Photocurable extracellular matrix sealant for cessation of venous hemorrhage.

Hemorrhage is the second leading cause of death in patients under 46 years of age in the United States. Cessation of hemorrhage prevents hemorrhagic shock and tissue hypoxia. Controlling the bleed via direct pressure or tourniquet is often the first line of defense, but long-term care requires staples, hemostatic agents, or sealants that seal the vessel and restore blood flow. Here, we compare a new photocurable extracellular matrix sealant (pcECM) with low, medium, and high crosslink density formulations to a commercially available fibrin-based sealant, TISSEEL®. pcECM has potential uses in surgical and remote settings due to room temperature storage conditions and fast preparation time. Here, we determine if pcECM sealant can stop venous hemorrhage in a murine model, adhere to the wound site in vivo throughout the wound-healing process, and has the mechanical properties necessary for stopping hemorrhage. Adjusting pcECM crosslinking density significantly affected viscosity, swelling, burst strength, tensile strength, and elasticity of the sealant. 3-Dimensional ultrasound volume segmentations showed pcECM degrades to 17 ± 8% of its initial implant volume by day 28. Initially, local hemodynamic changes were observed, but returned close to baseline levels by day 28. Acute inflammation was observed near the puncture site in pcECM implanted mice, and we observed inflammatory markers at the 14-day explant for both sealants. pcECM and fibrin sealant successfully sealed the vessel in all cases, and consistently degraded over 14-28 days. pcECM is a durable sealant with tunable mechanical properties and possible uses in hemorrhage control and other surgical procedures.

Active tissue adhesive activates mechanosensors and prevents muscle atrophy.

While mechanical stimulation is known to regulate a wide range of biological processes at the cellular and tissue levels, its medical use for tissue regeneration and rehabilitation has been limited by the availability of suitable devices. Here we present a mechanically active gel-elastomer-nitinol tissue adhesive (MAGENTA) that generates and delivers muscle-contraction-mimicking stimulation to a target tissue with programmed strength and frequency. MAGENTA consists of a shape memory alloy spring that enables actuation up to 40% strain, and an adhesive that efficiently transmits the actuation to the underlying tissue. MAGENTA activates mechanosensing pathways involving yes-associated protein and myocardin-related transcription factor A, and increases the rate of muscle protein synthesis. Disuse muscles treated with MAGENTA exhibit greater size and weight, and generate higher forces compared to untreated muscles, demonstrating the prevention of atrophy. MAGENTA thus has promising applications in the treatment of muscle atrophy and regenerative medicine.

Hemostatic Needles: Controlling Hemostasis Time by a Catecholamine Oxidative Pathway.

Most infectious human viruses are generally found in the bloodstream after being released by infected organs. Thus, hemorrhage in patients, whose blood contains infectious viruses might be a significant risk for secondary infections. In this work, a self-sealing hemostatic needle that causes no bleeding even after its removal is reported. The materials used for the self-sealing needles are inspired by mussel adhesive polysaccharide, chitosan-catechol, which shows a rapid phase transition from a solid phase (i.e., a thin film) to an adhesive gel upon coming into contact with blood. We found that the self-sealing time for the complete hemostasis depends on the oxidation pathway of the conjugated catechol. For high-temperature oxidation (i.e., 60 °C), Michael addition is a dominant oxidative coupling reaction, which weakens the chitosan-catechol attachment force on the needle surface. Thus, the film is easily transferred to the hemorrhaging sites, with the result that there is no bleeding even after a short injection time (<5 s). In contrast, during low-temperature oxidation (4 °C), Schiff base formation is dominant, which strengthens the film attachment force on the needle surface, resulting in continued bleeding owing to a dearth of tissue transfer after the injection.

A novel technique for preoperative localization of pulmonary nodules using a mixture of tissue adhesive and iohexol under computed tomography guidance: A 140 patient single-center study.

