3D-printed LEGO®-inspired titanium scaffolds for patient-specific regenerative medicine.

Despite the recent advances in 3D-printing, it is often difficult to fabricate implants that optimally fit a defect size or shape. There are some approaches to resolve this issue, such as patient-specific implant/scaffold designs based on CT images of the patients, however, this process is labor-intensive and costly. Especially in developing countries, affordable treatment options are required, while still not excluding these patient groups from potential material and manufacturing advances. Here, a selective laser melting (SLM) 3D-printing strategy was used to fabricate a hierarchical, LEGO®-inspired Assemblable Titanium Scaffold (ATS) system, which can be manually assembled in any shape or size with ease. A surgeon can quickly create a scaffold that would fit to the defect right before the implantation during the surgery. Additionally, the direct inclusion of micro- and macroporous structures via 3D-printing, as well as a double acid-etched surface treatment (ST) in the ATS, ensure biocompatibility, sufficient nutrient flow, cell migration and enhanced osteogenesis. Three different structures were designed (non-porous:NP, semi-porous:SP, ultra-porous:UP), 3D-printed with the SLM technique and then surface treated for the ST groups. After analyzing characteristics of the ATS such as printing quality, surface roughness and interconnected porosity, mechanical testing and finite element analysis (FEA) demonstrated that individual and stacked ATS have sufficient mechanical properties to withstand loading in a physiological system. All ATS showed high cell viability, and the SP and UP groups demonstrated enhanced cell proliferation rates compared to the NP group. Furthermore, we also verified that cells were well-attached and spread on the porous structures and successful cell migration between the ATS units was seen in the case of assemblies. The UP and SP groups exhibited higher calcium deposition and RT-qPCR proved higher osteogenic gene expression compared to NP group. Finally, we demonstrate a number of possible medical applications that reveal the potential of the ATS through assembly.

3D-Printed PEEK/Silicon Nitride Scaffolds with a Triply Periodic Minimal Surface Structure for Spinal Fusion Implants.

The issue of spine-related disorders is a global healthcare concern that requires effective solutions to restore normal spine functioning. Spinal fusion implants have become a standard approach for this purpose, making it crucial to develop biomaterials and structures that possess high osteogenic capacities and exhibit mechanical properties and dynamic responses similar to those of the host bone. This study focused on the fabrication of 3D-printed polyether ether ketone/silicon nitride (PEEK/SiN) scaffolds with a triply periodic minimal surface (TPMS) structure, which offers several advantages, such as a large surface area and uniform stress distribution under load. The mechanical properties and dynamic response of PEEK/SiN scaffolds with varying porosities were evaluated through mechanical testing and finite element analysis. The scaffold with 30% porosity exhibited a compressive strength (34.56 ± 1.91 MPa) and elastic modulus (734 ± 64 MPa) similar to those of trabecular bone. In addition, the scaffold demonstrated favorable damping properties. The biological data revealed that incorporating silicon nitride into the PEEK scaffold stimulated osteogenic differentiation. In light of these findings, it can be inferred that PEEK/SiN TPMS scaffolds exhibit significant potential for use in bone tissue engineering and represent a promising option as candidates for spinal fusion implants.

A 3D-Printed Assemblable Bespoke Scaffold as a Versatile Microcryogel Carrier for Site-Specific Regenerative Medicine.

Advances in additive manufacturing have led to diverse patient-specific implant designs utilizing computed tomography, but this requires intensive work and financial implications. Here, Digital Light Processing is used to fabricate a hive-structured assemblable bespoke scaffold (HIVE). HIVE can be manually assembled in any shape/size with ease, so a surgeon can create a scaffold that will best fit a defect before implantation. Simultaneously, it can have site-specific treatments by working as a carrier filled with microcryogels (MC) incorporating different biological factors in different pockets of HIVE. After characterization, possible site-specific applications are investigated by utilizing HIVE as a versatile carrier with incorporated treatments such as growth factors (GF), bioceramic, or cells. HIVE as a GF-carrier shows a controlled release of bone morphogenetic protein/vascular endothelial growth factor (BMP/VEGF) and induced osteogenesis/angiogenesis from human mesenchymal stem cells (hMSC)/human umbilical vein endothelial cells (HUVECs). Furthermore, as a bioceramic-carrier, HIVE demonstrates enhanced mineralization and osteogenesis, and as a HUVEC carrier, it upregulates both osteogenic and angiogenic gene expression of hMSCs. HIVE with different combinations of MCs yields a distinct local effect and successful cell migration is confirmed within assembled HIVEs. Finally, an in vivo rat subcutaneous implantation demonstrates site-specific osteogenesis and angiogenesis.

