Silver nitrate-assisted photo-crosslinking for enhancing the mechanical properties of an alginate/silk fibroin-based 3D scaffold.

Scaffolds play a pivotal role in tissue engineering and serve as vital biological substitutes providing structural support for cell adhesion and subsequent tissue development. An ideal scaffold must possess mechanical properties suitable for tissue function and exhibit biodegradability. Although synthetic polymer scaffolds offer high rigidity and elasticity owing to their reactive side groups, which facilitate tailored mechanical and rheological properties, they may lack biological cues and cause persistent side effects during degradation. To address these challenges, natural polymers have garnered attention owing to their inherent bioactivity and biocompatibility. However, natural polymers such as silk fibroin (SF) and tyramine-modified alginate-tyramine (AT) have limitations, including uncontrolled mechanical properties and weak structural integrity. In this study, we developed a blend of SF and AT as a printable biomaterial for extrusion-based 3D printing. The use of photocrosslinkable SF/AT inks facilitated the fabrication of complex scaffolds with high printability, thereby enhancing their structural stability. The incorporation of silver nitrate facilitated the tunability of mechanical and rheological behaviors. SF/AT scaffolds with varying stiffness in the physiologically relevant range for soft tissues (51-246 kPa) exhibited excellent biocompatibility, indicating their promising potential for diverse applications in tissue engineering.

Photo-/thermo-responsive bioink for improved printability in extrusion-based bioprinting.

Extrusion-based bioprinting has demonstrated significant potential for manufacturing constructs, particularly for 3D cell culture. However, there is a greatly limited number of bioink candidates exploited with extrusion-based bioprinting, as they meet the opposing requirements for printability with indispensable rheological features and for biochemical functionality with desirable microenvironment. In this study, a blend of silk fibroin (SF) and iota-carrageenan (CG) was chosen as a cell-friendly printable material. The SF/CG ink exhibited suitable viscosity and shear-thinning properties, coupled with the rapid sol-gel transition of CG. By employing photo-crosslinking of SF, the printability with Pr value close to 1 and structural integrity of the 3D constructs were significantly improved within a matter of seconds. The printed constructs demonstrated a Young's modulus of approximately 250 kPa, making them suitable for keratinocyte and myoblast cell culture. Furthermore, the high cell adhesiveness and viability (maximum >98%) of the loaded cells underscored the considerable potential of this 3D culture scaffold applied for skin and muscle tissues, which can be easily manipulated using an extrusion-based bioprinter.

Protective Topical Dual-Sided Nanofibrous Hemostatic Dressing Using Mussel and Silk Proteins with Multifunctionality of Hemostasis and Anti-Bacterial Infiltration.

Topical hemostatic agents are preferred for application to sensitive bleeding sites because of their immediate locoregional effects with less tissue damage. However, the majority of commercial hemostatic agents fail to provide stable tissue adhesion to bleeding wounds or act as physical barriers against contaminants. Hence, it has become necessary to investigate biologically favorable materials that can be applied and left within the body post-surgery. In this study, a dual-sided nanofibrous dressing for topical hemostasis is electrospun using a combination of two protein materials: bioengineered mussel adhesive protein (MAP) and silk fibroin (SF). The wound-adhesive inner layer is fabricated using dihydroxyphenylalanine (DOPA)-containing MAP, which promotes blood clotting by aggregation of hemocytes and activation of platelets. The anti-adhesive outer layer is composed of alcohol-treated hydrophobic SF, which has excellent spinnability and mechanical strength for fabrication. Because both proteins are fully biodegradable in vivo and biocompatible, the dressing would be suitable to be left in the body. Through in vivo evaluation using a rat liver damage model, significantly reduced clotting time and blood loss are confirmed, successfully demonstrating that the proposed dual-sided nanofibrous dressing has the right properties and characteristics as a topical hemostatic agent having dual functionality of hemostasis and physical protection.

