ions. Cross-linking gives the material sufficient spatial integrity to allow a holographic image to be recorded, while maintaining adequate porosity and elasticity of the polymer matrix for sensing applications. The new material has been characterized with respect to its response to pH and compared with a traditional gelatin holographic film. The response to some ions and small molecules typically found in analytical samples has also been measured. Functional groups introduced covalently into the poly(vinyl alcohol) matrix transform the base matrix into a pH-responsive polymer with predictable swelling properties and which can be further derivatized to incorporate specific ligands. A rationally designed holographic sensor for trypsin has been developed from chemically synthesized artificial polymers. A trypsin substrate, the poly(amino acid) poly(L-lysine), was incorporated into poly(vinyl alcohol) holograms to create a 'designed' holographic material which was degraded in a concentration-dependent manner by trypsin. Extensions of this approach to other hydrolytic enzymes are briefly discussed.     

Quantitative spatially resolved measurements of mass transfer through laryngeal cartilage.

The scanning electrochemical microscope (SECM) is a scanned probe microscope that uses the response of a mobile ultramicroelectrode (UME) tip to determine the reactivity, topography, and mass transport characteristics of interfaces with high spatial resolution. SECM strategies for measuring the rates of solute diffusion and convection through samples of cartilage, using amperometric UMEs, are outlined. The methods are used to determine the diffusion coefficients of oxygen and ruthenium(III) hexamine [Ru(NH3)6(3+)] in laryngeal cartilage. The diffusion coefficient of oxygen in cartilage is found to be approximately 50% of that in aqueous electrolyte solution, assuming a partition coefficient of unity for oxygen between cartilage and aqueous solution. In contrast, diffusion of Ru(NH3)6(3+) within the cartilage sample cannot be detected on the SECM timescale, suggesting a diffusion coefficient at least two orders of magnitude lower than that in solution, given a measured partition coefficient for Ru(NH3)6(3+) between cartilage and aqueous solution, Kp = [Ru(NH3)6(3+)]cartilage/[RU(NH3)6(3+)]solution = 3.4 +/- 0.1. Rates of Ru(NH3)6(3+) osmotically driven convective transport across cartilage samples are imaged at high spatial resolution by monitoring the current response of a scanning UME, with an osmotic pressure of approximately 0.75 atm across the slice. A model is outlined that enables the current response to be related to the local flux. By determining the topography of the sample from the current response with no applied osmotic pressure, local transport rates can be correlated with topographical features of the sample surface, at much higher spatial resolution than has previously been achieved.     

Activity and Stability of Native and Modified Subtilisins in Various Media

The activity and stability of native subtilisin 72, its complex with poly(acrylic acid), and subtilisin covalently attached to poly(vinyl alcohol) cryogel were studied in aqueous and organic media by hydrolysis of specific chromogenic peptide substrates. Kinetic parameters of the hydrolysis of Glp-Ala-Ala-Leu-pNA by native subtilisin and its complex with poly(acrylic acid) were determined. Based on the comparative study of stability of native and modified subtilisins in media of various compositions, it was established that covalent immobilization of subtilisin on poly(vinyl alcohol) cryogel is the most effective approach to improve enzyme stability in water as well as in mixtures with low water content.     

The influence of the fixed negative charges on mechanical and electrical behaviors of articular cartilage under unconfined compression

Unconfined compression test has been frequently used to study the mechanical behaviors of articular cartilage, both theoretically and experimentally. It has also been used in explant and gel-cell-complex studies in tissue engineering. In biphasic and poroelastic theories, the effect of charges fixed on the proteoglycan macromolecules in articular cartilage is embodied in the apparent compressive Young's modulus and the apparent Poisson's ratio of the tissue, and the fluid pressure is considered to be the portion above the osmotic pressure. In order to understand how proteoglycan fixed charges might affect the mechanical behaviors of articular cartilage, and in order to predict the osmotic pressure and electric fields inside the tissue in this experimental configuration, it is necessary to use a model that explicitly takes into account the charged nature of the tissue and the flow of ions within its porous interstices. In this paper, we used a finite element model based on the triphasic theory to study how fixed charges in the porous-permeable soft tissue can modulate its mechanical and electrochemical responses under a step displacement in unconfined compression. The results from finite element calculations showed that: 1) A charged tissue always supports a larger load than an uncharged tissue of the same intrinsic elastic moduli. 2) The apparent Young's modulus (the ratio of the equilibrium axial stress to the axial strain) is always greater than the intrinsic Young's modulus of an uncharged tissue. 3) The apparent Poisson's ratio (the negative ratio of the lateral strain to the axial strain) is always larger than the intrinsic Poisson's ratio of an uncharged tissue. 4) Load support derives from three sources: intrinsic matrix stiffness, hydraulic pressure and osmotic pressure. Under the unconfined compression, the Donnan osmotic pressure can constitute between 13%-22% of the total load support at equilibrium. 5) During the stress-relaxation process following the initial instant of loading, the diffusion potential (due to the gradient of the fixed charge density and the associated gradient of ion concentrations) and the streaming potential (due to fluid convection) compete against each other. Within the physiological range of material parameters, the polarity of the electric potential depends on both the mechanical properties and the fixed charge density (FCD) of the tissue. For softer tissues, the diffusion effects dominate the electromechanical response, while for stiffer tissues, the streaming potential dominates this response. 6) Fixed charges do not affect the instantaneous strain field relative to the initial equilibrium state. However, there is a sudden increase in the fluid pressure above the initial equilibrium osmotic pressure. These new findings are relevant and necessary for the understanding of cartilage mechanics, cartilage biosynthesis, electromechanical signal transduction by chondrocytes, and tissue engineering.     