BACKGROUND: The increase in the incidence of pulmonary nodules has made computed tomography (CT) screening a requirement for diagnosis and treatment. Small pulmonary nodule detection during video-assisted thoracoscopic surgery (VATS) or thoracotomy is frequently challenging; however, accurate and efficient localization of nodules is critical for precise resection. Herein, we introduce and evaluate the feasibility and safety of a novel technique for preoperative pulmonary nodule localization. METHODS: From March 2018 to December 2019, 140 patients with 153 pulmonary nodules measuring <2 cm in diameter were enrolled in this study. Preoperative, CT-guided localization was performed on each nodule with an injected mixture of tissue adhesive and iohexol. Patient and nodule characteristics, localization data, complications, surgical data, and pathological results were analyzed. RESULTS: All 153 nodules in 140 patients were successfully marked preoperatively and detected during surgery (n = 153/153). Mean nodule size was 8.7 ± 2.6 mm, and mean distance from nodule to pleura was 7.9 ± 8.2 mm. The mean procedural time was 8.7 ± 1.0 min. Nine patients (6.4%) underwent two simultaneous nodule localizations and two patients (1.4%) underwent three simultaneous nodule localizations. Pneumothorax (17/140, 12.1%), pain (6/140, 4.3%), and pungent odor (5/140, 3.6%) were the major complications. No patient required further treatment, and no allergic reactions or embolisms were observed. CONCLUSIONS: Preoperative CT-guided nodule localization using a mixture of tissue adhesive and iohexol is an efficient technique for localizing small and impalpable pulmonary lesions, including multiple pulmonary nodules. Our study demonstrates that this novel method is safe and straightforward to implement.

Multifunctional Tissue-Adhesive Cryogel Wound Dressing for Rapid Nonpressing Surface Hemorrhage and Wound Repair.

Cryogels with tissue adhesion have great potential as wound dressings for rapid hemostasis for uncontrollable nonpressing surface hemorrhage and wound healing, but their use has not been reported previously. Herein, we designed a series of antibacterial and antioxidant tissue-adhesive cryogels based on quaternized chitosan (QCS) and polydopamine (PDA). These cryogels had good blood cell and platelet adhesion, enrichment, and activation properties for rapid nonpressing surface hemostasis and wound healing. The cryogels exhibited outstanding mechanical strength and easy removability, antioxidant activity, and NIR photothermal-enhanced antibacterial performance. The cryogels showed much better hemostasis than gauze and gelatin sponge in a standardized strip rat liver injury model, a standardized circular rabbit liver section model, and a pig skin laceration model. Furthermore, the excellent hemostatic performance of the QCS/PDA2.0 cryogel (containing 20 mg/mL QCS and 2.0 mg/mL PDA) for coagulopathic hemorrhages was confirmed in a standardized coagulation disorder rabbit circular liver section model. In addition, the QCS/PDA2.0 cryogel promoted rapid hemostasis in a deep noncompressible wound and a much better wound healing effect than a chitosan sponge and Tegaderm film in a full-thickness skin defect model. Overall, these multifunctional tissue-adhesive cryogels with excellent hemostatic performance and enhanced wound healing properties are suitable candidates for tissue-adhesive hemostat and wound healing dressings.

Fourier transform infrared spectroscopic imaging of wear and corrosion products within joint capsule tissue from total hip replacements patients.

Implant debris generated by wear and corrosion is a prominent cause of joint replacement failure. This study utilized Fourier transform infrared spectroscopic imaging (FTIR-I) to gain a better understanding of the chemical structure of implant debris and its impact on the surrounding biological environment. Therefore, retrieved joint capsule tissue from five total hip replacement patients was analyzed. All five cases presented different implant designs and histopathological patterns. All tissue samples were formalin-fixed and paraffin-embedded. Unstained, 5 µm thick sections were prepared. The unstained sections were placed on BaF2 windows and deparaffinized with xylene prior to analysis. FTIR-I data were collected at a spectral resolution of 4 cm-1 using an Agilent Cary 670 spectrometer coupled with Cary 620 FTIR microscope. The results of study demonstrated that FTIR-I is a powerful tool that can be used complimentary to the existing histopathological evaluation of tissue. FTIR-I was able to distinguish areas with different cell types (macrophages, lymphocytes). Small, but distinct differences could be detected depending on the state of cells (viable, necrotic) and depending on what type of debris was present (polyethylene [PE], suture material, and metal oxides). Although, metal oxides were mainly below the measurable range of FTIR-I, the infrared spectra of tissues exhibited noticeable difference in their presence. Tens of micrometer sized polyethylene particles could be easily imaged, but also accumulations of submicron particles could be detected within macrophages. FTIR-I was also able to distinguish between PE debris, and other birefringent materials such as suture. Chromium-phosphate particles originating from corrosion processes within modular taper junctions of hip implants could be identified and easily distinguished from other phosphorous materials such as bone. In conclusion, this study successfully demonstrated that FTIR-I is a useful tool that can image and determine the biochemical information of retrieved tissue samples over tens of square millimeters in a completely label free, nondestructive, and objective manner. The resulting chemical images provide a deeper understanding of the chemical nature of implant debris and their impact on chemical changes of the tissue within which they are embedded.

