Glycidic Cues for Tissue Engineering: Synthesis and Characterization

Glycidic Cues for Tissue Engineering: Synthesis and Characterization | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Glycidic Cues for Tissue Engineering: Synthesis and Characterization FATMA Cetin TELLİ This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6678530/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The bioactivity of materials can be enhanced by incorporating adhesive cues and regulatory molecules. Carbohydrates are essential for numerous biological recognition processes. Recent evidence suggests that collagen glycosylation is crucial for maintaining the equilibrium between collagen deposition and turnover, with several implications for healthy and pathological conditions. In this study, we synthesized small glycidic cues for enrichment with collagen for this purpose. A relevant factor for successful biomaterial decoration pertains to the selection of appropriate chemistry. This research is strongly motivated by the need for innovative biomaterials in cartilage tissue engineering, especially for repairing osteoarthritis defects. Collagen Glycidic Cue Multifunctional Biomolecule Bioconjugation Tissue Engineering Highlights ►Glycidic clue was synthesized and characterized. ► Synthesis of small glycidic cues for enriching with collagen for the tissue engineering applications. ► The balance collagen turnover and deposition are aimed, a crucial factor in both healthy and pathological tissue states by using a chemoselective Michael reaction for bioconjugation in our future study. Introduction Osteoarthritis (OA) (Van der Kraan PM 2012 ) is the most prevalent form of degenerative joint disease and a primary cause of pain typically linked with aging (Helmick et. al. 2008 ). It affects over 50 and 25 million individuals in the EU and USA, accounting for approximately 25% of all visits to primary care physicians and half of the nonsteroidal anti-inflammatory drugs (NSAID) prescriptions. Globally, these figures are striking and projections indicate that the prevalence of OA-related disability will double by 2050, exacerbating its already onsiderableeconomic impact (Zhang and Jordan 2010 ). Despite centuries of scientific and medical advancements, a universally accepted and successful treatment for damaged articular cartilage remains elusive (Kalson et. al. 2010 ). Present pharmacological treatments are often limited in efficacy and are associated with substantial side effects or high costs. Furthermore, surgical interventions are frequently necessitated to repair cartilage and bone injuries, highlighting the imperative for novel and effective treatments (Lohmander and Roos 2007 ). Tissue engineering presents a comprehensive strategy for the structural and functional restoration of tissues. This field typically employs a combination of cells, biomaterials, and bioactive factors (signaling cues/regulators) (Kim et. al. 2012 ). to foster the regeneration of damaged or lost tissue, influencing cellular differentiation, proliferation, and tissue morphogenesis (Pashuck and Stevens. 2012 ). Repairing of articular cartilage represents a formidable challenge in musculoskeletal medicine owing to the limited intrinsic repair capacity of the tissue. Biomaterials crafted for cartilage tissue engineering are examined for their physical attributes, such as porosity and mechanical compressive strength, as well as their chemical properties, including degradation rates (Hutmacher 2000 ). Recent advances in the design of these biomaterials incorporated signaling cues within the synthetic microenvironment (Pashuck and Stevens. 2012 ; Keung et. al. 2010 ). In the fields of tissue engineering and regenerative medicine, the development of biomaterial matrices with covalently attached biomolecular cues that can influence cellular responses represents a cutting-edge direction (Benoit et. al. 2008 ; Chaudhuri and Mooney 2012 ). Specifically, the synthesis and integration of carbohydrate cues onto surfaces to confer specific biofunctionalities is a promising approach. Collagen is the most abundant protein in the body, serving as a principal element of the extracellular matrix (ECM). It forms fibrilar meshes and interacts with a multitude of adhesion proteins including fibronectin, integrins, and laminin. The biosynthesis of collagen initiates intracellularly at the ribosomal membranes and continues extracellularly. Owing to its dualistic properties, collagen can be characterized as a “block” copolymer, identified with favorable properties for tissue engineering (O’Leary et. al. 2011 ; Barry-Hamilton et. al. 2010 ; Abou Neel et. al. 2013 ) and cartilage repair (Wang et. al. 2007 ). The process of collagen glycosylation is highly conserved across the animal kingdom, observed in organisms ranging from simple sponges to mammals. It plays a critical role in the ECM breakdown and remodeling, influencing the dynamic equilibrium between turnover and collagen deposition in healthy and pathological states (Ohtsubo and Marth 2006 ; Jurgensen et. al. 2011 ). The saccharidic residues predominantly identified were β-galactosides or α-(1→2)-glucosyl-β-galactosides (Oikarinen et. al. 1976 ). Recent studies have demonstrated that lectin domains directly interact with glycosylated collagens (Jurgensen et. al. 2011 ). Lectins, proteins that bind with sugars, are implicated in signal transduction and carbohydrate recognition across various biological processes (Sharon and Lis 2004 ). Small saccharidic motifs are reportedly attached to material surfaces (Wendeln et. al. 2010 ). The versatility of this approach enables the ligation of diverse reducing sugars to adhesive groups or lipids without the need for protecting group chemistry, considerably expediting the development of new coating entities. This methodology applies to the synthesis of coating molecules that can be thoroughly characterized before their employment in surface functionalization. Biocompatible materials such as synthetic carbonate hydroxyapatite (CHA) and hydroxyapatite (HA) are extensively utilized in biomedical applications (Dorozhkin 2010 ). HA, the natural mineral component of bones, teeth, and calcified tissues in vertebrates, along with synthetic HA and CHA, are employed for human implant coatings owing to their osteoconductivity. The geometry of these materials can profoundly affect specific tissue responses, as observed in vascular in-growth (Karageorgiou and Kaplan 2005 ; Feng et. al. 2009 ; Houseman and Mrksich, 2002 ). Therefore, this study aimed to engineer a novel natural nanofibrous scaffold with surface-bound galactose ligands to augment the bioactivity and mechanical stability of primary hepatocytes in culture for liver regeneration. For carbohydrate grafting on polycaprolactone substrates, a distinct strategy was employed. Aliphatic polyesters, among synthetic polymers, are favored as biomaterials for scaffold design to support the regeneration of various tissue-engineered organs attributed to their unique biodegradability and biocompatibility (Houseman et. al. 2003 ; Brinkman et. al. 2003 ; Jurgensen et. al. 2010 ). However, similar to other synthetic polymers, they lack inherent molecular motifs for cellular biological recognition. Cell adhesion is intimately associated with the surface