Functional performance of a bi-layered chitosan-nano-hydroxyapatite osteochondral scaffold: a pre-clinical in vitro tribological study.

Osteochondral grafts are used for repair of focal osteochondral lesions. Autologous grafts are the gold standard treatment; however, limited graft availability and donor site morbidity restrict use. Therefore, there is a clinical need for different graft sources/materials which replicate natural cartilage function. Chitosan has been proposed for this application. The aim of this study was to assess the biomechanics and biotribology of a bioresorbable chitosan/chitosan-nano-hydroxyapatite osteochondral construct (OCC), implanted in an in vitro porcine knee experimental simulation model. The OCC implanted in different surgical positions (flush, proud and inverted) was compared to predicate grafts in current clinical use and a positive control consisting of a stainless steel graft implanted proud of the cartilage surface. After 3 h (10 800 cycles) wear simulation under a walking gait, subsidence occurred in all OCC samples irrespective of surgical positioning, but with no apparent loss of material and low meniscus wear. Half the predicate grafts exhibited delamination and scratching of the cartilage surfaces. No graft subsidence occurred in the positive controls but wear and deformation of the meniscus were apparent. Implanting a new chitosan-based OCC either optimally (flush), inverted or proud of the cartilage surface resulted in minimal wear, damage and deformation of the meniscus.

Development and in vitro assessment of a bi-layered chitosan-nano-hydroxyapatite osteochondral scaffold.

An innovative approach was developed to engineer a multi-layered chitosan scaffold for osteochondral defect repair. A combination of freeze drying and porogen-leaching out methods produced a porous, bioresorbable scaffold with a distinct gradient of pore size (mean = 160-275 µm). Incorporation of 70 wt% nano-hydroxyapatite (nHA) provided additional strength to the bone-like layer. The scaffold showed instantaneous mechanical recovery under compressive loading and did not delaminate under tensile loading. The scaffold supported the attachment and proliferation of human mesenchymal stem cells (MSCs), with typical adherent cell morphology found on the bone layer compared to a rounded cell morphology on the chondrogenic layer. Osteogenic and chondrogenic differentiation of MSCs preferentially occurred in selected layers of the scaffold in vitro, driven by the distinct pore gradient and material composition. This scaffold is a suitable candidate for minimally invasive arthroscopic delivery in the clinic with potential to regenerate damaged cartilage and bone.

Evaluating the cytotoxicity of Ge-Sb-Se chalcogenide glass optical fibres on 3T3 mouse fibroblasts.

In vivo cancer detection based on the mid-infrared molecular fingerprint of tissue is promising for the fast diagnosis and treatment of suspected cancer patients. Few materials are mid-infrared transmissive, even fewer, which can be converted into functional, low-loss optical fibres for in vivo non-invasive testing. Chalcogenide-based glass optical fibres are, however, one of the few. These glasses are transmissive in the mid-infrared and are currently under development for use in molecular sensing devices. The cytotoxicity of these materials is however unknown. The cytotoxicity of Ge-Sb-Se chalcogenide optical glass fibres on 3T3 mouse fibroblast cells is here investigated. Fibres exposed to four different pre-treatment conditions are used: as-drawn (AD), propylamine-etched (PE), oxidised-and-washed (OW) and oxidised (Ox). To achieve the latter two conditions, fibres are treated with H2O2(aqueous (aq.)) and dried to produce a surface oxide layer; this is either washed off (OW) or left on the glass surface (Ox). Cellular response is investigated via 3 day elution and 14 day direct contact trials. The concentration of the metalloids (Ge, Sb and Se) in each leachate was measured via inductively coupled plasma mass spectrometry. Cell viability is assessed using the neutral red assay and scanning electron microscopy. The concentration of Ge, Sb and Se ions after a 3 day dissolution was as follows. In AD leachates, Ge: 0.40 mg L-1, Sb: 0.17 mg L-1, and Se: 0.06 mg L-1. In PE leachates, Ge: 0.22 mg L-1, Sb: 0.15 mg L-1, and Se: 0.02 mg L-1. In Ox leachates, Ge: 823.8 mg L-1, Sb: 2586.6 mg L-1, and Se: 3750 mg L-1. Direct contact trials show confluent cell layers on AD, PE and OW fibres after 14 days, while no cells are observed on the Ox surfaces. A >50% cell viability is observed in AD, PE and OW eluates after 3 days, when compared with Ox eluates (<10% cell viability). Toxicity in Ox is attributed to the notable pH change, from neutral pH 7.49 to acidic pH 2.44, that takes place on dissolution of the surface oxide layer in the growth media. We conclude, as-prepared Ge-Sb-Se glasses are cytocompatible and toxicity arises when an oxide layer is forced to develop on the glass surface.