Bioadhesive and biodissolvable hydrogels consisting of water-swellable poly(acrylic acid)/poly(vinylpyrrolidone) complexes.

Films that can form bioadhesive hydrogels on wet biotissues absorbing blood or body fluids are useful for medical devices such as hemostats, adhesion barriers, wound dressings, and drug release devices. We focused on a hydrogen-bonding polymer complex consisting of poly(acrylic acid) (PAA) and poly(vinylpyrrolidone) (PVP). PAA is known as a tissue-adhesive polymer. However, simple mixing of aqueous PAA and PVP solutions resulted in the formation of an insoluble nonadhesive precipitate. We developed a novel solid/solution interface complexation method to afford a PAA/PVP complex that forms a strongly bioadhesive hydrogel with low cytotoxicity. The complex hydrogel can be slowly dissociated and dissolved in the body. The formation of the complexes as well as their swelling and degradation behavior depended strongly on the molecular weights and cross-linking densities of the component polymers. When the complex film was applied to a clipped incised jugular vein of a rat, it immediately formed a hydrogel and closed the incision. After removal of the clip, blood flowed through the vessel without any leakage. Application of the complex film to the surface of an incised mouse liver resulted in firm adhesion and the hemorrhage was effectively stopped. Such bioadhesive and biodissolvable materials consisting of low-toxicity synthetic polymers have high potential for implantable medical devices.

Elaboration of a thermosensitive smart biomaterial: From synthesis to the ex vivo bioadhesion evaluation.

Alginate and chitosan are polysaccharides that are widely used in the biomedical field, especially as wound dressings. Controlled bioadhesion is an advanced functionality that offers the potential to reduce injuries due to the stripping-off of the biomaterial. Herein, we report the efficient grafting of poly-N(isopropylacryamide) (PNIPAM), a thermosensitive polymer that exhibits a lower critical solution temperature (LCST) at 32 °C on the alginate/chitosan polyelectrolyte complex (PEC) surface. In vitro studies did not exhibit a cytotoxic effect, and cells adhered preferentially on the LCST on PNIPAM grafted surfaces, as reported in the literature. Ex vivo investigations revealed that the adhesive behavior of the biomaterials was not the same on the liver and pancreas. The effect of the temperature on the bioadhesion to organs was unexpected, as PNIPAM surfaces exhibited higher adhesion at low temperature. The PNIPAM was therefore able to confer PEC matrix thermosensitivity, but due to the application force, interactions between PNIPAM chains and their substrate could influence bioadhesion on tissues.

Evaluation of Tumor Tissue Fixation Effects of Formulation Modified Mohs Pastes in Mice and Their Water-Absorbing Properties.

Mohs paste (MP) is a hospital preparation containing zinc hydrochloride and zinc oxide starch. It is a topical medication used to fixate tissues for the removal of inoperable skin tumors and the management of hemorrhage and exudates, and to prevent foul odor resulting from secondary infections. However, it has problems, such as changes in hardness and viscoelasticity with time and liquefaction by exudate. It has been reported that the modified MP with D-sorbitol (S-MP) and the modified MP using the cellulose instead of starch (C-MP) have excellent physicochemical stability and better handling than original MP (O-MP). In this study, the effect of prescription improvement of MP on the pharmacological effect was examined with reference to water absorbing property, and its tumor tissue invasion fixation depth as an indicator. In the S-MP and C-MP, the amounts of water absorption did not differ significantly from those in the O-MP. The hardness of S-MP was decreased and liquefied like O-MP after absorbing water. In contrast, C-MP retained its form even after water absorption. The subcutaneous tumors in mice treated with modified MP formulations were measured for invasion fixation depth at 6 and 24 h after application. And the tissue status was observed using computed tomography. In all MPs, invasion fixation depth increased depending on application time. S-MP and O-MP depths did not differ significantly. The invasion depths of the C-MP significantly increased compared with those in the O-MP. These results suggest that C-MP had a high tissue fixation rate.

Functional Mechanics of a Pectin-Based Pleural Sealant after Lung Injury.