Analysis of the permeation of cryoprotectants in cartilage

Some tissues, such as cartilage and cornea, carry an internal fixed negative charge, leading to a swelling pressure that is balanced by tensile stress in the tissue matrix. During the addition and removal of cryoprotectants the changes in osmotic pressure will cause the tissue to deform. Because of the fixed charge and osmotic deformation, the permeation process in such tissues differs from ordinary diffusion processes. In this paper a biomechanical multi-solute theory is introduced to describe this process in cartilage tissue. Typical values for the physiological and biomechanical properties are used in the simulation. Several parameters - the aggregate modulus, the fixed charge density and the frictional parameter - are analyzed to show their impact on the process. It is shown that friction between water and cryoprotectant has the greatest influence but the fixed charge density is also important. The aggregate modulus and the frictional parameter between the cryoprotectant and the solid matrix have the least influence. Both the new biomechanical model and the conventional diffusion model were fitted to published experimental data concerning the time course of mean tissue cryoprotectant concentration when cartilage is immersed in solutions of dimethyl sulphoxide or propylene glycol: in all cases and with both models a good fit was obtained only when a substantial amount of non-solvent water was assumed.     

Studies of hydration and swelling pressure in normal and osteoarthritic cartilage

An experimental study was carried out which involved comparing cartilage from normal and osteoarthritic joints with respect to (a) swelling pressure and (b) variation of hydration with applied pressure. The main conclusion was that whilst osteoarthritic cartilage is undoubtedly less able to resist water loss under a given applied pressure than normal cartilage, this is not due to a change in the "quality" of the proteoglycans, resulting in a change in the osmotic pressure of the latter, but simply to a decreased fixed charge density. The latter decrease is either caused by an increase in the water content - and this we attribute to a weakened collagen network - and/or to a loss of part of the proteoglycans from the tissue.     

Nonuniform swelling-induced residual strains in articular cartilage

Swelling effects in cartilage originate from an interstitial osmotic pressure generated by the presence of negatively charged proteoglycans in the tissue. This swelling pressure gives rise to a non-zero residual strain in the cartilage solid matrix in the absence of externally applied loads. Previous studies have quantified swelling effects in cartilage as volumetric or dimensional change of excised samples in varying osmotically active solutions. This study presents a new optical technique for measuring two-dimensional swelling-induced residual strain fields in planar samples of articular cartilage attached to the bone (i.e., in situ). Osmotic loading was applied to canine cartilage bone samples by equilibration in external baths of varying NaCl concentration. Non-zero swelling-induced strains were measured in physiological saline, giving evidence of the existence of residual strains in articular cartilage. Only one component of planar strain (i.e., in thickness direction) was found to be non-zero. This strain was found to be highly non-uniform in the thickness direction, with evidence of compressive strain in the deep zone of cartilage and tensile strain in the middle and surface zones. The obtained results can be used to characterize the material properties of the articular cartilage solid matrix, with estimated values of 26 M Pa for the tensile modulus for middle zone cartilage. The method provides the basis to obtain material properties of the cartilage solid matrix from a simple, free-swelling test and may be useful for quantifying changes in cartilage properties with injury, degeneration and repair.     