Self-controllable proteinic antibacterial coating with bacteria-triggered antibiotic release for prevention of periprosthetic infection.

Periprosthetic infection is a devastating postimplantation complication in which a biofilm layer harboring invasive microorganisms forms around orthopedic implants, leading to severe implant failure and patient morbidity. Despite the development of several infection-triggered antibiotic release approaches, most current antibacterial coatings are susceptible to undesired antibiotic leakage or mechanical disintegration during prosthesis installation. Herein, we propose a self-controllable proteinic antibacterial coating capable of both long-lasting adherence onto titanium implant substrates over the implant fixation period and instantaneous bacterial eradication. Importantly, the pH-dependent reversible metal coordination of mussel adhesive protein (MAP) enabled bacterial concentration-dependent antibiotic delivery in response to infection-induced acidification. In addition, the MAP coating exhibited superior self-healable adhesive properties and scratch resistance, which enabled to avert issues associated with mechanical damages, including peeling and cracking, often occurring in conventional implant coating systems. The gentamicin-loaded MAP coating exhibited complete inhibition of bacterial growth in vivo against Staphylococcus aureus penetrations during implantation surgery (immediate infection) and even 4 weeks after implantation (delayed infection). Thus, our antibiotic-loaded MAP hydrogel coating can open new avenues for self-defensive antibiotic prophylaxis to achieve instant and sustainable bacteriocidal activity in orthopedic prostheses. © 2017 Elsevier Inc. All rights reserved.

Injectable alginate-based in situ self-healable transparent hydrogel as a vitreous substitute with a tamponading function.

Retinal detachment and other vision-threatening disorders often necessitate vitreous body removal and tamponade injection for retina stabilization. Current clinical tamponades such as silicone oil and expansile gases have drawbacks, including patient discomfort and the need for secondary surgery. We introduce a transparent alginate-phenylboronic acid/polyvinyl alcohol composite hydrogel (TALPPH) as a novel vitreous substitute with tamponading capabilities. In vitro physicochemical, rheological, and optical characterization of in situ self-healable TALPPH was performed, and long-term biocompatibility was assessed in a rabbit model of vitrectomy retinal detachment. In vivo evaluations confirmed TALPPH's ability to inhibit retinal detachment recurrence and preserve rabbit vision without adverse effects. TALPPH's close resemblance to the natural vitreous body suggests potential as a vitreous tamponade substitute for future ophthalmological applications.

A Customizable Proteinic Bioadhesive Patch with Water-Switchable Underwater Adhesiveness, Adjustable Biodegradability, and Modifiable Stretchability for Healing Diverse Internal Wounds.

Customizable bioadhesives for individual organ requirements, including tissue type and motion, are essential, especially given the rise in implantable medical device applications demanding adequate underwater adhesion. While synthetic bioadhesives are widely used, their toxicity upon degradation shifts focus to biocompatible natural biomaterials. However, enhancing the adhesive strengths of these biomaterials presents ongoing challenges while accommodating the unique properties of specific organs. To address these issues, three types of customized underwater bioadhesive patches (CUBAPs) with strong, water-responsive adhesion and controllable biodegradability and stretchability based on bioengineered mussel adhesive proteins conjugated with acrylic acid and/or methacrylic acid are proposed. The CUBAP system, although initially nonadhesive, shows strong underwater adhesion upon hydration, adjustable biodegradation, and adequate physical properties by adjusting the ratio of poly(acrylic acid) and poly(methacrylic acid). Through ex vivo and in vivo evaluations using defective organs and the implantation of electronic devices, the suitability of using CUBAPs for effective wound healing in diverse internal organs is demonstrated. Thus, this innovative CUBAP system offers strong underwater adhesiveness with tailored biodegradation timing and physical properties, giving it great potential in various biomedical applications.