Pleural injury and associated air leaks are a major influence on patient morbidity and healthcare costs after lung surgery. Pectin, a plant-derived heteropolysaccharide, has recently demonstrated potential as an adhesive binding to the glycocalyx of visceral mesothelium. Since bioadhesion is a process likely involving the interpenetration of the pectin-based polymer with the glycocalyx, we predicted that the pectin-based polymer may also be an effective sealant for pleural injury. To explore the potential role of an equal (weight%) mixture of high-methoxyl pectin and carboxymethylcellulose as a pleural sealant, we compared the yield strength of the pectin-based polymer to commonly available surgical products. The pectin-based polymer demonstrated significantly greater adhesion to the lung pleura than the comparison products (p < 0.001). In a 25 g needle-induced lung injury model, pleural injury resulted in an air leak and a loss of airway pressures. After application of the pectin-based polymer, there was a restoration of airway pressure and no measurable air leak. Despite the application of large sheets (50 mm2) of the pectin-based polymer, multifrequency lung impedance studies demonstrated no significant increase in tissue damping (G) or hysteresivity (η)(p > 0.05). In 7-day survival experiments, the application of the pectin-based polymer after pleural injury was associated with no observable toxicity, 100% survival (N = 5), and restored lung function. We conclude that this pectin-based polymer is a strong and nontoxic bioadhesive with the potential for clinical application in the treatment of pleural injuries.

Absence of conformational change in complement factor 3 and factor XII adsorbed to acrylate polymers is related to a high degree of polymer backbone flexibility.

In previous investigations, the authors have examined the adsorption of albumin, immunoglobulin, and fibrinogen to a series of acrylate polymers with different backbone and side-group flexibility. The authors showed that protein adsorption to acrylates with high flexibility, such as poly(lauryl methacrylate) (PLMA), tends to preserve native conformation. In the present study, the authors have continued this work by examining the conformational changes that occur during the binding of complement factor 3 (C3) and coagulation factor XII (FXII). Native C3 adsorbed readily to all solid surfaces tested, including a series of acrylate surfaces of varying backbone flexibility. However, a monoclonal antibody recognizing a "hidden" epitope of C3 (only exposed during C3 activation or denaturation) bound to the C3 on the rigid acrylate surfaces or on polystyrene (also rigid), but not to C3 on the flexible PLMA, indicating that varying degrees of conformational change had occurred with binding to different surfaces. Similarly, FXII was activated only on the rigid poly(butyl methacrylate) surface, as assessed by the formation of FXIIa-antithrombin (AT) complexes; in contrast, it remained in its native form on the flexible PLMA surface. The authors also found that water wettability hysteresis, defined as the difference between the advancing and receding contact angles, was highest for the PLMA surface, indicating that a dynamic change in the interface polymer structure may help protect the adsorbed protein from conformational changes and denaturation.

Silk-fibronectin protein alloy fibres support cell adhesion and viability as a high strength, matrix fibre analogue.

Silk is a natural polymer with broad utility in biomedical applications because it exhibits general biocompatibility and high tensile material properties. While mechanical integrity is important for most biomaterial applications, proper function and integration also requires biomaterial incorporation into complex surrounding tissues for many physiologically relevant processes such as wound healing. In this study, we spin silk fibroin into a protein alloy fibre with whole fibronectin using wet spinning approaches in order to synergize their respective strength and cell interaction capabilities. Results demonstrate that silk fibroin alone is a poor adhesive surface for fibroblasts, endothelial cells, and vascular smooth muscle cells in the absence of serum. However, significantly improved cell attachment is observed to silk-fibronectin alloy fibres without serum present while not compromising the fibres' mechanical integrity. Additionally, cell viability is improved up to six fold on alloy fibres when serum is present while migration and spreading generally increase as well. These findings demonstrate the utility of composite protein alloys as inexpensive and effective means to create durable, biologically active biomaterials.

Thermoreversible mucoadhesive in situ nasal gel for treatment of Parkinson's disease.

Parkinson's disease is a degenerative disorder of the central nervous system (CNS). The most obvious symptoms are movement-related such as shaking, rigidity, slowness of movement and difficulty with walking, rigid muscular movements and difficulty in chewing and swallowing especially solid dosage forms. Ropinirole is an anti-Parkinson drug that has low oral bioavailability which is primarily due to first-pass metabolism. The objective of proposed work was to increase bioavailability of ropinirole and avoid patient discomfort by formulating thermoreversible in situ nasal gel. Thermoreversible nasal gels were prepared by cold method using Pluronic F-127 and hydroxy methyl propyl cellulose (HPMC K4M) as gelling agents. Formulations were evaluated for various parameters such as drug content, pH, gelling time, gelling temperature, gel strength, mucoadhesive force, ex vivo diffusion, histological studies and in vivo bioavailability. Formulations displayed gelation at nasal temperature and the gelation time was found to be less than mucociliary clearance time. The nasal residence time was seen to be increased due to mucoadhesion and increased gel strength. The nasal gel formulations showed ex vivo drug release between 56-100% in 5 h. Histological study of sheep nasal mucosa revealed that the gel had a protective effect on the mucosa unlike plain ropinirole which showed evidence of moderate cellular damage. A fivefold increase in bioavailability in brain was observed on nasal administration as compared to IV route. Thermoreversible in situ nasal gel was found to a promising drug delivery for Parkinsonian patients.