Silicon Nitride as a Biomedical Material: An Overview.

Silicon nitride possesses a variety of excellent properties that can be specifically designed and manufactured for different medical applications. On the one hand, silicon nitride is known to have good mechanical properties, such as high strength and fracture toughness. On the other hand, the uniqueness of the osteogenic/antibacterial dualism of silicon nitride makes it a favorable bioceramic for implants. The surface of silicon nitride can simultaneously inhibit the proliferation of bacteria while supporting the physiological activities of eukaryotic cells and promoting the healing of bone tissue. There are hardly any biomaterials that possess all these properties concurrently. Although silicon nitride has been intensively studied as a biomedical material for years, there is a paucity of comprehensive data on its properties and medical applications. To provide a comprehensive understanding of this potential cornerstone material of the medical field, this review presents scientific and technical data on silicon nitride, including its mechanical properties, osteogenic behavior, and antibacterial capabilities. In addition, this paper highlights the current and potential medical use of silicon nitride and explains the bottlenecks that need to be addressed, as well as possible solutions.

Light-Based Printing of Leachable Salt Molds for Facile Shaping of Complex Structures.

3D printing is a powerful manufacturing technology for shaping materials into complex structures. While the palette of printable materials continues to expand, the rheological and chemical requisites for printing are not always easy to fulfill. Here, a universal manufacturing platform is reported for shaping materials into intricate geometries without the need for their printability, but instead using light-based printed salt structures as leachable molds. The salt structures are printed using photocurable resins loaded with NaCl particles. The printing, debinding, and sintering steps involved in the process are systematically investigated to identify ink formulations enabling the preparation of crack-free salt templates. The experiments reveal that the formation of a load-bearing network of salt particles is essential to prevent cracking of the mold during the process. By infiltrating the sintered salt molds and leaching the template in water, complex-shaped architectures are created from diverse compositions such as biomedical silicone, chocolate, light metals, degradable elastomers, and fiber composites, thus demonstrating the universal, cost-effective, and sustainable nature of this new manufacturing platform.

Comprehensive in vitro comparison of cellular and osteogenic response to alternative biomaterials for spinal implants.

A variety of novel biomaterials are emerging as alternatives to conventional metals and alloys, for use in spinal implants. These promise potential advantages with respect to e.g. elastic modulus compatibility with the host bone, improved radiological imaging or enhanced cellular response to facilitate osseointegration. However, to date there is scarce comparative data on the biological response to many of these biomaterials that would give insights into the relative level of bone formation, resorption inhibition and inflammation. Thus, in this study, we aimed to evaluate and compare the in vitro biological response to standard discs of four alternative biomaterials: polyether ether ketone (PEEK), zirconia toughened alumina (ZTA), silicon nitride (SN) and surface-textured silicon nitride (ST-SN), and the reference titanium alloy Ti6Al4V (TI). Material-specific characteristics of these biomaterials were evaluated, such as surface roughness, wettability, protein adsorption (BSA) and apatite forming capacity in simulated body fluid. The activity of pre-osteoblasts seeded on the discs was characterized, by measuring viability, proliferation, attachment and morphology. Then, the osteogenic differentiation of pre-osteoblasts was compared in vitro from early to late stage by Alizarin Red S staining and real-time PCR analysis. Finally, osteoclast activity and inflammatory response were assessed by real-time PCR analysis. Compared to TI, all other materials generally demonstrated a lower osteoclastic activity and inflammatory response. ZTA and SN showed generally an enhanced osteogenic differentiation and actin length. Overall, we could show that SN and ST-SN showed a higher osteogenic effect than the other reference groups, an inhibitive effect against bone resorption and low inflammation, and the results indicate that silicon nitride has a promising potential to be developed further for spinal implants that require enhanced osseointegration.

Addition of lactoferrin and substance P in a chitin/PLGA-CaSO4 hydrogel for regeneration of calvarial bone defects.