Self-Healable Adhesive Hydrogel with a Preserved Underwater Adhesive Ability Based on Histidine-Zinc Coordination and a Bioengineered Hybrid Mussel Protein.

Mussels are marine organisms that are capable of constructing an underwater adhesion between their bodies and rigid structures. It is well known that mussels achieve underwater adhesion through the presence of mussel adhesive proteins (MAPs) that contain high levels of 3,4-dihydroxyphenylalanine (DOPA). Although the extraordinary underwater adhesive properties of mussels are attributed to DOPA, its capacity to play a dual role in surface adhesion and internal cohesion is inherently limited. However, mussels employ a combination of chemical moieties, not just DOPA, along with anatomical components, such as plaque and byssus, in underwater adhesion. This also involves junction proteins that connect the plaque and byssus. In this study, a novel hybrid MAP was bioengineered via the fusion of the plaque protein (foot protein type 1) and the histidine-rich domain of the junction protein (foot protein type 4). To achieve direct adhesion underwater, the adhesive should maintain surface adhesion without disintegrating. Notably, the histidine-Zn-coordinated hybrid MAP hydrogel maintained a high surface adhesion ability even after cross-linking because of the preservation of its unoxidized and non-cross-linked DOPA moieties. The formulated adhesive hydrogel system based on the bioengineered hybrid MAP exhibited self-healing properties, owing to the reversible metal coordination bonds. The developed adhesive hydrogel exhibits outstanding levels of bulk adhesion in underwater environments, highlighting its potential as an effective adhesive biomaterial. Therefore, the introduction of histidine-rich domains into MAPs may be applied in various studies to formulate mussel-inspired adhesives with self-healing properties and to fully utilize the adhesive ability of DOPA.

Cell recognitive bioadhesive-based osteogenic barrier coating with localized delivery of bone morphogenetic protein-2 for accelerated guided bone regeneration.

Titanium mesh (Ti-mesh) for guided bone regeneration (GBR) approaches has been extensively considered to offer space maintenance in reconstructing the alveolar ridge within bone defects due to its superb mechanical properties and biocompatibility. However, soft tissue invasion across the pores of the Ti-mesh and intrinsically limited bioactivity of the titanium substrates often hinder satisfactory clinical outcomes in GBR treatments. Here, a cell recognitive osteogenic barrier coating was proposed using a bioengineered mussel adhesive protein (MAP) fused with Alg-Gly-Asp (RGD) peptide to achieve highly accelerated bone regeneration. The fusion bioadhesive MAP-RGD exhibited outstanding performance as a bioactive physical barrier that enabled effective cell occlusion and a prolonged, localized delivery of bone morphogenetic protein-2 (BMP-2). The MAP-RGD@BMP-2 coating promoted in vitro cellular behaviors and osteogenic commitments of mesenchymal stem cells (MSCs) via the synergistic crosstalk effects of the RGD peptide and BMP-2 in a surface-bound manner. The facile gluing of MAP-RGD@BMP-2 onto the Ti-mesh led to a distinguishable acceleration of the in vivo formation of new bone in terms of quantity and maturity in a rat calvarial defect. Hence, our protein-based cell recognitive osteogenic barrier coating can be an excellent therapeutic platform to improve the clinical predictability of GBR treatment.

Visible light-crosslinkable tyramine-conjugated alginate-based microgel bioink for multiple cell-laden 3D artificial organ.

While the natural carbohydrate alginate has enabled effective three-dimensional (3D) extrusion bioprinting, it still suffers from some issues such as low printability and resolution and limited cellular function due to ionic crosslinking dependency. Here, we prepared a harmless visible light-based photocrosslinkable alginate by chemically bonding tyrosine-like residues onto alginate chains to propose a new microgel manufacturing system for the development of 3D-printed bioinks. The photocrosslinkable tyramine-conjugated alginate microgel achieved both higher cell viability and printing resolution compared to the bulk gel form. This alginate-based jammed granular microgel bioink showed excellent 3D bioprinting ability with maintained structural stability. As a biocompatible material, the developed multiple cell-loaded photocrosslinkable alginate-based microgel bioink provided excellent proliferation and migration abilities of laden living cells, providing an effective strategy to construct implantable functional artificial organ structures for 3D bioprinting-based tissue engineering.

