ELSEVIER ARTICLE INFO Article history: Received 10 August 2020 Revised 28 September 2020 Accepted 29 September Available online 6 October 2020 Keywords: Bioadhesives Tissue adhesive Musculoskeletal regeneration Bioactive materials controlled

delivery Acta Biomaterialia 117 (2020) 77-92 Review article Bioadhesives for musculoskeletal tissue regeneration * Solaiman Tarafder, Ga Young Park, Jeffrey Felix, Chang H. Lee* Regenerative Engineering Laboratory, Center for Dental and Craniofacial Research, Columbia University Irving Medical Center, 630 West 168th Street, VC12-211B, New York, NY 10032, United States 1. Introduction Contents lists available at ScienceDirect Acta Biomaterialia journal homepage: www.elsevier.com/locate/actbio ABSTRACT Bioadhesives are often referred to as natural or synthetic ma- terials that adhere to biological components such as cells, tissues, and organs through physical or chemical conjugation. Bioadhesives have been widely applied as tissue adhesives to bind tissues to- gether in soft tissue wound healing [1-3]. Some types of bioadhe- sives have been used as a hemostatic agent to stop bleeding dur- ing surgical operations or as a tissue sealant for secure gaps or cracks to prevent leakage of liquid or air [3–5]. A number of dif- ferent bioadhesives have been investigated as a tissue glue for skin wound closure to replace suture or wound dressing [1,2,6]. Inter- nal medicine has also utilized various bioadhesives as hemostasis, graft fixation, and sealants in support of surgical treatments [3]. The key properties considered for such bioadhesives, include but not limited to, biocompatibility, biodegradability, toxicity, ad- hesion strength on target surface, and duration of cross-linking [1,3]. Although each aspect may be considered on different weight depending on target application, the adhesive property is likely the most important feature for the abovementioned applications Natural or synthetic materials designed to adhere to biological components, bioadhesives, have received significant attention in clinics and surgeries. As a result, there are several commercially available, FDA- approved bioadhesives used for skin wound closure, hemostasis, and sealing tissue gaps or cracks in soft tissues. Recently, the application of bioadhesives has been expanded to various areas including muscu- loskeletal tissue engineering and regenerative medicine. The instant establishment of a strong adhesion force on tissue surfaces has shown potential to augment repair of connective tissues. Bioadhesives have also been applied to secure tissue grafts to host bodies and to fill or seal gaps in musculoskeletal tis- sues caused by injuries or degenerative diseases. In addition, the injectability equipped with the instant adhesion formation may provide the great potential of bioadhesives as vehicles for localized delivery of cells, growth factors, and small molecules to facilitate tissue healing and regeneration. This review covers recent research progress in bioadhesives as focused on their applications in musculoskeletal tissue repair and regeneration. We also discuss the advantages and outstanding challenges of bioadhesives, as well as the future perspective toward regeneration of connective tissues with high mechanical demand. © 2020 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. C:\Users\Chang\OneDrive - cumc.columbia.edu Research & Study\LAB_Work \Papers\Bioadhesive Review 2020\Revision Bioadhesives Review_Outline Combined rev 2.docx * Corresponding author. E-mail address: chl2109@cumc.columbia.edu (C.H. Lee). Acta BioMaterialia Structure Property Function Relationships in materials https://doi.org/10.1016/j.actbio.2020.09.050 1742-7061/© 2020 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. Check for updates of bioadhesives [1,3]. Functionality of most bioadhesives highly attributed to its adhesion properties on tissues, grafts, or materi- als, providing secure graft fixation, wound closure or dressing. Re- cently, the application of bioadhesives has been expanded for tis- sue repair, tissue engineering, and regenerative medicine [7-14]. To support repair and healing, particularly for musculoskeletal tis- sues, the adhesion strength of bioadhesives is being challenged to move up to the next