Calcium-based injectable hydrogels with various bioactive active molecules possess a great potential for bone regeneration. Herein, we have synthesized a chitin-PLGA-calcium sulfate hydrogel (CSG) containing bioactive molecules - lactoferrin (LF) and substance P (SP). SEM and XRD analysis revealed that CS crystal growth was altered with the addition of LF. Rheological measurements indicated that the injectability of the hydrogels was maintained after the addition of LF, however, there was a reduction in storage modulus after LF addition. The addition of LF increased stem cell proliferation whereas, SP enhanced the cell migration. Osteogenic gene expression revealed that LF concentration at 25 µg/mg of CSG was optimal for a favourable outcome. To this optimized LF containing CSG, SP was incorporated and 0.05 µg/mg was found to be most effective (CSG-L3S2) in vitro studies. Further, the µ-CT and histological studies confirmed that CSG-L3S2 showed enhanced bone regeneration compared to the controls in critical-sized calvarial defect of mice. Thus the results indicate that a combination of the chemotactic agent (SP), pleiotropic growth protein (LF), and CS in the chitin-PLGA hydrogel could be a promising approach for non-load bearing bone defects.

A Biomimetic Macroporous Hybrid Scaffold with Sustained Drug Delivery for Enhanced Bone Regeneration.

Bone regeneration is a highly complex physiological process regulated by several factors. In particular, bone-mimicking extracellular matrix and available osteogenic growth factors such as bone morphogenetic protein (BMP) have been regarded as key contributors for bone regeneration. In this study, we developed a biomimetic hybrid scaffold (CEGH) with sustained release of BMP-2 that would result in enhanced bone formation. This hybrid scaffold, composed of BMP-2-loaded cryoelectrospun poly(ε-caprolactone) (PCL) (CE) surrounded by a macroporous gelatin/heparin cryogel (GH), is designed to overcome the drawbacks of the relatively weak mechanical properties of cryogels and poor biocompatibility and hydrophobicity of electrospun PCL. The GH component of the hybrid scaffold provides a hydrophilic surface to improve the biological response of the cells, while the CE component increases the mechanical strength of the scaffold to provide enhanced mechanical support for the defect area and a stable environment for osteogenic differentiation. After analyzing characteristics of the hybrid scaffold such as hydrophilicity, pore difference, mechanical properties, and surface charge, we confirmed that the hybrid scaffold shows enhanced cell proliferation rate and apatite formation in simulated body fluid. Then, we evaluated drug release kinetics of CEGH and confirmed the sustained release of BMP-2. Finally, the enhanced osteogenic differentiation of CEGH with sustained release of BMP-2 was confirmed by Alizarin Red S staining and real-time PCR analysis.

Partially Digested Osteoblast Cell Line-Derived Extracellular Matrix Induces Rapid Mineralization and Osteogenesis.

An extracellular matrix (ECM) utilized as a biomaterial can be obtained from organs of living organisms. Therefore, it has some limitations in its supply because of insufficient organs. Furthermore, therapeutic efficacy of ECMs varies depending on factors such as donor's health condition and age. For this reason, ECMs obtained from a cell line could be a good alternative because they can be produced under a controlled environment with uniform quality. Thus, the purpose of this study was to investigate the potential of the MC3T3-E1 cell line-derived ECM as bone graft. The optimized decellularization process was developed to separate the ECM from MC3T3-E1, osteoblast cell line, using Trypsin-EDTA and Triton X-100. The decellularized ECM was partially digested using pepsin. Also, human bone marrow-derived mesenchymal stem cells induced faster osteogenesis on the ECM-coated surface than on the collagen-coated surface. Partially digested ECM fragments were embedded on the polyethylene glycol scaffold without additional chemical modification or crosslinking. Micro-computed tomography and histological analysis results showed that the ECM in the scaffold promoted actual bone regeneration after in vivo implantation to a mouse calvarial defect model. This study suggests that the bone-specific ECM derived from the cell line can replace the ECM from organs for application in tissue engineering and regenerative medicine.

Combinatorial effect of nano whitlockite/nano bioglass with FGF-18 in an injectable hydrogel for craniofacial bone regeneration.