characteristics of biomaterials, influenced by surface charge, topography, wettability, roughness, etc. Introducing glucosamine is an innovative approach to enhance scaffold biocompatibility by increasing hydrophilicity. Notably, given the ubiquity of this monosaccharide in nature, including in humans, and its inclusion in biomedical formulations for osteoarthritis treatment, its application in biomaterial surface modification is considered a safe and promising strategy. The bioactivity of materials can be enhanced by incorporating adhesive cues and regulatory molecules. Carbohydrates are essential in numerous biological recognition processes. Recent findings indicate that collagen glycosylation is crucial for regulating the equilibrium between collagen deposition and turnover, with profound implications for healthy and pathological states (Brinkman et. al. 2003 ; Jurgensen et. al. 2010 ). However, only a handful of studies have investigated the impact of small carbohydrate epitopes on collagen (Andrés–Bergós et. al. 2012 ). Consequently, this study aims to augment collagen with small glycidic cues (vide infra). Present investigations at the host institution are assessing the effects of collagen glycosylation. The selection of appropriate chemistry is critical for the successful modification of biomaterials. We propose straightforward, dependable, and gentle bioconjugation steps, facilitating a chemoselective reaction between cues (multifunctional biomolecules) and the collagen matrix. This method utilizes a Michael reaction between a thiol-terminated peptide/carbohydrate and maleimido-modified scaffold (Jurgensen et. al. 2010 ). This study has undertaken the design and synthesis of biologically relevant glycidic structures. By focusing on the design and synthesis of collagen patches functionalized with such innovative signaling microenvironment cues, future research will be directed toward developing smart biomaterials based on collagen matrices for the repair of cartilage defects. This research is strongly motivated by the need for novel, promising, biomaterial-based cartilage tissue engineering strategies for the treatment of OA defects. The glycidic structures providing specific biological signals were characterized using spectroscopic methods ( 1 H and 13 C nuclear magnetic resonance (NMR), FTIR, Mass spectrum). Experimental Section Materials, Instrumentation and Characterization All reagents were procured commercially and utilized without modification. Column chromatography was conducted using silica gel G-60 (Merck 7734), while thin-layer chromatography employed silica gel 60 with a fluorescent indicator F 254 on precoated aluminum plates measuring 20 × 20 cm 2 (Merck 5554). Organic solvents were evaporated under reduced pressure at low temperatures. NMR spectra were acquired at 400 MHz for 1 H and 100 MHz for 13 C using a mercury FT NMR spectrometer (Varian AS 400+, Varian Inc., Palo Alto, CA, USA) at room temperature, with CDCl 3 as the solvent and tetramethylsilane (TMS) as the reference. Fourier-transform infrared (FT-IR) spectra were obtained within the wavenumber range of 400–4000 cm − 1 using KBr disks on a PerkinElmer 100 FTIR Model instrument (PerkinElmer, Norwalk, CT, USA). Synthesis of Allyl 2- O -acetyl-3,4,6-tri- O -benzyl-β-D-galactopyranoside (2) To a stirred solution of 1,2-di-O-acetyl-3,4,6-tri-O-benzyl-D-galactopyranose ( 1 ) (2.7 g, 5.0 mmol) in dry acetonitrile (50 mL) were added allyl alcohol (0.4 mL, 6 mmol) and BF 3 OEt (2.4 mL, 25 mmol) at -20°C under an atmosphere of argon. The reaction mixture was stirred for 18 h at -20°C and then it was neutralized with sat. Na 2 CO 3 solution. Afterward, acetonitrlie was evaporated and the water layer was extracted with EtOAc (3x100 mL). The organic layer was dried with Na 2 SO 4 and concentrated in vacuo. The residue was purified by flash chromatography (SiO2, Petroleum Ether:EtOAc, 4:1) to afford compound 2 (1.94 g, 70%) as a colorless syrup. 1 H NMR (CDCl 3 , 400 MHz): δ 7.34–7.24 (15H, m), 5.80 (1H, m), 5.39 (2H, dd, J = 12.0, 8.0 Hz), 5.25 (1H, dd, J = 16.0, 4.0 Hz), 5.13 (1H, dd, J = 12.0, 4.0 Hz), 4.93 (1H, d, J = 11.6 Hz), 4.68 (1H, d, J = 12.0 Hz), 4.60 (1H, d, J = 12.0 Hz), 4.55 (1H, d, J = 12.0 Hz), 4.43 (1H, d, J = 4.0 Hz), 4.40 (2H, d, J = 4.0 Hz), 4.31 (1H, dd, J = 12.0, 4.0 Hz), 4.04 (1H, dd, J = 12.0, 4.0 Hz), 3.95 (1H, d, J = 4.0 Hz), 3.65 (2H, dd, J = 8.0, 4.0 Hz), 3.55 (1H, dd, J = 12.0, 4.0 Hz), 2.05 (3H, s). 13 C (CDCl 3 , 100 MHz) δ 170.1, 138.4, 138.0, 137.8, 133.9, 128.5, 128.5, 128.4, 128.4, 128.3, 128.2, 128.2, 127.9, 127.8, 127.7, 127.6, 127.5, 127.4, 127.4, 116.9, 100.2, 80.3, 77.8, 76.7, 74.4, 73.6, 73.6, 72.5, 71.9, 71.3, 69.3, 21.1. m/z calculated for [M + Na] + C 32 H 36 O 7 555.6, found 555.2. Synthesis of Allyl 2-hydroxy - 3,4,6-tri- O -benzyl-β-D-galactopyranoside (3) To a stirred solution of compound 2 (1.7 g, 3.0 mmol) in dry MeOH (30 mL) was added Na metal (69.0 mg, 3.0 mmol) under an atmosphere of argon. The reaction mixture was stirred for 2 h at r.t.. Thereafter, the reaction mixture was quenched by addition of Amberlite IR-120 (H+) resin and the resulting mixture filtered. The filtrate was then concentrated in vacuo. The residue was purified by flash chromatography (SiO 2 , Petroleum Ether:EtOAc, 1:1) to afford compound 3 (1.24 g, 81%) as a colorless syrup. 1 H NMR (CDCl 3 , 400 MHz): δ 7.37–7.26 (15H, m), 5.94 (1H, m), 5.30 (1H, dd, J = 16.0, 1.2 Hz), 5.20 (1H, d, J = 12.0, 1.2 Hz), 4.90 (1H, d, J = 11.6 Hz), 4.70 (1H, dd, J = 12.0, J = 14.0 Hz ), 4.68 (1H, d, J = 14.0 Hz), 4.62 (1H, d, J = 12.0 Hz), 4.46 (2H, d, J = 6.8 Hz), 4.38 (1H, dd, J = 10.0, 4.8 Hz), 4.31 (1H, d, J = 7.6 Hz), 4.10 (1H, dd, J = 12.8, 6.4 Hz), 4.00 (1H, dd, J = 8.0, 8.0 Hz), 3.94 (1H, d, J = 2.4 Hz), 3.62 (2H, m), 3.58 (1H, dd, J = 5.2, 5.2 Hz), 3.45 (1H, dd, J = 9.6, 2.8 Hz), 2.23 (1H, br s). 13 C (CDCl 3 , 100 MHz) δ 138.5, 138.0, 137.8, 134.0, 128.5, 128.5, 128.4, 128.3, 128.3, 128.2, 128.2, 127.9, 127.9, 127.8, 128.8, 127.7, 127.7, 127.6, 117.1, 102.1, 82.0, 77.4, 76.7, 73.9, 72.5, 71.6, 70.9, 69.9, 69.3, 68.6. m/z calculated for [M + Na] + C 30 H 34 O 6 513.6, found 513.2. Synthesis of Allyl 3,4,6-tri- O -benzyl-β-D-galactopyranosyl-(1→2)-2,3,4,6-tetra- O -benzyl-β-D-glucopyranoside (5) To a stirred solution of compound 3 (1.54 g, 3.0 mmol) in dry DCM (30 mL) was added TMSOTf solution in DCM (c = 0.05 M, 0.06 mmol) at -20°C under an atmosphere of argon. The reaction mixture was stirred for 10 minutes at -20°C. Thereafter, the reaction mixture was added to a stirred solution of compound 4 (2.7 g, 3.9 mmol) in dry DCM (20 mL) -20°C under an atmosphere of argon.. Afterward, the reaction mixture was stirred for 30 minutes at -20°C and was neutralized with sat. Na 2 CO 3 solution. The water layer was extracted with DCM (3x100 mL). The organic layer was dried with Na 2 SO 4 and concentrated in vacuo at 35°C. The residue was purified by flash chromatography (SiO 2 , Petroleum Ether:EtOAc: Et 3 N, 7:3:0.1) to afford compound 5 (1.8 g, 60%) as a colorless syrup. 