Chitosan-catechol: a polymer with long-lasting mucoadhesive properties.

Numerous mucoadhesive polymers have been exploited for prolonging the residence time of formulated drugs or pharmaceuticals at specific delivery sites. However, it has been difficult to achieve satisfactory mucoadhesive properties. The two major modification strategies such as thiolation or lectin functionalization have been extensively studied, but disulfide bond reversibility in the case of thiolation and the toxicity of lectins have been problems. Thus, approaches for further improvement of mucoadhesive properties need to be developed. With an overwhelming library of mucoadhesive polymers, one practical way to improve mucoadhesion is chemical modification of existing mucoadhesive polymers. In other words, the method is based on utilizing the cooperative effect that might be achieved by chemical tethering of a small adhesive moiety to an available mucoadhesive polymer. Here, we conjugated catechols derived from mussel adhesive proteins to chitosan, which is a widely known mucoadhesive polymer. We demonstrated that the gastrointestinal (GI) tract retention of chitosan-catechol was improved compared to unmodified chitosan, which is due to the formation of irreversible catechol mediated-crosslinking with mucin. The results indicate that catechol modification of mucoadhesive polymers may possibly lead to a new generation of mucoadhesive polymers for mucosal drug delivery.

Mucoadhesive cellulosic derivative sponges as drug delivery system for vaginal application.

Vaginal delivery of active drugs has been largely studied for local and systemic applications. It is well known that vagina is a complex route, due to physiological and non-physiological changes. Therefore, in order to achieve a prolonged local effect, these variations have to be considered. The aim of this study was to formulate and to characterize a solid system, called sponges, obtained by lyophilization of cellulosic derivative (HEC 250M) hydrogels. These sponges have to meet particular criteria to be adapted for vaginal application: they have to adhere to the vaginal cavity and to be rehydrated by the small amount of vaginal fluids. Moreover, they have to be easily manipulated and to be stable. Three freezing temperatures have been tested to prepare sponges (-15°C, -25°C, -35°C). By SEM analyzes, it was observed that the pores into the sponges were smaller and numerous as the freezing temperature decreases. However, this temperature did not have any influence on the rehydration speed that was rather influenced by the HEC concentration. Viscosity and mucoadhesive strength of hydrogels and corresponding sponges were also measured. It appeared that these parameters are mainly dependent on the HEC concentration. These mucoadhesive sponges can be considered as potential drug delivery systems intended for vaginal application.

Bioadhesive vaginal tablets containing spray dried microspheres loaded with clotrimazole for treatment of vaginal candidiasis.

The aim of the present investigation was to prepare and evaluate novel bioadhesive vaginal tablets containing clotrimazole loaded microspheres in order to provide long-term therapeutic activity at the site of infection. Tablets were prepared by incorporating drug loaded microspheres and using bioadhesive polymers hydroxypropylmethylcellulose, sodium carboxymethylcellulose and Carbopol. Microspheres were prepared by the spray drying technique using Eudragit RS-100 and Eudragit RL-100. Microspheres were characterized by SEM, DSC, FTIR, particle size analysis and evaluated for percentage yield, drug loading, encapsulation efficiency and in vitro drug release. To achieve bioadhesion to the mucosal tissue, optimized microspheres were incorporated into bioadhesive tablets and were evaluated for in vitro drug release, in vitro and in vivo mucoadhesion. FTIR and DSC studies showed that no chemical interaction occurred between the drug and polymers. The sphericity factor indicated that the prepared microspheres were spherical. Formulation Mt6 indicated a controlled in vitro drug release and good bioadhesive strength. The in vivo images confirmed the bioadhesion and retention property of tablets up to 24 h. The results indicated that this drug delivery system can be explored for controlled intravaginal drug release.

Efficacy of mucoadhesive hydrogel microparticles of whey protein and alginate for oral insulin delivery.