Relative contribution of articular cartilage’s constitutive components to load support depending on strain rate

Cartilage is considered a biphasic material in which the solid is composed of proteoglycans and collagen. In biphasic tissue, the hydraulic pressure is believed to bear most of the load under higher strain rates and its dissipation due to fluid flow determines creep and relaxation behavior. In equilibrium, hydraulic pressure is zero and load bearing is transferred to the solid matrix. The viscoelasticity of the collagen network also contributes to its time-dependent behavior, and the osmotic pressure to load bearing in equilibrium. The aim of the present study was to determine the relative contributions of hydraulic pressure, viscoelastic collagen stress, solid matrix stiffness and osmotic pressure to load carriage in cartilage under transient and equilibrium conditions. Unconfined compression experiments were simulated using a fibril-reinforced poroviscoelastic model of articular cartilage, including water, fibrillar viscoelastic collagen and non-fibrillar charged glycosaminoglycans. The relative contributions of hydraulic and osmotic pressures and stresses in the fibrillar and non-fibrillar network were evaluated in the superficial, middle and deep zone of cartilage under five different strain rates and after relaxation. Initially upon loading, the hydraulic pressure carried most of the load in all three zones. The osmotic swelling pressure carried most of the equilibrium load. In the surface zone, where the fibers were loaded in tension, the collagen network carried 20 % of the load for all strain rates. The importance of these fibers was illustrated by artificially modifying the fiber architecture, which reduced the overall stiffness of cartilage in all conditions. In conclusion, although hydraulic pressure dominates the transient behavior during cartilage loading, due to its viscoelastic nature the superficial zone collagen fibers carry a substantial part of the load under transient conditions. This becomes increasingly important with higher strain rates. The interesting and striking new insight from this study suggests that under equilibrium conditions, the swelling pressure generated by the combination of proteoglycans and collagen reinforcement accounts cartilage stiffness for more than 90 % of the loads carried by articular cartilage. This finding is different from the common thought that load is transferred from fluid to solid and is carried by the aggregate modulus of the solid. Rather, it is transformed from hydraulic to osmotic swelling pressure. These results show the importance of considering both (viscoelastic) collagen fibers as well as swelling pressure in studies of the (transient) mechanical behavior of cartilage.     

比较三种防冰晶法对切片保存效果的影响

目的 比较冰冻切片技术中几种防冰晶方法对切片保存效果的影响。方法 分别使用液氮骤冷组织、高渗透压脱水法、单纯OCT胶包埋等几种方法后行冰冻切片、HE染色后,分别于当天、1、3、6个月后比较其染色效果,选出最佳保存方法。结果 单纯OCT包埋法在1个月后染色明显变浅,冰晶数量最多,而在3、6个月后染色基本接近背底色,冰晶面积已占据近半个视野,组织结构破坏极其严重;高渗透压脱水法在1个月后染色深度及冰晶数量上无明显变化,但3、6个月后则明显变浅,冰晶数量明显增加;而低温骤冷法在染色后的几个月,染色深度及冰晶的数量均无明显的变化,结构基本维持染色当天的状态。结论 低温骤冷法是这三种防冰晶法中最利于保存切片的方法。     

Comparison of the effects of mechanical and osmotic pressures on the collagen fiber architecture of intact and proteoglycan-depleted articular cartilage

One of the functions of articular cartilage is to withstand recurrent pressure applied in everyday life. In previous studies, osmotic pressure has been used to mimic the effects of mechanical pressure. In the present study, the response of the collagen network of intact and proteoglycans (PG)-depleted cartilage to mechanical and osmotic pressures is compared. The technique used is one-dimensional ²H double quantum filtered spectroscopic MRI, which gives information about the degree of order and the density of the collagen fibers at the different locations throughout the intact tissue. For the nonpressurized plugs, the depletion had no effect on these parameters. Major differences were found in the zones near the bone between the effects of the two types of application of pressure for both intact and depleted plugs. While the order is lost in these zones as a result of mechanical load, it is preserved under osmotic pressure. For both intact and PG-depleted plugs under osmotic stress most of the collagen fibers become disordered. Our results indicate that different modes of strain are produced by unidirectional mechanical load and the isotropic osmotic stress. Thus, osmotic stress cannot serve as a model for the effect of load on cartilage in vivo. This paper is presented in part in ISMRM 11th Scientific Meeting, p. 55, 2003 and ISMRM 14th Scientific Meeting, p. 59, 2006. Dedicated to Prof. K. Arnold on the occasion of his 65th birthday.     