Preclinical biological and physicochemical evaluation of two-photon engineered 3D biomimetic copolymer scaffolds for bone healing.

A major challenge in orthopedics is the repair of large non-union bone fractures. A promising therapy for this indication is the use of biodegradable bioinspired biomaterials that stabilize the fracture site, relieve pain and initiate bone formation and healing. This study uses a multidisciplinary evaluation strategy to assess immunogenicity, allergenicity, bone responses and physicochemical properties of a novel biomaterial scaffold. Two-photon stereolithography generated personalized custom-built scaffolds with a repeating 3D structure of Schwarz Primitive minimal surface unit cell with a specific pore size of ∼400 µm from three different methacrylated poly(d,l-lactide-co-ε-caprolactone) copolymers with lactide to caprolactone monomer ratios of 16 : 4, 18 : 2 and 9 : 1. Using in vitro and in vivo assays for bone responses, immunological reactions and degradation dynamics, we found that copolymer composition influenced the scaffold physicochemical and biological properties. The scaffolds with the fastest degradation rate correlated with adverse cellular effects and mechanical stiffness correlated with in vitro osteoblast mineralization. The physicochemical properties also correlated with in vivo bone healing and immune responses. Overall these observations provide compelling support for these scaffolds for bone repair and illustrate the effectiveness of a promising multidisciplinary strategy with great potential for the preclinical evaluation of biomaterials.

In vitro cellular testing of strontium/calcium substituted phosphate glass discs and microspheres shows potential for bone regeneration.

Phosphate-based glasses (PBGs) are ideal materials for regenerative medicine strategies because their composition, degradation rates, and ion release profiles can easily be controlled. Strontium has previously been found to simultaneously affect bone resorption and deposition. Therefore, by combining the inherent properties of resorbable PBG and therapeutic activity of strontium, these glasses could be used as a delivery device of therapeutic factors for the treatment of orthopaedic diseases such as osteoporosis. This study shows the cytocompatibility and osteogenic potential of PBGs where CaO is gradually replaced by SrO in the near invert glass system 40P2 O5 ·(16-x)CaO·20Na2 O·24MgO·xSrO (x = 0, 4, 8, 12, and 16 mol%). Direct seeding of MG63 cells onto glass discs showed no significant difference in cell metabolic activity and DNA amount measurement across the different formulations studied. Cell attachment and spreading was confirmed via scanning electron microscopy (SEM) imaging at Days 3 and 14. Alkaline phosphatase (ALP) activity was similarly maintained across the glass compositions. Follow-on studies explored the effect of each glass composition in microsphere conformation (size: 63-125 µm) on human mesenchymal stem cells (hMSCs) in 3D cultures, and analysis of cell metabolic activity and ALP activity showed no significant differences at Day 14 over the compositional range investigated, in line with the observations from MG63 cell culture studies. Environmental SEM and live cell imaging at Day 14 of hMSCs seeded on the microspheres showed cell attachment and colonisation of the microsphere surfaces, confirming these formulations as promising candidates for regenerative medicine strategies addressing compromised musculoskeletal/orthopaedic diseases.

Topographical and chemical effects of electrochemically assisted deposited hydroxyapatite coatings on osteoblast-like cells.

A recently commercialised hydroxyapatite electrochemically assisted chemical deposition technique (BoneMaster) has been shown to induce increased bone apposition; whether this response is caused by the surface topography or chemistry is unknown. An in-vitro examination using human osteoblast-like cells was performed on a series of BoneMaster-coated surfaces. The chemistry was separated from the topography using a thin gold coating; Thermanox coverslips were used as a control. BoneMaster surfaces showed significantly greater alkaline phosphatase activity and osteocalcin production compared with controls; however, no difference was found between the gold-coated and uncoated BoneMaster samples, indicating topography is the main contributing factor.

Development of an elastic cell culture substrate for a novel uniaxial tensile strain bioreactor.