Microporous Hemostatic Sponge Based on Silk Fibroin and Starch with Increased Structural Retentivity for Contact Activation of the Coagulation Cascade.

Hemostatic agents with diverse forms and materials are necessitated to control excessive bleeding to improve surgical site visibility during operation. The adequate use of hemostatic agents dramatically reduces the chance of dehydration, absence of oxygen, and, in severe cases, death. Polysaccharide-based hemostatic agents are widely used as they are safe for the human body. Among diverse polysaccharides, starch has exhibited a high swelling ability, but its powder formulation is limited during incompressible bleeding. Herein, starch was blended with silk protein and crosslinked using glycerol to improve structural integrity. The silk/starch solution was lyophilized to be a sponge with interconnected pores, which is beneficial to blood coagulation by increased swelling ratio and underwater retentivity to absorb blood plasma. The surface contact between the blood component and the sponge initiates clotting by intrinsic pathway activation and platelet activation without the hemolytic effect or cytotoxicity. The clinical effectiveness of the sponges as topical hemostatic agents was confirmed by animal bleeding model tests.

Hydrogel Microneedles Extracting Exosomes for Early Detection of Colorectal Cancer.

There are several methods for early diagnosis of tumors, such as detecting circulating tumor DNAs, detecting circulating tumor cells, or imaging with tumor-targeting contrast agents. However, these assays are time-consuming and may cause patient discomfort during the biopsy collecting process. Here, we develop a facile method for early diagnosis of tumors by extracting exosomes from interstitial fluid (ISF) using hydrogel microneedles (MNs). The hydrogel MNs expand in the skin to absorb the ISF, and the tumor exosomes contained in the ISF bind with the glypican-1 antibodies inside the hydrogel of MNs. After removing the hydrogel on the MNs, exosomes are separately purified from the ISF to analyze tumor-related biomarkers. Finally, colorectal cancer can be diagnosed by ELISA for the colorectal cancer-induced model mice. This noninvasive hydrogel MN system to obtain the exosome samples would play an important role in early cancer diagnosis.

A colorimetric lateral flow immunoassay based on oriented antibody immobilization for sensitive detection of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). The high human-to-human transmission and rapid evolution of SARS-CoV-2 have resulted in a worldwide pandemic. To contain SARS-CoV-2, it is essential to efficiently control the transmission of the virus through the early diagnosis of infected individuals, including asymptomatic people. Therefore, a rapid and accurate assay is vital for the early diagnosis of SARS-CoV-2 in suspected individuals. In this study, we developed a colorimetric lateral flow immunoassay (LFIA) in which a CBP31-BC linker was used to immobilize antibodies on a cellulose membrane in an oriented manner. The developed LFIA enabled sensitive detection of cultured SARS-CoV-2 in 15 min with a detection limit of 5 × 104 copies/mL. The clinical performance of the LFIA for detecting SARS-CoV-2 was evaluated using 19 clinical samples validated by reverse transcription-polymerase chain reaction (RT-PCR). The LFIA detected all the positive and negative samples accurately, corresponding to 100% accuracy. Importantly, patient samples with low viral loads were accurately identified. Thus, the proposed method can provide a useful platform for rapid and accurate point-of-care testing of SARS-CoV-2 in infected individuals to efficiently control the COVID-19 pandemic.

A Perfluoropolyether Microfluidic Device for Cell-Based Drug Screening with Accurate Quantitative Analysis.