1 H NMR (CDCl 3 , 400 MHz): δ 7.34–7.07 (35H, m), 5.89 (1H, m), 5.28 (1H, d, J = 16.0 Hz), 5.12 (2H, d, J = 12.0), 5.03 (1H, d, J = 4.0), 5.00 (1H, d, J = 16.0), 4.88 (1H, d, J = 8.0 Hz), 4.81 (2H, d, J = 12.0 Hz), 4.78 (1H, d, J = 4.0), 4.75 (2H, d, J = 12.0 Hz), 4.73 (2H, d, J = 16.0 Hz), 4.65 (1H, d, J = 16.0 Hz), 4.58 (2H, d, J = 8.0 Hz), 4.48 (1H, d, J = 8.0 Hz), 4.47 (2H, d, J = 7.6 Hz), 4.35 (1H, dd, J = 12.0, 4.0 Hz), 4.30 (2H, d, J = 12.0 Hz), 3.94 (1H, d, J = 2.4 Hz), 4.20 (1H, dd, J = 12.0, 4.0 Hz), 4.10 (1H, dd, J = 12.0, 4.0 Hz), 4.04 (1H, d, J = 2.4 Hz), 4.02 (2H, d, J = 2.4 Hz), 3.99 (1H, d, J = 4.0 Hz), 3.67 (1H, t, J = 4.0 Hz), 3.58 (1H, dd, J = 5.2, 5.2 Hz), 3.40 (1H, dd, J = 9.6, 2.8 Hz). 13 C (CDCl 3 , 100 MHz) δ 138.9, 138.8, 138.7, 138.4, 138.2, 137.9, 134.1, 134.1,128.7, 128.5, 128.4, 128.4, 128.3, 128.3, 128.3, 128.2, 128.2, 128.1, 128.1, 128.1, 128.0, 128.0, 127.9, 127.9, 127.8, 127.7, 127.6, 127.5, 127.5, 127.4, 127.3, 118.3, 106.7, 102.4, 101.5, 100.8, 95.4, 95.1, 95.0, 94.4, 84.9, 83.0, 82.2, 79.0, 77.7, 77.5, 77.4, 77.1, 76.8, 76.8, 74.8, 74.7, 73.7, 73.6, 73.3, 73.2, 71.8, 71.7, 70.1, 69.5, 69.4, 68.7, 68.7, 67.8. m/z calculated for [M + Na] + C 64 H 68 O 11 1035.2, founsd 1035.2. Synthesis of 1- O -(3-acetylthiopropane)-3,4,6-tri- O -benzyl-β-D-galactopyranosyl-(1→2)-2,3,4,6-tetra- O -benzyl-α-D-glucopyranoside (6) To a stirred solution of compound 5 (1.2 g, 1.2 mmol) and thioacetic acid (0.17 mL, 2.4 mmol) in dry 1,4-dioxane (0.3 mL), 2,2'-azobisisobutyronitrile (AIBN; 0.98 g, 6.0 mmol) was added at 50°C an atmosphere of argon. The reaction mixture was stirred for 3 h at 80°C, then cooled to room temperature. Cyclohexene (0.64 mL, 6.3 mmol) was added, and the reaction mixture was stirred at room temperature for 30 min. After evaporation, the residue was purified by flash chromatography (SiO 2 , Petroleum Ether:EtOAc, 8:2) to afford compound 6 (0.13 g, 10%) as a yellow syrup. 1 H NMR (CDCl 3 , 400 MHz): δ 7.36–7.06 (35H, m), 5.64 (1H, d, J = 3.6 Hz ), 4.96 (1H, d, J = 14.8 Hz), 4.86 (1H, d, J = 11.6), 4.83 (1H, d, J = 10.8), 4.80 (1H, d, J = 12.0), 4.78 (1H, d, J = 8.0 Hz), 4.68 (1H, d, J = 12.0 Hz), 4.65 (1H, d, J = 8.0), 4.58 (1H, d, J = 12.0 Hz), 4.56 (2H, d, J = 12.0 Hz), 4.51 (1H, d, J = 8.0 Hz), 4.48 (2H, d, J = 6.0 Hz), 4.42 (2H, d, J = 11.6 Hz), 4.29 (2H, t, J = 12.0 Hz), 4.12 (2H, d, J = 8.0 Hz), 3.98 (1H, d, J = 2.4 Hz), 3.97 (1H, d, J = 12.0 Hz), 3.88 (1H, m), 3.70 (1H, d, J = 8.0 Hz), 3.59 (6H, m), 3.36 (1H, d, J = 8.0 Hz), 2.86 (3H, m), 2.24 (3H, s). 13 C (CDCl 3 , 100 MHz) δ 204.2, 139.2, 138.8, 138.8, 138.6, 138.3, 137.9, 137.2, 128.8, 128.8, 128.8, 128.8, 128.8, 128.8, 128.8, 128.4, 128.4, 128.4, 128.4, 128.4, 128.4, 128.4, 128.4, 128.3, 128.3, 128.3, 128.3, 128.3, 128.3, 128.3, 128.2, 128.1, 128.1, 128.1, 128.1, 128.0, 128.0, 128.0, 127.9, 127.9, 127.9, 127.8, 127.7, 127.4, 103.1, 102.7, 97.2, 96.5, 90.0, 89.7, 83.5, 83.2, 78.0, 77.3, 77.3, 76.7, 75.0, 74.1, 73.4, 70.2, 69.9, 68.6, 68.3, 67.3, 29.8, 29.7, 25.3. m/z calculated for [M + Na] + C 66 H 72 O 12 S 1102.3, found 1102.1. Synthesis of 1- O -(3-mercaptopropyl)-β-D-galactopyranosyl-(1→2)-α-D-glucopyranoside (G1) To a stirred solution of compound 6 (100 mg,0.092 mmol) in dry MeOH (2 mL) were added Pd/ carbon (120 mg) and dry HCO 3 NH 4 (400 mg, 6.44 mmol) at r.t. under an atmosphere of argon. The reaction mixture was stirred and heated for 10 h at 100°C. Afterward, the resulting mixture filtered from Acrodise*Premium 25 mm Syringe Filter with 0.45 µm Nylon Membran. The filtrate was concentrated in vacuo to afford compound G1 (10 mg, 26%) as a colorless syrup. 1 H NMR (D 2 O, 400 MHz) δ 5.40 (1H, d, J = 3.6 Hz), 5.15 (1H, d, J = 3.6 Hz), 4.43 (1H, d, J = 8.0 Hz), 3.95 (1H, m), 3.86 (2H, m), 3.77–3.45 (10H, m), 3.30–3.22 (1H, m), 2.80 (1H, t, J = 7.2 Hz), 2.58 (1H, t, J = 7.2 Hz), 2.05–1.84 (2H, m). 13 C (CDCl 3 , 100 MHz) δ 110.4, 111.2, 83.6, 81.5, 81.2, 76.8, 76.0, 74.1, 71.8, 71.5, 67.4, 62.9, 62.2, 30.3, 20.9. m/z calculated for [M + Na] + C 15 H 28 O 11 S 416.4, found 416.2. Results and Discussion The synthesis of glycidic cues poses a considerable challenge. Unlike proteins and nucleic acids, carbohydrate derivatives are complex to synthesize because of the absence of generalized methods for their routine preparation, often necessitating multiple selective protection and deprotection steps. The selection of glycidic cues is informed by their natural occurrence in collagen; the synthesis of relevant glycidic structures eliciting specific biological signals has been accomplished. Concurrently, the production of suitable linkers for the biodecoration process is underway. These heterobifunctional linkers are designed to attach to the material on one end and to the cues on the other. Considerably, the glycidic cue G1 (Scheme 1) has been selected for biomaterial functionalization. G1 , a disaccharide present in embryonic collagen, has been suitably modified for conjugation to materials via an appropriate linker. The synthesis of individual monosaccharides is undertaken to elucidate the specific role of each sugar unit. Moreover, the synthesis of the target disaccharidic cue G1 (Scheme 2) is particularly demanding. The critical synthesis aspects include (i) protection/deprotection steps, leading to a glycosyl acceptor (Gal unit) with selective deprotection at the 2-position for the glycosylation reaction; (ii) formation of glycosidic bond with alpha-stereoselection, necessitating a nonparticipating protecting group at the 2-OH of the glucose unit. dedicated to the development of intelligent biomaterials based on collagen matrices for repairing cartilage defects, with a focus on designing and synthesizing collagen patches functionalized with innovative signaling microenvironment cues, such as biologically pertinent glycidic structures. The impetus for this research is the demand for novel and effective biomaterial-based strategies for OA defect repair. The relevant glycidic structures that provide specific biological signals were characterized using spectroscopic techniques ( 1 H and 13 C NMR, FTIR, Mass spectrometry). In the 1 H NMR spectra of the synthesized glycidic cue G1 , the anomeric protons are discernible at 5.40 and 5.15 ppm. The remaining sugar protons are evident at 4.43–3.45 ppm. Notably, the characteristic aromatic peaks of compound 6 are absent in the 1 H NMR spectra of the synthesized glycidic cue G1, following the hydrolysis reaction, indicating the successful synthesis of glycidic cue G1 . In the 13 C NMR spectrum, anomeric carbons C-1 of glycidic cue G1 are observed at 111.1 and 110.4 ppm. Additionally, the alkyl group carbon peaks of glycidic cue G1 are present at 30.3–20.4 ppm. Conclusion This investigation executed the design and synthesis of glycidic structures with biological relevance. Focusing on the design and synthesis of collagen patches, functionalized with such pioneering signaling microenvironment cues, will guide subsequent research toward the creation of intelligent biomaterials derived from collagen matrices for the remediation of cartilage defects. The driving force behind this endeavor is the necessity for inventive and promising biomaterial-based strategies in cartilage tissue engineering to address OA defects. Declarations Acknowledgement I received a grant from the TUBITAK (Scientific and Technical Research Council of Turkey) BIDEB-2219 Programme for a postdoctoral fellowship. Conflict of interest: The authors have no conflict of interest to declare. References Abou Neel EA, Bozec L, Knowles JC, Syed O, Mudera V, Day R, Hyun JK (2013) Collagen--emerging collagen based therapies hit the patient. 