level [8,9,14-16]. The majority of bioadhe- sives have adhesion strength at a magnitude of kPa but connec- tive tissues such as tendon and knee meniscus show tensile mod- ulus and strength in a MPa range [8,9,14-16]. Such high mechani- cal demands in musculoskeletal tissues also requires a further im- provement not only in the adhesion strength but also in bulk me- chanical properties of bioadhesives [8,9,14-16]. Besides the phys- ical support, bioadhesives have been evolved to deliver bioactive cues and/or cells in addition to provide tissue adhesion [13,14,17– 19]. Bioadhesives applied on injured site can serve as effective lo- calized delivery vehicles as secured in situ [13,14,17-19] or provid- ing physical and/or biochemical environment promoting tissue re- pair [20,21]. Recent improvements in the design and synthesis of bioadhesives have made steps closer to successful applications for musculoskeletal tissues in high mechanical demand. As per PubMed literature search from 2000 to 2019, the num- ber of peer-reviewed publications hit by a keyword "tissue adhe- sives" had increased by 2012 and started to decline from 2013 (Fig. 1A). The number of papers with a keyword "tissue adhesives S. Tarafder, G.Y. Park, J. Felix et al. # of publications A 1000 800 600 400 200 02 2000 2001 2002 2003 Tissue adhesive in vivo 2004 2005 2006 2007 2008 2009 2010 2016 2017 2018 2019 # of publications 250 200 150 100 50 B Bioadhesives in vivo 2000 2001 2002 2003 2004 2005 in vivo" appears to be steady over last decade but only account for -6.7% of total papers searched by "tissue adhesives" (Fig. 1A). The yearly number of publications hit by "bioadhesives" shows a steady increase in last 20 years (Fig. 1B). The number of papers hit by "bioadhesives in vivo" also show a continuous increase (Fig. 1B) that accounts for over 20% of total papers with "bioadhesives". These observations in literature search may suggest a growing re- search interest in adhesive biomaterials containing a designed bi- ological function beyond the traditional role as "tissue adhesive" providing physical bonding, hemostasis, or sealing. Tissue specifi- cally, the publication number of tissue adhesives and bioadhesives was the highest for bone, followed by cartilage, tendon, meniscus and intervertebral discs (IVD) (Fig. 1C). This review summarizes various types of existing bioadhesives and their adhesion mechanisms. It covers the recent advancements in bioadhesives for tissue repair and regeneration, focusing on musculoskeletal tissues. The advantages and outstanding limita- tions of bioadhesives in musculoskeletal repair and regeneration are also discussed with regard to potential and perspective. 2007 2. Types of adhesive 2.1. Cyanoacrylates Cyanoacrylates or acrylic tissue adhesives, synthesized by con- densation of a cyanoacetate with formaldehyde [22,23], have been used as a surgical glue for over 50 years [22,24]. The cyanoacrylate monomers polymerize very rapidly (5-60 s) on contact with tis- sue surfaces to form a film that bonds the apposed wound edges. As summarized in Fig. 2, this polymerization is an exothermic reaction triggered by the hydroxyl groups present on the tissue surface or from the moisture [1,25,26]. Participation from amino groups on the tissue surface can also take place during the poly- merization resulting in a strong bond with the tissue. The gen- eral chemical name and formula of cyanoacrylates are alkyl-2- cyanoacrylates and CH₂=C(CN)-COOR, respectively, where R could be any alkyl group ranging from methyl to decyl [25,27]. The first developed cyanoacrylate adhesive was methyl-2-cyanoacrylate (R= -CH3) (known as Eastman 910) with the shortest chain deriva- tive [22]. It was found that the longer the alkyl chain (the R group) the lower the tissue toxicity from the cyanoacrylate ad- hesives [3,28,29]. While the cyanoacrylate with the shortest alkyl group (-CH3) produces a rigid polymer, flexibility can be improved with the longer alkyl chain and adding plasticizer as well. As a result, many cyanoacrylates with longer chain derivatives have been developed such as ethyl-2-cyanoacrylate (EpigluⓇ; Meyer- Haake, Ober-Morlen, Germany) & Krazy Glue® (Elmer's Products Inc, Columbus, OH), butyl-2-cyanoacrylate (Trufill®; Codman & Years 78 2013 