Microfluidic drug screening technologies have been extensively explored to evaluate the pharmacology and therapeutic implications of promising chemical compounds in multiplexed physiological microenvironments in vivo. However, conventional poly(dimethylsiloxane) microchips are susceptible to adsorption by hydrophobic molecules on channel surfaces and permeation in the matrix. These can significantly compromise the drug availability and accuracy of dose-dependent quantitative analyses. Here, we prepared a perfluorinated polyether (PFPE) microchip via digital light processing 3D printing as a quantitative drug screening platform for precise concentration-dependent pharmaceutical assays. Cells cultured on PFPE microchips exhibited excellent viability with a spread morphology as well as superior proliferative capability. Importantly, PFPE constructions with a low surface energy significantly prevented the nonspecific molecular adsorption into their surfaces or permeation into the matrix. In particular, the PFPE multibranched channel preserved the concentration of the pharmaceutical drug during the perfusion process and generated a linear concentration gradient, resulting in a dose-dependent chemotherapeutic effect. We suggest that the biocompatible and nonadsorbing PFPE microchannel can provide a cell-based drug screening platform for concentration-dependent quantitative analyses.

Thiol-Rich fp-6 Controls the Tautomer Equilibrium of Oxidized Dopa in Interfacial Mussel Foot Proteins.

3,4-Dihydroxyphenylalanine (Dopa) is a versatile molecule that enables marine mussels to achieve successful underwater adhesion. However, due to its complicated redox chemistry and vulnerability to oxidation, controlling surface adhesion and cohesion has been a challenging issue to overcome. Foot protein type 6 (fp-6), a thiol-rich interfacial mussel adhesive protein, has been reported as a proteinaceous antioxidant for mussels that helps Dopa maintain surface adhesion ability. In this study, we focused on the role of fp-6 in oxidized Dopa. The effect on the tautomer equilibrium of oxidized Dopa was investigated using recombinant fp-6 (rfp-6) and Dopa-incorporated foot protein type 3 fast variant (drfp-3F), which were produced in bacterial cells. The redox chemistry of Dopa in drfp-3F and the role of rfp-6 were observed using a UV-vis spectrophotometer and a surface forces apparatus (SFA). We discovered that rfp-6 shifts the tautomer equilibrium to ΔDopa as a preferred tautomer for oxidized Dopa in drfp-3F and makes drfp-3F better on underwater surface adhesion.

Double-layered adhesive microneedle bandage based on biofunctionalized mussel protein for cardiac tissue regeneration.

Heart failure following myocardial infarction (MI), the primary cause of mortality worldwide, is the consequence of cardiomyocyte death or dysfunction. Clinical efforts involving the delivery of growth factors (GFs) and stem cells with the aim of regenerating cardiomyocytes for the recovery of structural and functional integrity have largely failed to deliver, mainly due to short half-lives and rapid clearance in in vivo environments. In this work, we selected and genetically fused four biofunctional peptides possessing angiogenic potential, originating from extracellular matrix proteins and GFs, to bioengineered mussel adhesive protein (MAP). We found that MAPs fused with vascular endothelial growth factor (VEGF)-derived peptide and fibronectin-derived RGD peptide significantly promoted the proliferation and migration of endothelial cells in vitro. Based on these characteristics, we fabricated advanced double-layered adhesive microneedle bandages (DL-AMNBs) consisting of a biofunctional MAP-based root and a regenerated silk fibroin (SF)-based tip, allowing homogeneous distribution of the regenerative factor via swellable microneedles. Our developed DL-AMNB system clearly demonstrated better preservation of cardiac muscle and regenerative effects on heart remodeling in a rat MI model, which might be attributed to the prolonged retention of therapeutic peptides as well as secure adhesion between the patch and host myocardium by MAP-inherent strong underwater adhesiveness.

Tunicate-Inspired Photoactivatable Proteinic Nanobombs for Tumor-Adhesive Multimodal Therapy.