Bioreactors can be used for mechanical conditioning and to investigate the mechanobiology of cells in vitro. In this study a polyurethane (PU), Chronoflex AL, was evaluated for use as a flexible cell culture substrate in a novel bioreactor capable of imparting cyclic uniaxial tensile strain to cells. PU membranes were plasma etched, across a range of operating parameters, in oxygen. Contact angle analysis and X-ray photoelectron spectroscopy showed increases in wettability and surface oxygen were related to both etching power and duration. Atomic force microscopy demonstrated that surface roughness decreased after etching at 20 W but was increased at higher powers. The etching parameters, 20 W 40 s, produced membranes with high surface oxygen content (21%), a contact angle of 66° ± 7° and reduced topographical features. Etching and protein conditioning membranes facilitated attachment, and growth to confluence within 3 days, of MG-63 osteoblasts. After 2 days with uniaxial strain (1%, 30 cycles/min, 1500 cycles/day), cellular alignment was observed perpendicular to the principal strain axis, and found to increase after 24 h. The results indicate that the membrane supports culture and strain transmission to adhered cells.

Effect of boron addition on the thermal, degradation, and cytocompatibility properties of phosphate-based glasses.

In this study eight different phosphate-based glass compositions were prepared by melt-quenching: four in the (P2O5)45-(CaO)16-(Na2O)15-x -(MgO)24-(B2O3) x system and four in the system (P2O5)50-(CaO)16-(Na2O)10-x -(MgO)24-(B2O3) x , where x = 0,1, 5 and 10 mol%. The effect of B2O3 addition on the thermal properties, density, molar volume, dissolution rates, and cytocompatibility were studied for both glass systems. Addition of B2O3 increased the glass transition (T(g)), crystallisation (T(c)), melting (T(m)), Liquidus (T(L)) and dilatometric softening (T(d)) temperature and molar volume (V(m)). The thermal expansion coefficient (α) and density (ρ) were seen to decrease. An assessment of the thermal stability of the glasses was made in terms of their processing window (crystallisation onset, T(c,ons) minus glass transition temperature, T(g)), and an increase in the processing window was observed with increasing B2O3 content. Degradation studies of the glasses revealed that the rates decreased with increasing B2O3 content and a decrease in degradation rates was also observed as the P2O5 content reduced from 50 to 45 mol%. MG63 osteoblast-like cells cultured in direct contact with the glass samples for 14 days revealed comparative data to the positive control for the cell metabolic activity, proliferation, ALP activity, and morphology for glasses containing up to 5 mol% of B2O3.

Investigating the use of coupling agents to improve the interfacial properties between a resorbable phosphate glass and polylactic acid matrix.

Eight different chemicals were investigated as potential candidate coupling agents for phosphate glass fibre reinforced polylactic acid composites. Evidence of reaction of the coupling agents with phosphate glass and their effect on surface wettability and glass degradation were studied along with their principle role of improving the interface between glass reinforcement and polymer matrix. It was found that, with an optimal amount of coupling agent on the surface of the glass/polymer, interfacial shear strength improved by a factor of 5. Evidence of covalent bonding between agent and glass was found for three of the coupling agents investigated, namely: 3-aminopropyltriethoxysilane; etidronic acid and hexamethylene diisocyanate. These three coupling agents also improved the interfacial shear strength and increased the hydrophobicity of the glass surface. It is expected that this would provide an improvement in the macroscopic properties of full-scale composites fabricated from the same materials which may also help to retain these properties for the desired length of time by retarding the breakdown of the fibre/matrix interface within these composites.

Influence of screw holes and gamma sterilization on properties of phosphate glass fiber-reinforced composite bone plates.