Functional regeneration of bone defects, especially critical-sized, in the craniofacial region remains a major clinical challenge that needs intervention. To address this, the present work focuses on the development of an injectable chitin-PLGA hydrogel (CG) containing bioglass nanoparticles (nBG) or whitlockite nanoparticles (nWH) with FGF-18, and compares the osteogenic and neo-bone formation potential against commercially available hydroxyapatite nanoparticles (nHAP) with FGF-18 fortified CG hydrogel in the critical-sized defect region. The developed CG was injectable and the incorporation of bio-ceramics didn't affect the injectability. Sustained release of FGF-18 was achieved in bio-ceramic containing CG hydrogel systems, while CG hydrogel alone displayed rapid release. In addition, the nBG or nWH containing CG hydrogel groups showed in vitro angiogenic potential. Furthermore, ALP activity, BMP-2 quantification and osteogenic gene expression assays were conducted to ascertain the osteogenic differentiation potential of the hydrogels. In the combination groups, CGnWHF (nWH + FGF-18 containing CG) showed highest osteogenic potential with a synergistic effect, compared to all other groups studied. In vivo bone regeneration studies displayed near-complete bone regeneration for CGnWHF, where its BV/TV% was the highest (synergistic effect) compared to CGnBGF (nBG + FGF-18 in CG) and nHAP with FGF-18 (additive effect) after 8 weeks of implantation. Thus, the use of CGnWHF in irregular craniofacial bone defects could be an attractive option.

Silicon Nitride, a Bioceramic for Bone Tissue Engineering: A Reinforced Cryogel System With Antibiofilm and Osteogenic Effects.

Silicon nitride (SiN [Si3N4]) is a promising bioceramic for use in a wide variety of orthopedic applications. Over the past decades, it has been mainly used in industrial applications, such as space shuttle engines, but not in the medical field due to scarce data on the biological effects of SiN. More recently, it has been increasingly identified as an emerging material for dental and orthopedic implant applications. Although a few reports about the antibacterial properties and osteoconductivity of SiN have been published to date, there have been limited studies of SiN-based scaffolds for bone tissue engineering. Here, we developed a silicon nitride reinforced gelatin/chitosan cryogel system (SiN-GC) by loading silicon nitride microparticles into a gelatin/chitosan cryogel (GC), with the aim of producing a biomimetic scaffold with antibiofilm and osteogenic properties. In this scaffold system, the GC component provides a hydrophilic and macroporous environment for cells, while the SiN component not only provides antibacterial properties and osteoconductivity but also increases the mechanical stiffness of the scaffold. This provides enhanced mechanical support for the defect area and a better osteogenic environment. First, we analyzed the scaffold characteristics of SiN-GC with different SiN concentrations, followed by evaluation of its apatite-forming capacity in simulated body fluid and protein adsorption capacity. We further confirmed an antibiofilm effect of SiN-GC against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) as well as enhanced cell proliferation, mineralization, and osteogenic gene upregulation for MC3T3-E1 pre-osteoblast cells. Finally, we developed a bioreactor to culture cell-laden scaffolds under cyclic compressive loading to mimic physiological conditions and were able to demonstrate improved mineralization and osteogenesis from SiN-GC. Overall, we confirmed the antibiofilm and osteogenic effect of a silicon nitride reinforced cryogel system, and the results indicate that silicon nitride as a biomaterial system component has a promising potential to be developed further for bone tissue engineering applications.

Sequential growth factor releasing double cryogel system for enhanced bone regeneration.

Bone regeneration is a complicated physiological process regulated by several growth factors. In particular, vascular endothelial growth factor (VEGF) and bone morphogenetic protein-4 (BMP-4) are regarded as key factors that induce bone regeneration by angiogenesis and osteogenesis. In this study, we developed a double cryogel system (DC) composed of gelatin/chitosan cryogel (GC) surrounded by gelatin/heparin cryogel (GH) for dual drug delivery with different release kinetics. VEGF was loaded in GH (outer layer of DC) for the initial release of VEGF to induce angiogenesis and provide blood supply in the defect area, while BMP-4 was loaded in GC (inner layer of DC) that leads to sustained release for continuous osteogenic induction. After analyzing characteristics of the double cryogel system such as porosity, degradation rate, swelling ratio, and mechanical properties, we evaluated release kinetics of VEGF (initial release) and BMP-4 (sustained-release) by ELISA. Then, the timely release of VEGF and BMP from DC synergistically induced in vitro osteogenic differentiation as confirmed by alkaline phosphatase staining, Alizarin Red S staining, and real-time PCR analysis. Finally, a critical-sized cranial defect model confirmed the enhanced bone regeneration as a result of dual release growth factor mechanisms.