Near-IR (NIR) light-responsive multimodal nanotherapeutics have been proposed to achieve improved therapeutic efficacy and high specificity in cancer therapy. However, their clinical application is still elusive due to poor biometabolization and short retention at the target site. Here, innovative photoactivatable vanadium-doped adhesive proteinic nanoparticles (NPs) capable of allowing biological photoabsorption and NIR-responsive anticancer therapeutic effects to realize trimodal photothermal-gas-chemo-therapy treatments in a highly biocompatible, site-specific manner are proposed. The photoactivatable tumor-adhesive proteinic NPs can enable efficient photothermal conversion via tunicate-inspired catechol-vanadium complexes as well as prolonged tumor retention by virtue of mussel protein-driven distinctive adhesiveness. The incorporation of a thermo-sensitive nitric oxide donor and doxorubicin into the photoactivatable adhesive proteinic NPs leads to synergistic anticancer therapeutic effects as a result of photothermal-triggered "bomb-like" multimodal actions. Thus, this protein-based phototherapeutic tumor-adhesive NPs have great potential as a spatiotemporally controllable therapeutic system to accomplish effective therapeutic implications for the complete ablation of cancer.

Diverse silk and silk-like proteins derived from terrestrial and marine organisms and their applications.

Organisms develop unique systems in a given environment. In the process of adaptation, they employ materials in a clever way, which has inspired mankind extensively. Understanding the behavior and material properties of living organisms provides a way to emulate these natural systems and engineer various materials. Silk is a material that has been with human for over 5000 years, and the success of mass production of silkworm silk has realized its applications to medical, pharmaceutical, optical, and even electronic fields. Spider silk, which was characterized later, has expanded the application sectors to textile and military materials based on its tough mechanical properties. Because silk proteins are main components of these materials and there are abundant creatures producing silks that have not been studied, the introduction of new silk proteins would be a breakthrough of engineering materials to open innovative industry fields. Therefore, in this review, we present diverse silk and silk-like proteins and how they are utilized with respect to organism's survival. Here, the range of organisms are not constrained to silkworms and spiders but expanded to other insects, and even marine creatures which produce silk-like proteins that are not observed in terrestrial silks. This viewpoint broadening of silk and silk-like proteins would suggest diverse targets of engineering to design promising silk-based materials. STATEMENT OF SIGNIFICANCE: Silk has been developed as a biomedical material due to unique mechanical and chemical properties. For decades, silks from various silkworm and spider species have been intensively studied. More recently, other silk and silk-like proteins with different sequences and structures have been reported, not only limited to terrestrial organisms (honeybee, green lacewing, caddisfly, and ant), but also from marine creatures (mussel, squid, sea anemone, and pearl oyster). Nevertheless, there has hardly been well-organized literature on silks from such organisms. Regarding the relationship among sequence-structure-properties, this review addresses how silks have been utilized with respect to organism's survival. Finally, this information aims to improve the understanding of diverse silk and silk-like proteins which can offer a significant interest to engineering fields.

Oriented in situ immobilization of a functional tyrosinase on microcrystalline cellulose effectively incorporates DOPA residues in bioengineered mussel adhesive protein.

BACKGROUND: Catechol-containing polymers such as mussel adhesive proteins (MAPs) are attractive as biocompatible adhesive biomaterials, and the catecholic amino acid 3,4-dihydroxyphenyl-L-alanine (DOPA) is considered a key molecule in underwater mussel adhesion. Tyrosinases can specifically convert tyrosine to DOPA without any cofactors. However, their catalytic properties still need to be adjusted to minimize unwanted DOPA oxidation via their diphenolase activity and catechol instability at neutral and basic pH values in the reaction products. METHODS AND RESULTS: In this work, we constructed a novel functional tyrosinase, mTyr-CNK_CBM, by fusion of mTyr-CNK with a cellulose-binding motif (CBM) for oriented in situ immobilization on microcrystalline cellulose via the C-terminal CBM without any additional purification steps. mTyr-CNK_CBM showed optimal catalytic activity at pH 4.5-6.5 and room temperature and had a high monophenolase/diphenolase activity ratio (Vmax mono/Vmax di = 2.08 at pH 6 and 25°C). mTyr-CNK_CBM exhibited 2.17-fold higher (as a unimmobilized free enzyme) and similarly high (upon immobilization) in vitro DOPA modification of a bioengineered MAP compared to a commercially available mushroom tyrosinase. Moreover, the immobilized mTyr-CNK_CBM showed long-term storability and improved reusability. CONCLUSIONS: These results clearly demonstrate a strong potential for practical use of immobilized mTyr-CNK_CBM as a monophenol monooxygenase in preparing biocompatible DOPA-tethered biomaterials and other catechol-containing polymers.