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Annual Review of Cell and Developmental Biology. 26:533–556. https://doi.org/10.1146/annurev-cellbio-100109-104042 Kim TG, Shin H, Lim DW (2012) Biomimetic Scaffolds for Tissue Engineering. Advanced Functional Materials. 22:2446–2468. https://doi.org/10.1002/adfm.201103083 Lohmander LS, Roos EM (2007) Clinical update: treating osteoarthritis. Lancet 370:2082-2084. https://doi.org/10.1016/S0140-6736(07)61879-0 Jurgensen HJ, Madsen DH, Ingvarsen S, Melander MC, Gårdsvoll H, Patthy L, Engelholm LH, Behrendt N (2011) A Novel Functional Role of Collagen Glycosylation Journal of Biological Chemistry . 286:32736–32748. https://doi.org/10.1074/jbc.M111.266692 Ohtsubo K, Marth JD (2006) Glycosylation in cellular mechanisms of health and disease. Cell 126:855–867. https://doi.org/10.1016/j.cell.2006.08.019 Oikarinen A, Anttinen H, Kivirikko KI (1976) Hydroxylation of lysine and glycosylation of hydroxylysine during collagen biosynthesis in isolated chick-embryo cartilage cells. Biochemistry Journal 156:545–551. https://doi.org/10.1042/bj1560545 O’Leary LER., Fallas JA, Bakota EL, Kang MK, Hartgerink JD (2011). Multi hierarchical self assembly of a collagen mimetic peptide from triple helix to nanofibre and hydrogel. Nature Chemistry. 3:821−828. https://doi.org/10.1038/nchem.1123 Pashuck ET, Stevens MM (2012) Designing regenerative biomaterial therapies for the clinic. Science Translational Medicine. 4:160–164. https://doi.org/10.1126/scitranslmed.3002717 Sharon N, Lis H (2004) History of lectins: from hemagglutinins to biological recognition molecules. Glycobiology 14:53–62. https://doi.org/10.1093/glycob/cwh122 Van der Kraan PM (2012) Osteoarthritis year 2012 in review: biology. Osteoarthritis and Cartilage. 20:1447–1450. https://doi.org/10.1016/j.joca.2012.07.010 Wang DA, Varghese S, Sharma B, Strehin I, Fermanian S, Gorham J, Fairbrother DH, Cascio B, Elisseeff, JH (2007) Multifunctional chondroitin sulphate for cartilage tissue biomaterial integration. . Nature Materials. 6:385–392. https://doi.org/10.1038/nmat1890 Wendeln C, Rinnen S, Schulz C, Arlinghaus HF, Ravoo BJ (2010) Photochemical microcontact printing by thiol-ene and thiol-yne click chemistry. Langmuir 26:15966-15971. https://doi.org/10.1021/la102966j Zhang Y, Jordan JM (2010) Epidemiology of osteoarthritis. Clinics in Geriatric Medicine, 26: 355-369. https://doi.org/10.1016/j.cger.2010.03.001 Schemes Schemes 1 and 2 are available in the Supplementary Files section Supplementary Files SchemesandGA.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6678530","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":462618427,"identity":"dae9aa58-7cca-4399-8a0c-3d283ca6bfa3","order_by":0,"name":"FATMA Cetin TELLİ","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9klEQVRIiWNgGAWjYBAC9gYYi5mB8QGElYBfC88BZiiDmYHZAK7lAFFaGBjYJIjTwn7+6IYfDHZy9uy8xyo+/LJh4GfPMWD+uAePFp5ktps9DMnGPMx8aTdn9qUxSPa8MWA48Ay3FnuGZLYbPAwHEnuYecxu8/YcZjC4kQPUgsdlPPyP2W7+YThQD9JS/LfnP4M9QS0SyWy3gbYk8AC1MDP8OMBgIEFQy2Oz2zIGyYY9h3mMJXsbknkkzjwrOHAGr8MSn918U2Enz95/xvDDjz92cvztyRsfVODRAgHQOGRgbAPFD/5oQQd/SFA7CkbBKBgFIwYAAKOhTUulOjq9AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-2302-6409","institution":"Ege University: Ege Universitesi","correspondingAuthor":true,"prefix":"","firstName":"FATMA","middleName":"Cetin","lastName":"TELLİ","suffix":""}],"badges":[],"createdAt":"2025-05-16 08:01:54","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6678530/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6678530/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84173231,"identity":"5eaa7083-c4e1-4c96-aca6-6817722459d1","added_by":"auto","created_at":"2025-06-09 00:58:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":578572,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6678530/v1/629ed630-2254-4ba5-afec-5de903cf8ffb.pdf"},{"id":83622867,"identity":"e0a3d93d-1028-499e-9e53-fa7f5c9c490f","added_by":"auto","created_at":"2025-05-29 15:44:16","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":148529,"visible":true,"origin":"","legend":"","description":"","filename":"SchemesandGA.docx","url":"https://assets-eu.researchsquare.com/files/rs-6678530/v1/58637b9bf9dca33257d3a7c6.docx"}],"financialInterests":"","formattedTitle":"Glycidic Cues for Tissue Engineering: Synthesis and Characterization","fulltext":[{"header":"Highlights","content":"\u003cp\u003e►Glycidic clue was synthesized and characterized.\u003c/p\u003e\n\u003cp\u003e►\u0026nbsp;Synthesis of small glycidic cues for enriching with collagen for the tissue engineering applications.\u003c/p\u003e\n\u003cp\u003e► The balance collagen turnover and deposition are aimed, a crucial factor in both healthy and pathological tissue states by using a chemoselective Michael reaction for bioconjugation in our future study.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eOsteoarthritis (OA) (Van der Kraan PM \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) is the most prevalent form of degenerative joint disease and a primary cause of pain typically linked with aging (Helmick et. al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). It affects over 50 and 25\u0026nbsp;million individuals in the EU and USA, accounting for approximately 25% of all visits to primary care physicians and half of the nonsteroidal anti-inflammatory drugs (NSAID) prescriptions. Globally, these figures are striking and projections indicate that the prevalence of OA-related disability will double by 2050, exacerbating its already onsiderableeconomic impact (Zhang and Jordan \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Despite centuries of scientific and medical advancements, a universally accepted and successful treatment for damaged articular cartilage remains elusive (Kalson et. al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Present pharmacological treatments are often limited in efficacy and are associated with substantial side effects or high costs. Furthermore, surgical interventions are frequently necessitated to repair cartilage and bone injuries, highlighting the imperative for novel and effective treatments (Lohmander and Roos \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Tissue engineering presents a comprehensive strategy for the structural and functional restoration of tissues. This field typically employs a combination of cells, biomaterials, and bioactive factors (signaling cues/regulators) (Kim et. al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). to foster the regeneration of damaged or lost tissue, influencing cellular differentiation, proliferation, and tissue morphogenesis (Pashuck and Stevens. \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2012\u003c/span\u003e). Repairing of articular cartilage represents a formidable challenge in musculoskeletal medicine owing to the limited intrinsic repair capacity of the tissue.