PURPOSE: To evaluate the efficacy of mucoadhesive insulin-loaded whey protein (WP) /alginate (ALG) microparticles (MP) for oral insulin administration. METHODS: Insulin-loaded microparticles (ins-MP) made of whey protein and alginate were prepared by a cold gelation technique and an adsorption method, without adjunction of organic solvent in order to develop a biocompatible vehicle for oral administration of insulin. In vitro characterization, evaluations of ins-MP in excised intestinal tissues and hypoglycaemic effects after intestinal administration in healthy rats were performed RESULTS: The release properties and swelling behaviors, investigated in different pH buffers, demonstrated a release based on diffusion mechanism following matrix swelling. Mucoadhesion studies in rabbits and insulin transport experiments with excised intestinal rat tissues revealed that encapsulation in microparticles with mucoadhesive properties promotes insulin absorption across duodenal membranes and bioactivity protection. In vivo experiments reinforced the interest of encapsulation in whey protein/alginate combination. Confocal microscopic observations associated with blood glucose levels bring to light duodenal absorption of insulin biologically active following in vivo administration. CONCLUSIONS: Insulin-loaded WP/ALG MP with high quantities of drug entrapped, in vitro matrix swelling and protective effect as well as excellent mucohadesive properties was developped. Improvement of intestinal delivery of insulin and increased in bioavailability were recorded.

An orthopedic tissue adhesive for targeted delivery of intraoperative biologics.

Tissue adhesives can bind together damaged tissues and serve as tools to deliver and localize therapeutics to facilitate regeneration. One emerging therapeutic trend in orthopedics is the use of intraoperative biologics (IOB), such as bone marrow (BM) and platelet-rich plasma (PRP), to stimulate healing. Here, we introduce the application of the biomaterial chondroitin sulfate succinimidyl succinate (CS-NHS) to deliver IOB in a hydrogel adhesive. We demonstrate the biomaterial's ability to bind various tissue types and its cellular biocompatibility with encapsulated human mesenchymal stem cells (hMSCs). Further, we examine in detail the CS-NHS adhesive combined with BM aspirate for use in bone applications. hMSCs were encapsulated in CS-BM and cultured for 5 weeks in osteogenic medium. Quantitative RT-PCR demonstrated osteogenesis via upregulation of the osteogenic transcription factor Runx2 and bone markers alkaline phosphatase and osteocalcin. Significant deposition of calcium and osteocalcin was detected using biochemical, histological, and immunohistochemical techniques. Shear testing demonstrated that the CS-BM adhesive exhibited an adhesive strength approximately an order of magnitude stronger than fibrin glue and approaching that of a cyanoacrylate adhesive. These results indicate that CS-NHS is a promising delivery tool for IOB in orthopedic applications requiring a strong, degradable, and biocompatible adhesive that supports bone growth.

Identification, characterization, and expression levels of putative adhesive proteins from the tube-dwelling polychaete Sabellaria alveolata.

The shelter of the tube-dwelling polychaete Sabellaria alveolata is composed of mineral particles assembled with spots of a proteinaceous cement. The adhesive proteins constituting the cement were identified on the basis of their sequence similarity with proteins of a phylogenetically related species, Phragmatopoma californica. Two positively charged proteins, Sa-1 and Sa-2, share common features: they both have a mass of 22 kDa; are rich in glycine, tyrosine and basic residues; and show repeated peptide motifs. The consensus repeat of Sa-1 is KGAYGAKGLGYGNKAGYGAYG (occurring 6-8 times), while Sa-2 displays the consensus heptapeptide VHKAAWG (5 times) and undecapeptide VHKAAGYGGYG (8 times). Two variants of a serine-rich protein, Sa-3A (22 kDa) and Sa-3B (21 kDa), were also identified. Their serine residues account for 75 mol% and are probably phosphorylated, meaning that Sa-3 is very acidic and negatively charged. Moreover, tyrosine residues of all adhesive proteins are presumably modified into DOPA. Although protein sequences are not well-conserved between S. alveolata and P. californica, their main characteristics (including amino acid composition, post-translational modifications, repeated patterns, isoelectric point, and mass) are shared by both species. This suggests that these features are more important for their function than the primary structure of the proteins. The mRNA abundance for each protein was estimated by quantitative real-time PCR, revealing relative expression levels of about 5, 11, 1.5, and 1 for Sa-1, -2, -3A, and -3B, respectively. These levels could be indicative of charge neutralization phenomena or could reflect their function (interface vs. bulk) in the cement.