2015 2016 2.2. Fibrin 2017 2018 2019 C # of publications 2000 1500 1000 500 Years Fig. 1. PubMed literature search from 2000 to 2019 with selected key words (A and B) and specific tissue target (C). Bone Acta Biomaterialia 117 (2020) 77-92 IVD Cartilage Tendon Meniscus Shurtleff, Inc., Raynham MA), Indermil® (Connexicon Medical Ltd., Dublin, Ireland), Histoacryl® (B. Braun AG, Melsungen, Germany), and 2-octyl-cyanoacrylate (Dermabond®; Ethicon US, LLC., a John- son & Johnson Company, Cincinnati, OH) & Surgiseal (Adhezion Biomedical, Wyomissing, PA)[22, 29, 30]. Although cyanoacrylates I have been used as tissue adhesives or sealants for decades out- side the U.S., the first cyanoacrylate that was approved by FDA (in 1998) to be used as tissue adhesive was 2-octyl-cyanoacrylate (Dermabond®) [1,22]. Some of the benefits of cyanoacrylate tissue adhesives are the ease of application for first aid, quick adhesion or sealing of wounded tissues, excellent hemostasis, and potential bacterio- static or microbial barrier properties [1,31]. Despite these benefits, cyanoacrylate and its degradation by-products may cause cytotox- icity, foreign body reactions, tissue necrosis, and inflammatory re- sponses [32,33]. Cyanoacrylates degrade via hydrolysis resulting in toxic cyanoacetates and formaldehydes as degradation by-products [26,33]. The inherent brittleness is another setback for cyanoacry- lates. Significant efforts have been made to mitigate such brittle- ness and cytotoxicity by introducing longer alkyl chain derivatives. Because of their cytotoxic and inflammation prone nature, a lim- ited number of cyanoacrylates are approved by FDA, predominately for topical use. Fibrin tissue adhesives or tissue sealants are the most widely used bioadhesives in the U.S. since their first approval by FDA in 1998 [27,30,34,35]. Fibrin sealants, also known as fibrin glue, con- tain two key components derived from plasma coagulation pro- teins, (i) fibrinogen and (ii) thrombin. Upon mixture, these two components mimic the body's natural blood clotting cascades, as thrombin converts soluble fibrinogen into crosslinked, insoluble fibrin [1,34]. Calcium is often added to thrombin to further cat- alyze the clot formation. Although clotting occurs rapidly (within seconds), the clotting time can vary depending on the concentra- tions of fibrinogen and thrombin and the presence of other cat- alyzing and stabilizing components. Fibrin is the only material that is currently FDA approved for use as a hemostat, tissue adhesive, and tissue sealant [36]. Fibrin sealant has wide range of applications. For exam- ple, orthopaedic surgeons frequently use fibrin sealant in autol- ogous chondrocyte implantation (ACI) treatment, where culture- expanded chondrocytes are delivered into a cartilage defect con- fined by a periosteal or collagenous membrane fixated by sutures, followed by sealing the defect boundaries with fibrin sealant. In the suture-free matrix-induced ACI (MACI), type I/type III colla- gen bilayer seeded with chondrocytes is secured directly to the S. Tarafder, G.Y. Park, J. Felix et al. (i) n/H₂C=C1 (ii) Alkyl-2-cyanoacrylates (R could be any alkyl group ranging from methyl to decyl) R H₂C Shorter alkyl (R) group on (i) Cyanoacetate tő-c Poly(methyl-2-cyanoacrylate) (Eastman 910) CH3 OH™ tő-c t-c Toxicity Polycyanoacrylates (i) H Poly(ethyl-2-cyanoacrylate) (EpigluⓇ & Kragy glue®) defect site by fibrin glue [37,38]. Repair of delaminated acetabular articular cartilage using fibrin adhesive was shown to be a useful technique for the early cartilage damage treatment strategy [23]. A cadaveric study showed that improved press-fit fixation of os- teochondral scaffolds can be achieved using fibrin glue [39]. Fibrin glue can improve the meniscus healing when applied to outer zone meniscus defect compared to defect only repair [40,41]. Even bet- ter meniscus healing was observed when fibrin was mixed with bone marrow cells in a rabbit model [40]. A long term follow- up (average of 8 years) of 40 patients showed better repair and healing of arthroscopically repaired meniscal tears using fibrin glue with comparable recurrence rate (10%) compared to repair with su- turing [40]. Fibrin is a unique biopolymer with unique biological and physi- cal