\u003c/p\u003e \u003cp\u003eBiomaterials crafted for cartilage tissue engineering are examined for their physical attributes, such as porosity and mechanical compressive strength, as well as their chemical properties, including degradation rates (Hutmacher \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Recent advances in the design of these biomaterials incorporated signaling cues within the synthetic microenvironment (Pashuck and Stevens. \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2012\u003c/span\u003e ; Keung et. al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In the fields of tissue engineering and regenerative medicine, the development of biomaterial matrices with covalently attached biomolecular cues that can influence cellular responses represents a cutting-edge direction (Benoit et. al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Chaudhuri and Mooney \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Specifically, the synthesis and integration of carbohydrate cues onto surfaces to confer specific biofunctionalities is a promising approach.\u003c/p\u003e \u003cp\u003eCollagen is the most abundant protein in the body, serving as a principal element of the extracellular matrix (ECM). It forms fibrilar meshes and interacts with a multitude of adhesion proteins including fibronectin, integrins, and laminin. The biosynthesis of collagen initiates intracellularly at the ribosomal membranes and continues extracellularly. Owing to its dualistic properties, collagen can be characterized as a \u0026ldquo;block\u0026rdquo; copolymer, identified with favorable properties for tissue engineering (O\u0026rsquo;Leary et. al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Barry-Hamilton et. al. \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2010\u003c/span\u003e; Abou Neel et. al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) and cartilage repair (Wang et. al. \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2007\u003c/span\u003e). The process of collagen glycosylation is highly conserved across the animal kingdom, observed in organisms ranging from simple sponges to mammals. It plays a critical role in the ECM breakdown and remodeling, influencing the dynamic equilibrium between turnover and collagen deposition in healthy and pathological states (Ohtsubo and Marth \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Jurgensen et. al. \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2011\u003c/span\u003e). The saccharidic residues predominantly identified were β-galactosides or α-(1\u0026rarr;2)-glucosyl-β-galactosides (Oikarinen\u003c/p\u003e \u003cp\u003eet. al. \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e1976\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRecent studies have demonstrated that lectin domains directly interact with glycosylated collagens (Jurgensen et. al. \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2011\u003c/span\u003e). Lectins, proteins that bind with sugars, are implicated in signal transduction and carbohydrate recognition across various biological processes (Sharon and Lis \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Small saccharidic motifs are reportedly attached to material surfaces (Wendeln et. al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The versatility of this approach enables the ligation of diverse reducing sugars to adhesive groups or lipids without the need for protecting group chemistry, considerably expediting the development of new coating entities. This methodology applies to the synthesis of coating molecules that can be thoroughly characterized before their employment in surface functionalization.\u003c/p\u003e \u003cp\u003eBiocompatible materials such as synthetic carbonate hydroxyapatite (CHA) and hydroxyapatite (HA) are extensively utilized in biomedical applications (Dorozhkin \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). HA, the natural mineral component of bones, teeth, and calcified tissues in vertebrates, along with synthetic HA and CHA, are employed for human implant coatings owing to their osteoconductivity. The geometry of these materials can profoundly affect specific tissue responses, as observed in vascular in-growth (Karageorgiou and Kaplan \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Feng et. al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Houseman and Mrksich, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Therefore, this study aimed to engineer a novel natural nanofibrous scaffold with surface-bound galactose ligands to augment the bioactivity and mechanical stability of primary hepatocytes in culture for liver regeneration.\u003c/p\u003e \u003cp\u003eFor carbohydrate grafting on polycaprolactone substrates, a distinct strategy was employed. Aliphatic polyesters, among synthetic polymers, are favored as biomaterials for scaffold design to support the regeneration of various tissue-engineered organs attributed to their unique biodegradability and biocompatibility (Houseman et. al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Brinkman et. al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Jurgensen et. al. \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2010\u003c/span\u003e). However, similar to other synthetic polymers, they lack inherent molecular motifs for cellular biological recognition. Cell adhesion is intimately associated with the surface characteristics of biomaterials, influenced by surface charge, topography, wettability, roughness, etc. Introducing glucosamine is an innovative approach to enhance scaffold biocompatibility by increasing hydrophilicity. Notably, given the ubiquity of this monosaccharide in nature, including in humans, and its inclusion in biomedical formulations for osteoarthritis treatment, its application in biomaterial surface modification is considered a safe and promising strategy.\u003c/p\u003e \u003cp\u003eThe bioactivity of materials can be enhanced by incorporating adhesive cues and regulatory molecules. Carbohydrates are essential in numerous biological recognition processes. Recent findings indicate that collagen glycosylation is crucial for regulating the equilibrium between collagen deposition and turnover, with profound implications for healthy and pathological states (Brinkman et. al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Jurgensen et. al. \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2010\u003c/span\u003e). However, only a handful of studies have investigated the impact of small carbohydrate epitopes on collagen (Andr\u0026eacute;s\u0026ndash;Berg\u0026oacute;s et. al. \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2012\u003c/span\u003e). Consequently, this study aims to augment collagen with small glycidic cues (vide infra). Present investigations at the host institution are assessing the effects of collagen glycosylation. The selection of appropriate chemistry is critical for the successful modification of biomaterials. We propose straightforward, dependable, and gentle bioconjugation steps, facilitating a chemoselective reaction between cues (multifunctional biomolecules) and the collagen matrix. This method utilizes a Michael reaction between a thiol-terminated peptide/carbohydrate and maleimido-modified scaffold (Jurgensen et. al. \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis study has undertaken the design and synthesis of biologically relevant glycidic structures. By focusing on the design and synthesis of collagen patches functionalized with such innovative signaling microenvironment cues, future research will be directed toward developing smart biomaterials based on collagen matrices for the repair of cartilage defects. This research is strongly motivated by the need for novel, promising, biomaterial-based cartilage tissue engineering strategies for the treatment of OA defects. The glycidic structures providing specific biological signals were characterized using spectroscopic methods (\u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e13\u003c/sup\u003eC nuclear magnetic resonance (NMR), FTIR, Mass spectrum).