Polymers prepared from polylactic acid (PLA) have found a multitude of uses as medical devices. For a material that degrades, the main advantage is that an implant would not necessitate a second surgical event for removal. In this study, fibers produced from a quaternary phosphate-based glass (PBG) in the system 50P2O5-40CaO-5Na2O-5Fe2O3 were used to reinforce PLA polymer. The purpose of this study was to assess the effect of screw holes in a range of PBG-reinforced PLA composites with varying fiber layup and volume fraction. The flexural properties obtained showed that the strength and modulus values increased with increasing fiber volume fraction; from 96 MPa to 320 MPa for strength and between 4 GPa and 24 GPa for modulus. Furthermore, utilizing a larger number of thinner unidirectional (UD) fiber prepreg layers provided a significant increase in mechanical properties, which was attributed to enhanced wet out and thus better fiber dispersion during production. The effect of gamma sterilization via flexural tests showed no statistically significant difference between the sterilized and nonsterilized samples, with the exception of the modulus values for samples with screw holes. Degradation profiles revealed that samples with screw holes degraded faster than those without screw holes due to an increased surface area for the plates with screw holes in PBS up to 30 days. Scanning electron microscope (SEM) analysis revealed fiber pullout before and after degradation. Compared with various fiber impregnation samples, with 25% volume fraction, 8 thinner unidirectional prepreg stacked samples had the shortest fiber pull-out lengths in comparison to the other samples investigated.

Mechanical properties of epidermal cells of whole living roots of Arabidopsis thaliana: an atomic force microscopy study.

The knowledge of mechanical properties of root cell walls is vital to understand how these properties interact with relevant genetic and physiological processes to bring about growth. Expansion of cell walls is an essential component of growth, and the regulation of cell wall expansion is one of the ways in which the mechanics of growth is controlled, managed and directed. In this study, the inherent surface mechanical properties of living Arabidopsis thaliana whole-root epidermal cells were studied at the nanoscale using the technique of atomic force microscopy (AFM). A novel methodology was successfully developed to adapt AFM to live plant roots. Force-Indentation (F-I) experiments were conducted to investigate the mechanical properties along the length of the root. F-I curves for epidermal cells of roots were also generated by varying turgor pressure. The F-I curves displayed a variety of features due to the heterogeneity of the surface. Hysteresis is observed. Application of conventional models to living biological systems such as cell walls in nanometer regimes tends to increase error margins to a large extent. Hence information from the F-I curves were used in a preliminary semiquantitative analysis to infer material properties and calculate two parameters. The work done in the loading and unloading phases (hysteresis) of the force measurements were determined separately and were expressed in terms of "Index of Plasticity" (η), which characterized the elasticity properties of roots as a viscoelastic response. Scaling approaches were used to find the ratio of hardness to reduced modulus (H/E(*)).

Mono-functional aminosilanes as primers for peptide functionalization.

When covalently attaching biomolecules to surfaces such as titanium, trifunctional silanes are commonly used as primers to produce surface amine groups. However, these primed surfaces are rarely uniform in structure due to networking of the silane. Mono-functional aminosilanes may result in more uniform structures, although their long-term stability and effect on osteoblast cell responses are possible issues for orthopedic applications. This study examines for the first time the optimization of peptide coupling to titanium using mono-functional aminosilane reaction chemistry. The resultant surface topography, chemistry, and thicknesses were characterized showing improved surface uniformity compared with trifunctional silanized surfaces. The stability of the coatings was examined over a period of 8 days in environments of varying pH, temperature, and humidity. In addition, human osteosarcoma (HOS) cell adhesion and spreading on the samples was examined; adhesion was minimal on silanized surfaces, but after functionalization with cysteine the cell density was greater than the titanium control and showed no overall detrimental effect on initial cell responses.

In vitro study of hydroxyapatite-based photocurable polymer composites prepared by laser stereolithography and supercritical fluid extraction.

The fabrication of three-dimensional (3-D) structures using computer-controlled ultraviolet (UV) photopolymerization of acrylates (laser stereolithography) often results in the trapping of residual unreacted monomer and initiator. These residuals can leach from the finished structure and affect the biological response of cells and tissues. Thus the potential applications of these structures for tissue engineering have not been fully realized. In this paper we demonstrate that conventional post-lithography treatments followed by processing in the environmentally benign solvent, supercritical carbon dioxide (scCO(2)), dramatically increased biocompatibility. The scCO(2) processing of pure polyacrylate and polyacrylate/hydroxyapatite composite structures extracts residuals from all structures including those that had received full conventional post-lithography treatment (acetone washing/UV drying). Human osteoblast cells seeded on the extracted surfaces of these structures demonstrated increased cell attachment and proliferation on the scCO(2)-treated materials.

The effect of different surface morphology and roughness on osteoblast-like cells.