characteristics. Fibrin sealants exhibit excellent biocompatibil- Formaldehyde H₂ C-CH3 N 79 R H₂ Longer alkyl (R) group on (i) H-c] C C CH3 H₂ H₂ H₂ Poly(octyl-2-cyanoacrylate) (Dermabond® & Surgiseal®) CH3 Hydrolysis (Degradation) Acta Biomaterialia 117 (2020) 77-92 H₂ H₂ Poly(butyl-2-cyanoacrylate) (Histoacryl®, Indermil® & Trufill®) H₂ Fig. 2. Cyanoacrylate adhesivies: formation of polycyanoacrylates from alkyl-2-cyanoacrylate monomeric units, and the resulting byproducts from the degradation of poly- cyanoacrylates (i); some of the shorter and longer alkyl chain derivatives and their commercial names (ii). CH3 ity, biodegradability, deformability, and elasticity. In addition to that, fibrin adhesives do not trigger any inflammatory responses, foreign body reactions, tissue necrosis, or extensive fibrosis. How- ever, in spite of having all these benefits, fibrin glues have low bond strengths (0.005-0.17 MPa) compared to synthetic tissue ad- hesives [1,35]. This limits their application to the defect site under- going significant tensile loads. Fibrin glue can degrade very rapidly even before the healing process begins because of the proteolytic activity in the musculoskeletal joints [42,43]. This is one of the ma- jor reasons because of their limited applicability for musculoskele- tal tissue repair or regeneration associated with synovial joints. In addition to their uses as sealants and tissue adhesives, fibrin alone, or in combination with other polymers, has also been ex- tensively used for tissue engineering and regenerative medicine applications. S. Tarafder, G.Y. Park, J. Felix et al. 2.3. Aldehyde based bioadhesives Another family of commonly used bioadhesive is based on alde- hyde. For example, gelatin-resorcinol cross-linked with formalde- hyde (GRF) and GRFG (GRF with glutaraldehyde) adhesives are the most commonly applied aldehyde based formulations. Originally developed in Europe in the 1960s, GRF/GRFG have been widely used in Europe and Japan for the past few decades for vascular, thoracoscopic, gastrointestinal, lever, and urinary track surgeries (i) (ii) (iii) (iv) H₂N- H₂N- S H₂N- Gelatin H₂N- Gelatin OH OH -NH₂ + -NH₂ + OH H H Formaldehyde bychom OH Resorcin Formaldehyde Formaldehyde H₂N- H₂N- Ho S... R H Glutaraldehyde H₂N- Glutaraldehyde H₂N- [1,44,45]. The idea of having both formaldehyde and glutaralde- hyde in the same formulation is to obtain the initial strong bond- ing from formaldehyde and the high in vivo stability from glu- taraldehyde. Gelatin contributes to the biodegradability and elas- ticity of the GRF/GRFG glue. Since gelatin crosslinked by formalde- hyde/glutaraldehyde performs poorly in wet condition, resorcin, a phenolic component (1,3-benzenediol), is added to GRFG formula- tion to improve its strength by minimizing the negative effect from an aqueous environment [1] (Fig. 3). Formaldehyde and (or) glu- Schil + Resorcin Formaldehyde Tissue H₂N- H H ΝΗ NH- 3x4 ( t usl +H₂N- H₂N- Tissue H₂N- 80 НО H H NH HN- HO H₂C -N=CH HC=N- R H₂N- H₂N- OH OH CH₂ H₂C NH H₂C fo HO HO H₂C -NH₂ ΝΗ -NH₂ -NH₂ -NH₂ |N=CH HC_N R OH OH OH OH H₂ CH₂ OH OH CH₂ CH, HỌC OH OH HN CH₂ OH Acta Biomaterialia 117 (2020) 77-92 OH CH₂ ΗΝ Fig. 3. Aldehyde based adhesives: formaldehyde and glutaraldehyde act as cross-linkers between gelatin molecules (i) and gelatin and tissue or biological surfaces (ii); crosslinking between resorcin and formaldehyde (iii) and resorcin, formaldehyde and tissue (iv). S. Tarafder, G.Y. Park, J. Felix et al. taraldehyde act as crosslinking agents for both gelatin and resor- cin. Aldehyde groups from formaldehyde and glutaraldehyde re- act with the amine group from gelatin, in addition to the amine group present in the tissue, and thus form a strong bond be- tween GRF/GRFG and tissue. Bonding strength of GRF/GRFG can be achieved to the level of cyanoacrylates. In spite of its ex- cellent hemostatic and adhesive properties and widespread us- age in Europe and Japan for decades, GRF/GRFG glues have not been approved by FDA to be used for clinical applications in the U.S.