\u003c/p\u003e"},{"header":"Experimental Section","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials, Instrumentation and Characterization\u003c/h2\u003e \u003cp\u003eAll reagents were procured commercially and utilized without modification. Column chromatography was conducted using silica gel G-60 (Merck 7734), while thin-layer chromatography employed silica gel 60 with a fluorescent indicator F\u003csub\u003e254\u003c/sub\u003e on precoated aluminum plates measuring 20 \u0026times; 20 cm\u003csup\u003e2\u003c/sup\u003e (Merck 5554). Organic solvents were evaporated under reduced pressure at low temperatures.\u003c/p\u003e \u003cp\u003eNMR spectra were acquired at 400 MHz for \u003csup\u003e1\u003c/sup\u003eH and 100 MHz for \u003csup\u003e13\u003c/sup\u003eC using a mercury FT NMR spectrometer (Varian AS 400+, Varian Inc., Palo Alto, CA, USA) at room temperature, with CDCl\u003csub\u003e3\u003c/sub\u003e as the solvent and tetramethylsilane (TMS) as the reference. Fourier-transform infrared (FT-IR) spectra were obtained within the wavenumber range of 400\u0026ndash;4000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e using KBr disks on a PerkinElmer 100 FTIR Model instrument (PerkinElmer, Norwalk, CT, USA).\u003c/p\u003e \u003cp\u003e \u003cb\u003eSynthesis of Allyl 2-\u003c/b\u003e \u003cb\u003eO\u003c/b\u003e \u003cb\u003e-acetyl-3,4,6-tri-\u003c/b\u003e \u003cb\u003eO\u003c/b\u003e \u003cb\u003e-benzyl-β-D-galactopyranoside (2)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo a stirred solution of 1,2-di-O-acetyl-3,4,6-tri-O-benzyl-D-galactopyranose (\u003cb\u003e1\u003c/b\u003e) (2.7 g, 5.0 mmol) in dry acetonitrile (50 mL) were added allyl alcohol (0.4 mL, 6 mmol) and BF\u003csub\u003e3\u003c/sub\u003eOEt (2.4 mL, 25 mmol) at -20\u0026deg;C under an atmosphere of argon. The reaction mixture was stirred for 18 h at -20\u0026deg;C and then it was neutralized with sat. Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e solution. Afterward, acetonitrlie was evaporated and the water layer was extracted with EtOAc (3x100 mL). The organic layer was dried with Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e and concentrated in vacuo. The residue was purified by flash chromatography (SiO2, Petroleum Ether:EtOAc, 4:1) to afford compound \u003cb\u003e2\u003c/b\u003e (1.94 g, 70%) as a colorless syrup. \u003csup\u003e1\u003c/sup\u003eH NMR (CDCl\u003csub\u003e3\u003c/sub\u003e, 400 MHz): δ 7.34\u0026ndash;7.24 (15H, m), 5.80 (1H, m), 5.39 (2H, dd, J\u0026thinsp;=\u0026thinsp;12.0, 8.0 Hz), 5.25 (1H, dd, J\u0026thinsp;=\u0026thinsp;16.0, 4.0 Hz), 5.13 (1H, dd, J\u0026thinsp;=\u0026thinsp;12.0, 4.0 Hz), 4.93 (1H, d, J\u0026thinsp;=\u0026thinsp;11.6 Hz), 4.68 (1H, d, J\u0026thinsp;=\u0026thinsp;12.0 Hz), 4.60 (1H, d, J\u0026thinsp;=\u0026thinsp;12.0 Hz), 4.55 (1H, d, J\u0026thinsp;=\u0026thinsp;12.0 Hz), 4.43 (1H, d, J\u0026thinsp;=\u0026thinsp;4.0 Hz), 4.40 (2H, d, J\u0026thinsp;=\u0026thinsp;4.0 Hz), 4.31 (1H, dd, J\u0026thinsp;=\u0026thinsp;12.0, 4.0 Hz), 4.04 (1H, dd, J\u0026thinsp;=\u0026thinsp;12.0, 4.0 Hz), 3.95 (1H, d, J\u0026thinsp;=\u0026thinsp;4.0 Hz), 3.65 (2H, dd, J\u0026thinsp;=\u0026thinsp;8.0, 4.0 Hz), 3.55 (1H, dd, J\u0026thinsp;=\u0026thinsp;12.0, 4.0 Hz), 2.05 (3H, s). \u003csup\u003e13\u003c/sup\u003eC (CDCl\u003csub\u003e3\u003c/sub\u003e, 100 MHz) δ 170.1, 138.4, 138.0, 137.8, 133.9, 128.5, 128.5, 128.4, 128.4, 128.3, 128.2, 128.2, 127.9, 127.8, 127.7, 127.6, 127.5, 127.4, 127.4, 116.9, 100.2, 80.3, 77.8, 76.7, 74.4, 73.6, 73.6, 72.5, 71.9, 71.3, 69.3, 21.1. \u003cem\u003em/z\u003c/em\u003e calculated for [M\u0026thinsp;+\u0026thinsp;Na]\u003csup\u003e+\u003c/sup\u003e C\u003csub\u003e32\u003c/sub\u003eH\u003csub\u003e36\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e 555.6, found 555.2.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSynthesis of Allyl 2-hydroxy\u003c/b\u003e \u003cb\u003e-\u003c/b\u003e \u003cb\u003e3,4,6-tri-\u003c/b\u003e \u003cb\u003eO\u003c/b\u003e \u003cb\u003e-benzyl-β-D-galactopyranoside (3)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo a stirred solution of compound \u003cb\u003e2\u003c/b\u003e (1.7 g, 3.0 mmol) in dry MeOH (30 mL) was added Na metal (69.0 mg, 3.0 mmol) under an atmosphere of argon. The reaction mixture was stirred for 2 h at r.t.. Thereafter, the reaction mixture was quenched by addition of Amberlite IR-120 (H+) resin and the resulting mixture filtered. The filtrate was then concentrated in vacuo. The residue was purified by flash chromatography (SiO\u003csub\u003e2\u003c/sub\u003e, Petroleum Ether:EtOAc, 1:1) to afford compound \u003cb\u003e3\u003c/b\u003e (1.24 g, 81%) as a colorless syrup. \u003csup\u003e1\u003c/sup\u003eH NMR (CDCl\u003csub\u003e3\u003c/sub\u003e, 400 MHz): δ 7.37\u0026ndash;7.26 (15H, m), 5.94 (1H, m), 5.30 (1H, dd, J\u0026thinsp;=\u0026thinsp;16.0, 1.2 Hz), 5.20 (1H, d, J\u0026thinsp;=\u0026thinsp;12.0, 1.2 Hz), 4.90 (1H, d, J\u0026thinsp;=\u0026thinsp;11.6 Hz), 4.70 (1H, dd, J\u0026thinsp;=\u0026thinsp;12.0, J\u0026thinsp;=\u0026thinsp;14.0 Hz ), 4.68 (1H, d, J\u0026thinsp;=\u0026thinsp;14.0 Hz), 4.62 (1H, d, J\u0026thinsp;=\u0026thinsp;12.0 Hz), 4.46 (2H, d, J\u0026thinsp;=\u0026thinsp;6.8 Hz), 4.38 (1H, dd, J\u0026thinsp;=\u0026thinsp;10.0, 4.8 Hz), 4.31 (1H, d, J\u0026thinsp;=\u0026thinsp;7.6 Hz), 4.10 (1H, dd, J\u0026thinsp;=\u0026thinsp;12.8, 6.4 Hz), 4.00 (1H, dd, J\u0026thinsp;=\u0026thinsp;8.0, 8.0 Hz), 3.94 (1H, d, J\u0026thinsp;=\u0026thinsp;2.4 Hz), 3.62 (2H, m), 3.58 (1H, dd, J\u0026thinsp;=\u0026thinsp;5.2, 5.2 Hz), 3.45 (1H, dd, J\u0026thinsp;=\u0026thinsp;9.6, 2.8 Hz), 2.23 (1H, br s). \u003csup\u003e13\u003c/sup\u003eC (CDCl\u003csub\u003e3\u003c/sub\u003e, 100 MHz) δ 138.5, 138.0, 137.8, 134.0, 128.5, 128.5, 128.4, 128.3, 128.3, 128.2, 128.2, 127.9, 127.9, 127.8, 128.8, 127.7, 127.7, 127.6, 117.1, 102.1, 82.0, 77.4, 76.7, 73.9, 72.5, 71.6, 70.9, 69.9, 69.3, 68.6. \u003cem\u003em/z\u003c/em\u003e calculated for [M\u0026thinsp;+\u0026thinsp;Na]\u003csup\u003e+\u003c/sup\u003e C\u003csub\u003e30\u003c/sub\u003eH\u003csub\u003e34\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e 513.6, found 513.2.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSynthesis of Allyl 3,4,6-tri-\u003c/b\u003e \u003cb\u003eO\u003c/b\u003e \u003cb\u003e-benzyl-β-D-galactopyranosyl-(1\u0026rarr;2)-2,3,4,6-tetra-\u003c/b\u003e \u003cb\u003eO\u003c/b\u003e \u003cb\u003e-benzyl-β-D-glucopyranoside (5)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo a stirred solution of compound \u003cb\u003e3\u003c/b\u003e (1.54 g, 3.0 mmol) in dry DCM (30 mL) was added TMSOTf solution in DCM (c\u0026thinsp;=\u0026thinsp;0.05 M, 0.06 mmol) at -20\u0026deg;C under an atmosphere of argon. The reaction mixture was stirred for 10 minutes at -20\u0026deg;C. Thereafter, the reaction mixture was added to a stirred solution of compound \u003cb\u003e4\u003c/b\u003e (2.7 g, 3.9 mmol) in dry DCM (20 mL) -20\u0026deg;C under an atmosphere of argon.. Afterward, the reaction mixture was stirred for 30 minutes at -20\u0026deg;C and was neutralized with sat. Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e solution. The water layer was extracted with DCM (3x100 mL). The organic layer was dried with Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e and concentrated in vacuo at 35\u0026deg;C. The residue was purified by flash chromatography (SiO\u003csub\u003e2\u003c/sub\u003e, Petroleum Ether:EtOAc: Et\u003csub\u003e3\u003c/sub\u003eN, 7:3:0.1) to afford compound \u003cb\u003e5\u003c/b\u003e (1.8 g, 60%) as a colorless syrup. \u003csup\u003e1\u003c/sup\u003eH NMR (CDCl\u003csub\u003e3\u003c/sub\u003e, 400 MHz): δ 7.34\u0026ndash;7.07 (35H, m), 5.89 (1H, m), 5.28 (1H, d, J\u0026thinsp;=\u0026thinsp;16.0 Hz), 5.12 (2H, d, J\u0026thinsp;=\u0026thinsp;12.0), 5.03 (1H, d, J\u0026thinsp;=\u0026thinsp;4.0), 5.00 (1H, d, J\u0026thinsp;=\u0026thinsp;16.0), 4.88 (1H, d, J\u0026thinsp;=\u0026thinsp;8.0 Hz), 4.81 (2H, d, J\u0026thinsp;=\u0026thinsp;12.0 Hz), 4.78 (1H, d, J\u0026thinsp;=\u0026thinsp;4.0), 4.75 (2H, d, J\u0026thinsp;=\u0026thinsp;12.0 Hz), 4.73 (2H, d, J\u0026thinsp;=\u0026thinsp;16.0 Hz), 4.65 (1H, d, J\u0026thinsp;=\u0026thinsp;16.0 Hz), 4.58 (2H, d, J\u0026thinsp;=\u0026thinsp;8.0 Hz), 4.48 (1H, d, J\u0026thinsp;=\u0026thinsp;8.0 Hz), 4.47 (2H, d, J\u0026thinsp;=\u0026thinsp;7.6 Hz), 4.35 (1H, dd, J\u0026thinsp;=\u0026thinsp;12.0, 4.0 Hz), 4.30 (2H, d, J\u0026thinsp;=\u0026thinsp;12.0 Hz), 3.94 (1H, d, J\u0026thinsp;=\u0026thinsp;2.4 Hz), 4.20 (1H, dd, J\u0026thinsp;=\u0026thinsp;12.0, 4.0 Hz), 4.10 (1H, dd, J\u0026thinsp;=\u0026thinsp;12.0, 4.0 Hz), 4.04 (1H, d, J\u0026thinsp;=\u0026thinsp;2.4 Hz), 4.02 (2H, d, J\u0026thinsp;=\u0026thinsp;2.4 Hz), 3.99 (1H, d, J\u0026thinsp;=\u0026thinsp;4.0 Hz), 3.67 (1H, t, J\u0026thinsp;=\u0026thinsp;4.0 Hz), 3.58 (1H, dd, J\u0026thinsp;=\u0026thinsp;5.2, 5.2 Hz), 3.40 (1H, dd, J\u0026thinsp;=\u0026thinsp;9.6, 2.8 Hz). \u003csup\u003e13\u003c/sup\u003eC (CDCl\u003csub\u003e3\u003c/sub\u003e, 100 MHz) δ 138.9, 138.8, 138.7, 138.4, 138.2, 137.9, 134.1, 134.1,128.7, 128.5, 128.4, 128.4, 128.3, 128.3, 128.3, 128.2, 128.2, 128.1, 128.1, 128.1, 128.0, 128.0, 127.9, 127.9, 127.8, 127.7, 127.6, 127.5, 127.5, 127.4, 127.3, 118.3, 106.7, 102.4, 101.5, 100.8, 95.4, 95.1, 95.0, 94.4, 84.9, 83.0, 82.2, 79.0, 77.7, 77.5, 77.4, 77.1, 76.8, 76.8, 74.8, 74.7, 73.7, 73.6, 73.3, 73.2, 71.8, 71.7, 70.1, 69.5, 69.4, 68.7, 68.7, 67.8. \u003cem\u003em/z\u003c/em\u003e calculated for [M\u0026thinsp;+\u0026thinsp;Na]\u003csup\u003e+\u003c/sup\u003e C\u003csub\u003e64\u003c/sub\u003eH\u003csub\u003e68\u003c/sub\u003eO\u003csub\u003e11\u003c/sub\u003e 1035.2, founsd 1035.2.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSynthesis of 1-\u003c/b\u003e \u003cb\u003eO\u003c/b\u003e \u003cb\u003e-(3-acetylthiopropane)-3,4,6-tri-\u003c/b\u003e \u003cb\u003eO\u003c/b\u003e \u003cb\u003e-benzyl-β-D-galactopyranosyl-(1\u0026rarr;2)-2,3,4,6-tetra-\u003c/b\u003e \u003cb\u003eO\u003c/b\u003e \u003cb\u003e-benzyl-α-D-glucopyranoside (6)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo a stirred solution of compound \u003cb\u003e5\u003c/b\u003e (1.2 g, 1.2 mmol) and thioacetic acid (0.17 mL, 2.4 mmol) in dry 1,4-dioxane (0.3 mL), 2,2'-azobisisobutyronitrile (AIBN; 0.98 g, 6.0 mmol) was added at 50\u0026deg;C an atmosphere of argon. The reaction mixture was stirred for 3 h at 80\u0026deg;C, then cooled to room temperature. Cyclohexene (0.64 mL, 6.3 mmol) was added, and the reaction mixture was stirred at room temperature for 30 min. After evaporation, the residue was purified by flash chromatography (SiO\u003csub\u003e2\u003c/sub\u003e, Petroleum Ether:EtOAc, 8:2) to afford compound \u003cb\u003e6\u003c/b\u003e (0.13 g, 10%) as a yellow syrup. \u003csup\u003e1\u003c/sup\u003eH NMR (CDCl\u003csub\u003e3\u003c/sub\u003e, 400 MHz): δ 7.36\u0026ndash;7.06 (35H, m), 5.64 (1H, d, J\u0026thinsp;=\u0026thinsp;3.6 Hz ), 4.96 (1H, d, J\u0026thinsp;=\u0026thinsp;14.8 Hz), 4.86 (1H, d, J\u0026thinsp;=\u0026thinsp;11.6), 4.83 (1H, d, J\u0026thinsp;=\u0026thinsp;10.8), 4.80 (1H, d, J\u0026thinsp;=\u0026thinsp;12.0), 4.78 (1H, d, J\u0026thinsp;=\u0026thinsp;8.0 Hz), 4.68 (1H, d, J\u0026thinsp;=\u0026thinsp;12.0 Hz), 4.65 (1H, d, J\u0026thinsp;=\u0026thinsp;8.0), 4.58 (1H, d, J\u0026thinsp;=\u0026thinsp;12.0 Hz), 4.56 (2H, d, J\u0026thinsp;=\u0026thinsp;12.0 Hz), 4.51 (1H, d, J\u0026thinsp;=\u0026thinsp;8.0 Hz), 4.48 (2H, d, J\u0026thinsp;=\u0026thinsp;6.0 Hz), 4.42 (2H, d, J\u0026thinsp;=\u0026thinsp;11.6 Hz), 4.29 (2H, t, J\u0026thinsp;=\u0026thinsp;12.0 Hz), 4.12 (2H, d, J\u0026thinsp;=\u0026thinsp;8.0 Hz), 3.98 (1H, d, J\u0026thinsp;=\u0026thinsp;2.4 Hz), 3.97 (1H, d, J\u0026thinsp;=\u0026thinsp;12.0 Hz), 3.88 (1H, m), 3.70 (1H, d, J\u0026thinsp;=\u0026thinsp;8.0 Hz), 3.59 (6H, m), 3.36 (1H, d, J\u0026thinsp;=\u0026thinsp;8.0 Hz), 2.86 (3H, m), 2.24 (3H, s). \u003csup\u003e13\u003c/sup\u003eC (CDCl\u003csub\u003e3\u003c/sub\u003e, 100 MHz) δ 204.2, 139.2, 138.8, 138.8, 138.6, 138.3, 137.9, 137.2, 128.8, 128.8, 128.8, 128.8, 128.8, 128.8, 128.8, 128.4, 128.4, 128.4, 128.4, 128.4, 128.4, 128.4, 128.4, 128.3, 128.3, 128.3, 128.3, 128.3, 128.3, 128.3, 128.2, 128.1, 128.1, 128.1, 128.1, 128.0, 128.0, 128.0, 127.9, 127.9, 127.9, 127.8, 127.7, 127.4, 103.1, 102.7, 97.2, 96.5, 90.0, 89.7, 83.5, 83.2, 78.0, 77.3, 77.3, 76.7, 75.0, 74.1, 73.4, 70.2, 69.9, 68.6, 68.3, 67.3, 29.8, 29.7, 25.3. \u003cem\u003em/z\u003c/em\u003e calculated for [M\u0026thinsp;+\u0026thinsp;Na]\u003csup\u003e+\u003c/sup\u003e C\u003csub\u003e66\u003c/sub\u003eH\u003csub\u003e72\u003c/sub\u003eO\u003csub\u003e12\u003c/sub\u003eS 1102.3, found 1102.1.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSynthesis of 1-\u003c/b\u003e \u003cb\u003eO\u003c/b\u003e \u003cb\u003e-(3-mercaptopropyl)-β-D-galactopyranosyl-(1\u0026rarr;2)-α-D-glucopyranoside (G1)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo a stirred solution of compound \u003cb\u003e6\u003c/b\u003e (100 mg,0.092 mmol) in dry MeOH (2 mL) were added Pd/ carbon (120 mg) and dry HCO\u003csub\u003e3\u003c/sub\u003eNH\u003csub\u003e4\u003c/sub\u003e (400 mg, 6.44 mmol) at r.t. under an atmosphere of argon. The reaction mixture was stirred and heated for 10 h at 100\u0026deg;C. Afterward, the resulting mixture filtered from Acrodise*Premium 25 mm Syringe Filter with 0.45 \u0026micro;m Nylon Membran. The filtrate was concentrated in vacuo to afford compound \u003cb\u003eG1\u003c/b\u003e (10 mg, 26%) as a colorless syrup. \u003csup\u003e1\u003c/sup\u003eH NMR (D\u003csub\u003e2\u003c/sub\u003eO, 400 MHz) δ 5.40 (1H, d, J\u0026thinsp;=\u0026thinsp;3.6 Hz), 5.15 (1H, d, J\u0026thinsp;=\u0026thinsp;3.6 Hz), 4.43 (1H, d, J\u0026thinsp;=\u0026thinsp;8.0 Hz), 3.95 (1H, m), 3.86 (2H, m), 3.77\u0026ndash;3.45 (10H, m), 3.30\u0026ndash;3.22 (1H, m), 2.80 (1H, t, J\u0026thinsp;=\u0026thinsp;7.2 Hz), 2.58 (1H, t, J\u0026thinsp;=\u0026thinsp;7.2 Hz), 2.05\u0026ndash;1.84 (2H, m). \u003csup\u003e13\u003c/sup\u003eC (CDCl\u003csub\u003e3\u003c/sub\u003e, 100 MHz) δ 110.4, 111.2, 83.6, 81.5, 81.2, 76.8, 76.0, 74.1, 71.8, 71.5, 67.4, 62.9, 62.2, 30.3, 20.9. \u003cem\u003em/z\u003c/em\u003e calculated for [M\u0026thinsp;+\u0026thinsp;Na]\u003csup\u003e+\u003c/sup\u003e C\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e28\u003c/sub\u003eO\u003csub\u003e11\u003c/sub\u003eS 416.4, found 416.2.