Increased magnitude of biomaterial surface roughness and micromachined-grooved surfaces has both been shown to stimulate osteoblast activity, but have not been compared in the same study quantitatively. A series of titanium alloy (Ti6Al4V) samples were prepared using simple machining techniques to undertake such a comparison. Samples were either grit blasted (Gb) or shot peened (Sp) to give random discontinuities, or silicon carbide ground (SiC) to produce ordered grooves. These were compared with micropolished samples (Mp). The samples were coated with a 1 mum continuous coating of hydroxyapatite to remove differences in surface chemistry. Human osteoblast-like cells were seeded onto the materials and metabolic activity, proliferation, alkaline phosphatase activity, and osteocalcin production assessed. Cell responses were highly dependent on the substrate that they were cultured on. Cells cultured on the smooth and ordered (Mp and SiC, respectively) samples had higher metabolic activity and a more elongated morphology than those cultured on the randomly structured Gb or Sp samples. Over 21 days, cell metabolic activity peaked relative to the control between 7 and 14 days on the Mp sample, and between 14 and 21 days on the Gb, Sp, and SiC samples. In common with other researchers, we note that micron scale topography may have potential for influencing osseointegration. More importantly, as the magnitude of the discontinuities on SiC, Gb, and Sp were similar, the differences in cell responses does not appear to lie with the size of the features, but whether the features showed an ordered or disordered structure.

Human osteoblast cell spreading and vinculin expression upon biomaterial surfaces.

Any biomaterial implanted within the human body is influenced by the interactions that take place between its surface and the surrounding biological milieu. These interactions are known to influence the tissue interface dynamic, and thus act to emphasize the need to study cell-surface interactions as part of any biomaterial design process. The work described here investigates the relationship between human osteoblast attachment, spreading and focal contact formation on selected surfaces using immunostaining and digital image processing for vinculin, a key focal adhesion component. Our observations show that a relationship exists between levels of cell attachment, the degree of vinculin-associated plaque formation and biocompatibility. It also suggests that cell adhesion is not indicative of how supportive a substrate is to cell spreading, and that cell spreading does not correlate with focal contact formation.

In vitro assessment of cell penetration into porous hydroxyapatite scaffolds with a central aligned channel.

There is a clinical need for synthetic scaffolds that promote bone regeneration. A common problem encountered when using scaffolds in tissue engineering is the rapid formation of tissue on the outer edge of the scaffold whilst the tissue in the centre becomes necrotic. To address this, the scaffold design should improve nutrient and cell transfer to the scaffold centre. In this study, hydroxyapatite scaffolds with random, open porosity (average pore size of 282+/-11microm, average interconnecting window size of 72+/-4microm) were manufactured using a modified slip-casting methodology with a single aligned channel inserted into the centre. By varying the aligned channel diameter, a series of scaffolds with channel diameters ranging from 170 to 421microm were produced. These scaffolds were seeded with human osteosarcoma (HOS TE85) cells and cultured for 8 days. Analysis of cell penetration into the aligned channels revealed that cell coverage increased with increasing channel diameter; from 22+/-3% in the 170microm diameter channel to 38+/-6% coverage in the 421microm channel. Cell penetration into the middle section of the 421microm diameter channel (average cell area coverage 121x10(3)+/-32x10(3)microm(2)) was significantly greater than that observed within the 170microm channel (average cell area coverage 26x10(3)+/-6x10(3)microm(2)). In addition, the data presented demonstrates that the minimum channel (or pore) diameter required for cell penetration into such scaffolds is approximately 80microm. These results will direct the development of scaffolds with aligned macroarchitecture for tissue engineering bone.

Comparison of environmental scanning electron microscopy with high vacuum scanning electron microscopy as applied to the assessment of cell morphology.

The efficacy of conventional high vacuum scanning electron microscopy (SEM), environmental SEM (ESEM), and confocal laser scanning microscopy techniques in the assessment of cell-material interactions is compared. Specific attention is given to the application of these techniques in the assessment of the early morphological response of human osteoblast-like cells cultured on titanium dioxide. The processing of cells cultured for conventional high vacuum SEM leads to the loss of morphological features that are retained when using ESEM. The use of cytoskeletal labeling, viewed with confocal laser scanning microscopy, in conjunction with ESEM gives an indication of the changes to cell morphology as a consequence of incubation time in response to interactions at the biological/material interface.

Use of a novel carbon fibre composite material for the femoral stem component of a THR system: in vitro biological assessment.