Hemoglobin and myoglobin embedded in dry poly(vinyl alcohol) film for X-ray absorption studies

Solid hemoprotein samples are prepared by embedding proteins in thin poly(vinyl alcohol) films. These film samples have several unique qualities: in the solid poly(vinyl alcohol) matrix, hemoproteins have the same properties as in frozen buffer solution (proven by optical absorption, ligand recombination kinetics and EXAFS); they are very stable, easy to store and resistant to radiation; damage; protein concentration can be as high as 15 mM; light transparency is as good as liquid solution samples; and they can be made as thin as 20 microns, so that 100% photolysis across a film, even with a high protein concentration, is easily achievable. The film samples are ideal for X-ray studies of optically illuminated hemoproteins.     

High osmotic stress behavior of hyaluronate and heparin.

Using polyethylene glycol and dextran as osmotic stressing agents, the concentrations of hyaluronate and heparin were measured as a function of osmotic pressure II over the range of 0.03 to nearly 50 atmospheres. The experimental results were analyzed in terms of the Donnan osmotic pressure, the virial expansion, and Flory's first neighbor interaction parameter. In addition, II was looked at as a function of the reciprocal cube root of the concentration, which represents an average intermonomer spacing at high concentrations. The decay lengths in the so-called hydration region were found to be around 2.6 A and negligibly salt dependent. In the electrostatically dominated region the decay lengths were found to be dependent on the ionic strength, but not simply so. The osmotic compressibilities were also calculated, and were compared to compressibility data of corneal stroma and articular cartilage. These latter compressibilities were close to those for the pure hyaluronate and heparin, strengthening the evidence that glycosaminoglycans (GAGs) are largely responsible for connective tissue compressibility. Higher compressibilities for previously reported GAG data is thought to be related to the protein content of those samples.     

Friction coefficients for mechanically damaged bovine articular cartilage

We used a pin-on-disc tribometer to measure the friction coefficient of both pristine and mechanically damaged cartilage samples in the presence of different lubricant solutions. The experimental set up maximizes the lubrication mechanism due to interstitial fluid pressurization. In phosphate buffer solution (PBS), the measured friction coefficient increases with the level of damage. The main result is that when poly(ethylene oxide) (PEO) or hyaluronic acid (HA) are dissolved in PBS, or when synovial fluid (SF) is used as lubricant, the friction coefficients measured for damaged cartilage samples are only slightly larger than those obtained for pristine cartilage samples, indicating that the surface damage is in part alleviated by the presence of the various lubricants. Among the lubricants considered, 100 mg/mL of 100,000 Da MW PEO in PBS appears to be as effective as SF. We attempted to discriminate the lubrication mechanism enhanced by the various compounds. The lubricants viscosity was measured at shear rates comparable to those employed in the friction experiments, and a quartz crystal microbalance with dissipation monitoring was used to study the adsorption of PEO, HA, and SF components on collagen type II adlayers pre-formed on hydroxyapatite. Under the shear rates considered the viscosity of SF is slightly larger than that of PBS, but lower than that of lubricant formulations containing HA or PEO. Neither PEO nor HA showed strong adsorption on collagen adlayers, while evidence of adsorption was found for SF. Combined, these results suggest that synovial fluid is likely to enhance boundary lubrication. It is possible that all three formulations enhance lubrication via the interstitial fluid pressurization mechanism, maximized by the experimental set up adopted in our friction tests.     

Poly(vinyl alcohol) hydrogel as a biocompatible viscoelastic mimetic for articular cartilage

The prevalence of suboptimal outcome for surgical interventions in the treatment of full-thickness articular cartilage damage suggests that there is scope for a materials-based strategy to deliver a more durable repair. Given that the superficial layer of articular cartilage creates and sustains the tribological function of synovial joints, it is logical that candidate materials should have surface viscoelastic properties that mimic native articular cartilage. The present paper describes force spectroscopy analysis by nano-indentation to measure the elastic modulus of the surface of a novel poly(vinyl alcohol) hydrogel with therapeutic potential as a joint implant. More than 1 order of magnitude decrease in the elastic modulus was detected after adsorption of a hyaluronic acid layer onto the hydrogel, bringing it very close to previously reported values for articular cartilage. Covalent derivatization of the hydrogel surface with fibronectin facilitated the adhesion and growth of cultured rat tibial condyle chondrocytes as evidenced morphologically and by the observance of metachromatic staining with toluidine blue dye. The present results indicate that hydrogel materials with potential therapeutic benefit for injured and diseased joints can be engineered with surfaces with biomechanical properties similar to those of native tissue and are accepted as such by their constituent cell type.     