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eThe synthesis of glycidic cues poses a considerable challenge. Unlike proteins and nucleic acids, carbohydrate derivatives are complex to synthesize because of the absence of generalized methods for their routine preparation, often necessitating multiple selective protection and deprotection steps. The selection of glycidic cues is informed by their natural occurrence in collagen; the synthesis of relevant glycidic structures eliciting specific biological signals has been accomplished. Concurrently, the production of suitable linkers for the biodecoration process is underway. These heterobifunctional linkers are designed to attach to the material on one end and to the cues on the other.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026lt;Insert\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eScheme 1\u003c/strong\u003e\u003cstrong\u003e\u0026gt;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConsiderably, the glycidic cue \u003cstrong\u003eG1\u003c/strong\u003e (Scheme 1) has been selected for biomaterial functionalization. \u003cstrong\u003eG1\u003c/strong\u003e, a disaccharide present in embryonic collagen, has been suitably modified for conjugation to materials via an appropriate linker. The synthesis of individual monosaccharides is undertaken to elucidate the specific role of each sugar unit. Moreover, the synthesis of the target disaccharidic cue \u003cstrong\u003eG1\u003c/strong\u003e (Scheme 2) is particularly demanding. The critical synthesis aspects include (i) protection/deprotection steps, leading to a glycosyl acceptor (Gal unit) with selective deprotection at the 2-position for the glycosylation reaction; (ii) formation of glycosidic bond with alpha-stereoselection, necessitating a nonparticipating protecting group at the 2-OH of the glucose unit.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026lt;Insert\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eScheme 2\u003c/strong\u003e\u003cstrong\u003e\u0026gt;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ededicated to the development of intelligent biomaterials based on collagen matrices for repairing cartilage defects, with a focus on designing and synthesizing collagen patches functionalized with innovative signaling microenvironment cues, such as biologically pertinent glycidic structures. The impetus for this research is the demand for novel and effective biomaterial-based strategies for OA defect repair. The relevant glycidic structures that provide specific biological signals were characterized using spectroscopic techniques (\u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e13\u003c/sup\u003eC NMR, FTIR, Mass spectrometry).\u003c/p\u003e\n\u003cp\u003eIn the \u003csup\u003e1\u003c/sup\u003eH NMR spectra of the synthesized glycidic cue \u003cstrong\u003eG1\u003c/strong\u003e, the anomeric protons are discernible at 5.40 and 5.15 ppm. The remaining sugar protons are evident at 4.43–3.45 ppm. Notably, the characteristic aromatic peaks of compound \u003cstrong\u003e6\u003c/strong\u003e are absent in the \u003csup\u003e1\u003c/sup\u003eH NMR spectra of the synthesized glycidic cue \u003cstrong\u003eG1,\u003c/strong\u003e following the hydrolysis reaction, indicating the successful synthesis of glycidic cue \u003cstrong\u003eG1\u003c/strong\u003e. In the \u003csup\u003e13\u003c/sup\u003eC NMR spectrum, anomeric carbons C-1 of glycidic cue \u003cstrong\u003eG1\u003c/strong\u003e are observed at 111.1 and 110.4 ppm. Additionally, the alkyl group carbon peaks of glycidic cue \u003cstrong\u003eG1\u003c/strong\u003e are present at 30.3–20.4 ppm.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis investigation executed the design and synthesis of glycidic structures with biological relevance. Focusing on the design and synthesis of collagen patches, functionalized with such pioneering signaling microenvironment cues, will guide subsequent research toward the creation of intelligent biomaterials derived from collagen matrices for the remediation of cartilage defects. The driving force behind this endeavor is the necessity for inventive and promising biomaterial-based strategies in cartilage tissue engineering to address OA defects.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u0026nbsp;\u003c/strong\u003eI received a grant from the TUBITAK (Scientific and Technical Research Council of Turkey) BIDEB-2219 Programme for a postdoctoral fellowship.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest:\u0026nbsp;\u003c/strong\u003eThe authors have no conflict of interest to declare.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbou Neel EA, Bozec L, Knowles JC, Syed O, Mudera V, Day R, Hyun JK (2013) Collagen--emerging collagen based therapies hit the patient. Drug Delivery Review. 65:429\u0026ndash;456. https://doi.org/10.1016/j.addr.2012.08.010\u003c/li\u003e\n\u003cli\u003eAndr\u0026eacute;s\u0026ndash;Berg\u0026oacute;s J, Tardio L, Larranaga\u0026ndash;Vera A, G\u0026oacute;mez R, Herrero\u0026ndash;Beaumont G, Largo R (2012) The Increase in \u003cem\u003eO\u003c/em\u003e-Linked \u003cem\u003eN\u003c/em\u003e-Acetylglucosamine Protein Modification Stimulates Chondrogenic Differentiation Both \u003cem\u003ein Vitro\u003c/em\u003e and \u003cem\u003ein Vivo\u003c/em\u003e. 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Clinics in Geriatric Medicine, 26: 355-369. https://doi.org/10.1016/j.cger.2010.03.001\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Schemes","content":"\u003cp\u003eSchemes 1 and 2 are available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Collagen, Glycidic Cue, Multifunctional Biomolecule, Bioconjugation, Tissue Engineering","lastPublishedDoi":"10.21203/rs.3.rs-6678530/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6678530/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe bioactivity of materials can be enhanced by incorporating adhesive cues and regulatory molecules. Carbohydrates are essential for numerous biological recognition processes. Recent evidence suggests that collagen glycosylation is crucial for maintaining the equilibrium between collagen deposition and turnover, with several implications for healthy and pathological conditions. In this study, we synthesized small glycidic cues for enrichment with collagen for this purpose. A relevant factor for successful biomaterial decoration pertains to the selection of appropriate chemistry. This research is strongly motivated by the need for innovative biomaterials in cartilage tissue engineering, especially for repairing osteoarthritis defects.\u003c/p\u003e","manuscriptTitle":"Glycidic Cues for Tissue Engineering: Synthesis and Characterization","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-29 15:44:12","doi":"10.21203/rs.3.rs-6678530/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2fa732e3-9337-465a-89f8-e221c2850769","owner":[],"postedDate":"May 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-09T00:50:25+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-29 15:44:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6678530","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6678530","identity":"rs-6678530","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}