Sutureless Transplantation of Amniotic Membrane Using a Visible Light-Curable Protein Bioadhesive for Ocular Surface Reconstruction.

The conjunctiva is a thin mucous membrane of the eye. Pterygium, a commonly appearing disease on the ocular surface, requires surgery to excise the conjunctiva to prevent visual deterioration. Recently, transplantation of the amniotic membrane (AM), which is the innermost membrane of the placenta, has been highlighted as an efficient method to cure conjunctiva defects because of its advantages of no side effects compared to mitomycin C treatment and not leaving additional scars on donor site compared to conjunctival autografting. However, to minimize additional damage to the ocular surface by suturing, AM transplantation (AMT) needs to be simplified by using a less invasive, time-saving method. In this work, a visible light-curable protein bioadhesive (named FixLight) for efficient sutureless AMT is applied. FixLight, which is based on bioengineered mussel adhesive protein (MAP), is easily applied between damaged ocular surfaces and transplanted AM, and rapidly cured by harmless blue light activation. Through in vivo evaluation using a rabbit model, the authors demonstrated that FixLight enabled facile, fast, and strong attachment of AM on sclera and promoted ocular surface reconstruction with good biocompatibility. Thus, FixLight can be successfully used as a promising clinical bioadhesive in opthalmological surgeries that require sutureless and rapid operation.

Adhesive protein-based angiogenesis-mimicking spatiotemporal sequential release of angiogenic factors for functional regenerative medicine.

Damaged vascular structures after critical diseases are difficult to completely restore to their original conditions without specific treatments. Thus, therapeutic angiogenesis has been spotlighted as an attractive strategy. However, effective strategies for mimicking angiogenic processes in the body have not yet been developed. In the present work, we developed a bioengineered mussel adhesive protein (MAP)-based novel therapeutic angiogenesis platform capable of spatiotemporally releasing angiogenic growth factors to target disease sites with high viscosity and strong adhesiveness in a mucus-containing environment with curvature. Polycationic MAP formed complex coacervate liquid microdroplets with polyanionic hyaluronic acid and subsequently gelated into microparticles. Platelet-derived growth factor (PDGF), which is a late-phase angiogenic factor, was efficiently encapsulated during the process of coacervate microparticle formation. These PDGF-loaded microparticles were blended with vascular endothelial growth factor (VEGF), which is the initial-phase angiogenic factor, in MAP-based pregel solution and finally crosslinked in situ into a hydrogel at the desired site. The microparticle-based angiogenic-molecule spatiotemporal sequential (MASS) release platform showed good adhesion and underwater durability, and its elasticity was close to that of target tissue. Using two in vivo critical models, i.e., full-thickness excisional wound and myocardial infarction models, the MASS release platform was evaluated for its in vivo feasibility as an angiogenesis-inducing platform and demonstrated effective angiogenesis as well as functional regenerative efficacy. Based on these superior physicochemical characteristics, the developed MASS release platform could be successfully applied in many biomedical practices as a waterproof bioadhesive with the capability for the spatiotemporal delivery of angiogenic molecules in the treatment of ischemic diseases.