A novel, low elastic modulus femoral component for THR has been developed using a composite of polyetheretherketone and carbon fibre. The objectives of this study were to investigate human osteoblast-like cell and macrophage responses to this material in vitro. Cells were grown on composite discs and controls. Osteoblast attachment and proliferation was not significantly different to that on Ti6Al4V. The levels of alkaline phosphatase activity, Type I collagen production and osteocalcin production were not significantly different to that on Ti6Al4V by the end of the experimental period. Hydrogen peroxide production by macrophages was significantly less than that detected for cells cultured on copper, but was still greater than that detected for cells cultured on tissue culture plastic and Ti6Al4V. Beta-glucoronidase activity was not significantly different to that detected for cells cultured on tissue culture plastic. The in vitro biocompatibility assessment of this composite undertaken in this study showed initial osteoblast attachment at least comparable to that of the tissue culture plastic and Ti6Al4V controls, with proliferation similar to the controls at all time points up to 11 days. Alkaline phosphatase activity was similar to that of Ti6Al4V but reduced compared to tissue culture plastic controls. Whilst hydrogen peroxide production by macrophages was raised on composite surfaces compared to controls, beta-glucoronidase activity and osteoblastic production of Type I collagen and osteocalcin were similar to levels detected on Ti6Al4V.

Cytotoxicity of glutaraldehyde crosslinked collagen/poly(vinyl alcohol) films is by the mechanism of apoptosis.

Collagen has been investigated as a potential natural biomaterial, because of its occurrence in the extracellular matrix. Collagen requires crosslinking in this context, by reagents that are often cytotoxic. Glutaraldehyde is one such agent that is potentially cytotoxic. The aim of this study was to determine the cause of poor cell attachment and growth on collagen/poly(vinyl alcohol) bioartificial composite films, when crosslinked with glutaraldehyde. Dehydrothermal crosslinking was used as a comparison. Human osteoblasts were observed to undergo apoptosis on glutaraldehyde crosslinked films dependent on concentration of collagen present. Higher collagen content resulted in higher levels of apoptosis with poor cell attachment and spreading of remaining cells. Post-treatment of films with 8% L-glutamic acid prevented the apoptotic response of osteoblasts and allowed attachment and spreading. The addition of 100 nM insulin-like growth factor-1 to the culture medium also prevented apoptosis. Glutaraldehyde toxicity of crosslinked collagen has been demonstrated in this study, the mechanism of which is apoptosis. This study indicates that poor biocompatibility and induction of apoptosis on collagen/poly(vinyl alcohol) films crosslinked by glutaraldehyde are attributed to glutaraldehyde components on the surface of the films (not residual glutaraldehyde), whose effects can be quenched by glutamic acid, and prevented by insulin-like growth factor-1.

Protein adsorption and human osteoblast-like cell attachment and growth on alkylthiol on gold self-assembled monolayers.

Protein adsorption and growth of primary human osteoblasts on self-assembled monolayers of alkylthiols on gold (SAMs) with carboxylic acid and hydroxyl and methyl termini were investigated. Single-component SAMs and SAMs patterned by photolithographic techniques were used. Cell growth on patterned SAMs demonstrated preferences for one pattern region in all combinations of alkylthiols, with the hierarchical preference COOH > OH > CH(3). Patterned SAMs and immunochemistry were used to investigate adsorption of fibronectin and albumin with respect to different alkylthiol termini. Fibronectin adsorption from both pure solution and serum containing cell culture medium (SDMEM) followed the sequence COOH > OH > CH(3). Albumin adsorption from pure solution followed the sequence OH > COOH > CH(3); from SDMEM the sequence was CH(3) > OH > COOH. Cell attachment to SAMs with the above termini, after preadsorption with fibronectin, albumin, or mixtures of fibronectin and albumin, was measured. Attachment was maximal on COOH-terminated SAMs precoated with fibronectin. Attachment to COOH was significantly reduced only when fibronectin was omitted from the protein preadsorption solution. On OH and CH(3) SAMs increasing the proportion of albumin in the solution was sufficient to significantly reduce cell attachment. The distribution vinculin and the integrins alpha(5)beta(1) and alpha(v)beta(3) indicated that focal contact formation by cells varied with alkylthiol termini in the following sequence: COOH > OH > CH(3).