[3] This is likely due to the potential cytotoxicity, mutagenicity, and carcinogenicity caused by formaldehydes, which either can be caused by the residue of unreacted formaldehyde molecules or by the degradation byproducts [46,47]. As a result, some formulations with less toxic glutaraldehyde glyoxal or glutaric acid may improve the biosafety of GRF/GRFG [1,48,49]. BioGlue® (Cryolife, Kennesaw, GA), a protein-aldehyde system (PAS), is a commercially available glutaraldehyde-based formula- tion. It has two components, bovine serum albumin (BSA) and glu- taraldehyde, and the gluing mechanism is similar to GRF/GRFG. BioGlue® has been approved by FDA in 1999 to be used in the U.S. as adjunct to suturing or stapling for acute thoracic aortic dissection and cardiac surgery [27,35]. In vivo degradation rate of BioGlue® is slower than GRF/GRFG. However, the potential cytotoxicity of glutaraldehyde has led to the use of alternative crosslinking agents in other albumin based formulations. For in- stance, PreveLeak™ (Baxter Healthcare, Deerfield, IL) is composed of BSA and polyaldehyde [50] and Progel® (Neomend, Inc., Irvine, CA) is composed of human serum albumin and a polyethyleneg- lycol (PEG) crosslinker functionalized with succinate groups (PEG- (SS)₂), where N-hydroxysuccinimide (NHS) ester groups are at- tached to each end of the PEG [51]. Both PreveLeak and Progel® (i) Tetra-sussinimidyl polyethyleneglycol(4S-PEG): LICHA BOICH SCHO) H₂O) n -OC(CH 2) 3CO(CH₂CH₂O) n Tissue HSCH₂CH₂O(CH2CH₂O) n CH₂ HSCH₂CH₂O(CH2CH₂O) n (ii) Tetra-thiol polyethyleneglycol (4T-PEG): CH₂ H₂C CH₂ H₂C #CHOICH=CH-01/1/20 CH₂ CH₂ (OCH₂CH₂) OC( (CH₂CH₂), (OCH2CH₂) OC(CH2) 3CO- CH₂ CH₂ (OCH2CH₂) OCH₂CH₂SH CH₂ , (OCH2CH₂) OCH₂CH₂SH (1904, 04, DEICH) CH₂ N CH₂ C-SCH2CH₂O(CH2CH₂O) n are FDA approved for vascular reconstructions and intraoperative use during pulmonary resection, respectively. 2.4. Polyethylene glycol (PEG) based adhesives Polyethylene glycol (PEG) based adhesives are a highly water- absorptive hydrogel which have been widely used as fluid barri- ers and hemostatic adhesives. The first commercially available PEG based adhesive was FocalSealⓇ® (Genzyme Biosurgery Inc., Cam- bridge, MA) [52]. It was activated by light and intended to be used as a lung sealant. However, FocalSeal® is no longer available in the market due to its difficulty to use. Currently there are two FDA ap- proved PEG based adhesives available in the market, Coseal (Bax- ter International Inc., Deerfield, IL) and DuraSeal® (Integra Life- Sciences, Princeton, NJ) [53–55]. Coseal is a fully synthetic adhesive that contains two biocompatible functionalized polyethylene gly- cols (PEG), tetra-succinimidyl (4S) and tetra-thiol (4T)-derivatized polyethyleneglycol (4S-PEG and 4T-PEG) [52]. A covalently bonded hydrogel forms when 4S-PEG and 4T-PEG are mixed together. Gel formation occurs through the reaction between the thiol groups and the carbonyl groups of the succinimidyl esters resulting in the formation of a thio-ester covalent network between PEG molecules (Fig. 4). Free N-hydroxy-succinimide molecules are liberated from the reactions. It is indicated for use in vascular reconstructions to achieve adjunctive hemostasis by mechanically sealing areas of leakage. Duraseal contains polyethylene glycol (PEG) ester solution and a trilysine amine solution. This has been used as an adjunct for dural closure to prevent cerebrospinal fluid (CSF) leakage dur- ing brain and spine surgeries. PEG based adhesives are hydrophilic, biocompatible, and biodegradable. However, these adhesives ex- hibit high swelling ratio of up to 400% and thus need to be very (iii) Schematic representation of how a covalently bonded hydrogel forms when 4S-PEG and 4T-PEG are mixed together: CH₂ H₂C CH₂ CH₂ (iv) Schematic representation of how the hydrogel formed from 4S-PEG and 4T-PEG interacts with the biological tissue: (OCH_CH),OCH/CH,S-CICH+;) C&CH/C+01 CH₂CH₂S- 81 H₂C (OCH2CH₂) OCH₂CH₂SH CH₂ CH₂ 4T-PEG (OCH=CH), OCH(CHS C&H) C&CH CHOIR CH₂ CH₂ H₂C Acta Biomaterialia 117 (2020) 77-92 CH₂ LỊCH LỊCH CHỌN CH₂ CH₂ CH₂ H₂C CH₂ (OCH₂CH₂),OC(CH 2) 3C- CH₂ H₂C CH₂ CH₂ 4S-PEG -OH N-hydroxy-succinimide Tissue Fig. 4. Polyethylene glycol (PEG) based adhesives: Chemical structure of tetra-succinimidyl (4S) and tetra-thiol (4T)-derivatized polyethyleneglycol 4S-PEG (i) and 4T-PEG (ii); crosslinking between 4S-PEG and 4T-PEG (iii); and interactions between tissue/biological surfaces with the hydrogel formed from 4S-PEG and 4T-PEG (iv).