Modification of cellulosic fabric using polyvinyl alcohol—Part-I: Physicochemical properties

A series of poly(vinyl alcohol) of different commercial grades were prepared and applied onto the surfaces of cotton and blends of cotton/polyester fibers. The molecular structure was confirmed using Fourier Transform Infrared spectroscopy. Physicochemical properties such as viscosity and solid contents (%) were determined and discussed. Factors affecting the performance properties of the finished substrate such as post-treatment with poly(vinyl alcohol) of different grades, concentration and dilutions were studied. Fixation of the poly(vinyl alcohol) onto/or within the cellulose structure is accompanied by the formation of semi-inter-penetrated network structure thereby enhancing the association as well as providing very high stiffness. The results revealed that applications of poly(vinyl alcohol) on the textile fabrics in the finishing processes enables to enhance the stiffness as well as helps to improve its pilling resistance.     

Measurement of the water potential of stored potato tubers

A method of measuring the water potential of stored potato tubers (Solanum tuberosum L.) was needed to investigate the relationship of bacterial soft rot in tubers to water potential. Pressure chamber measurements, while useful for tubers with functional stolons, cannot be made on stored tubers. Measurements could be made on excised tissue pieces in a hygrometer chamber and with hygrometers implanted into tubers. We report here our evaluation of these hygrometric methods using a comparison with the pressure chamber on tubers harvested with stolons intact.

In tubers of high water potential, measurements on excised tissue were as much as 0.5 megapascals lower than the pressure chamber, probably due to turgor-driven expansion of the sample when freed from constraints imposed by surrounding tissue. Good agreement (±0.05 megapascals) was found between the implanted hygrometer and the pressure chamber at potentials higher than −0.5 megapascals. At lower water potentials, both hygrometer measurements were higher than the pressure chamber. Respirational heating of the tissue contributed to the increase in the excised tissue samples, but not with the implanted hygrometers because of the hygrometer design. The osmotic pressure balanced the pressure chamber measurement of potential at −0.7 megapascals, but was too small to do so at lower potentials. At most, 25% of this discrepancy can be accounted for by dilution by apoplastic water. We believe that the pressure chamber measurement is too low at low water potentials and that the error is associated with air bubbles in the xylem. At low potentials air emerged from xylem vessels along with sap, and fewer xylem emitted sap as potentials decreased.

     

The effect of osmotic and mechanical pressures on water partitioning in articular cartilage

X-ray diffraction measurements on native and proteoglycan-free articular cartilage have been made in order to test the dependence of the lateral packing of the collagen molecules on the osmotic pressure gradient, either naturally occurring or externally applied, between the intra- and extrafibrillar compartments. From the information on collagen packing we have been able to calculate, albeit with several assumptions, the amount of intrafibrillar water as a function of pressure. In parallel with the above measurements, we have quantitated, using serum albumin partitioning, the intrafibrillar water in proteoglycan-free cartilage, as a function of mechanically applied pressure. The results of both sets of experiments lead to the conclusion that the molecular packing density, and hence the intrafibrillar water content, are a function of the osmotic pressure difference between the extrafibrillar and intrafibrillar spaces or the equivalent mechanically applied pressure. The determination of intrafibrillar water has enabled us to calculate, from measured values of fixed charge density, the internal osmotic pressure of cartilage specimens, both in compressed and uncompressed states.     

Non-mulberry silk sericin/poly (vinyl alcohol) hydrogel matrices for potential biotechnological applications

This study reports a novel biopolymeric matrix fabricated by chemically cross-linking poly (vinyl alcohol) with silk sericin protein obtained from cocoons of the tropical tasar silkworm Antheraea mylitta. Glutaraldehyde was used as a cross-linking agent with hydrochloric acid acting as an initiator. The matrices were biophysically characterized and the cytocompatibility of the matrices was evaluated for their suitability as biomaterials. The surface morphology was assessed using atomic force microscopy while the changes taking place after cross-linking were confirmed by Fourier transform infrared spectroscopy. The enhanced thermal stability of the constructs was assessed by thermogravimetric and differential scanning calorimetry. Fourier transform infrared spectroscopy analysis showed that sericin was chemically cross-linked with poly (vinyl alcohol) using glutaraldehyde. Silk sericin protein demonstrated a favorable effect on animal cell culture by successfully improving the adhering and spreading of cells on the poorly adhering surface of poly (vinyl alcohol). Confocal microscopy revealed cell spreading and actin filament development in sericin/poly (vinyl alcohol) hydrogel matrices. These findings prove the potential of non-mulberry silk sericin/poly (vinyl alcohol) hydrogel matrices to be used as biocompatible and biopolymeric material for tissue-engineering and biotechnological applications.