Ectopic transient overexpression of OCT-4 facilitates BMP4-induced osteogenic transdifferentiation of human umbilical vein endothelial cells.

Limitation in cell sources for autologous cell therapy has been a recent focus in stem cell therapy and tissue engineering. Among various research advances, direct conversion, or transdifferentiation, is a notable and feasible strategy for the generation and acquirement of wanted cell source. So far, utilizing cell transdifferentiation technology in tissue engineering was mainly restricted at achieving single wanted cell type from diverse cell types with high efficiency. However, regeneration of a complete tissue always requires multiple cell types which poses an intrinsic complexity. In this study, enhanced osteogenic differentiation was achieved by transient ectopic expression of octamer-binding transcription factor 4 (OCT-4) gene followed by bone morphogenetic protein 4 treatment on human umbilical vein endothelial cells. OCT-4 transfection and bone morphogenetic protein 4 treatment resulted in enhanced expression of osteogenic markers such as core-binding factor alpha 1, alkaline phosphatase, and collagen 1 compared with bone morphogenetic protein 4 treatment alone. Furthermore, we employed gelatin-heparin cryogel in cranial defect model for in vivo bone formation. Micro-computed tomography and histological analysis of in vivo samples showed that OCT-4 transfection followed by bone morphogenetic protein 4 treatment resulted in efficient transdifferentiation of endothelial cells to osteogenic cells. These results suggest that the combination of OCT-4 and bone morphogenetic protein 4 on endothelial cells would be a reliable multicellular transdifferentiation model which could be applied for bone tissue engineering.

Osteogenic Effects of VEGF-Overexpressed Human Adipose-Derived Stem Cells with Whitlockite Reinforced Cryogel for Bone Regeneration.

Bone is a vascularized tissue that is comprised of collagen fibers and calcium phosphate crystals such as hydroxyapatite (HAp) and whitlockite (WH). HAp and WH are known to elicit bone regeneration by stimulating osteoblast activities and osteogenic commitment of stem cells. In addition, vascular endothelial growth factor (VEGF) is shown to promote osteogenesis and angiogenesis which is considered as an essential process in bone repair by providing nutrients. In this study, VEGF-secreting human adipose-derived stem cells (VEGF-ADSCs) are developed by transducing ADSCs with VEGF-encoded lentivirus. Additionally, WH-reinforced gelatin/heparin cryogels (WH-C) are fabricated by loading WH into gelatin/heparin cryogels. VEGF-ADSC secrete tenfold more VEGF than ADSC and show increased VEGF secretion with cell growth. Also, incorporation of WH into cryogels provides a mineralized environment with ions secreted from WH. When the VEGF-ADSCs are seeded on WH-C, sustained release of VEGF is observed due to the specific affinity of VEGF to heparin. Finally, the synergistic effect of VEGF-ADSC and WH on osteogenesis is successfully confirmed by alkaline phosphatase and real-time polymerase chain reaction analysis. In vivo bone formation is demonstrated via implantation of VEGF-ADSC seeded WH-C into mouse calvarial bone defect model, resulted in enhanced bone development with the highest bone volume/total volume.

Heparin Functionalized Injectable Cryogel with Rapid Shape-Recovery Property for Neovascularization.

Cryogel based scaffolds have high porosity with interconnected macropores that may provide cell compatible microenvironment. In addition, cryogel based scaffolds can be utilized in minimally invasive surgery due to its sponge-like properties, including rapid shape recovery and injectability. Herein, we developed an injectable cryogel by conjugating heparin to gelatin as a carrier for vascular endothelial growth factor (VEGF) and fibroblasts in hindlimb ischemic disease. Our gelatin/heparin cryogel showed gelatin concentration-dependent mechanical properties, swelling ratios, interconnected porosities, and elasticities. In addition, controlled release of VEGF led to effective angiogenic responses both in vitro and in vivo. Furthermore, its sponge-like properties enabled cryogels to be applied as an injectable carrier system for in vivo cells and growth factor delivery. Our heparin functionalized injectable cryogel facilitated the angiogenic potential by facilitating neovascularization in a hindlimb ischemia model.

Enhanced osteogenic commitment of murine mesenchymal stem cells on graphene oxide substrate.

BACKGROUND: Tissue engineering is an interdisciplinary field that attempts to restore or regenerate tissues and organs through biomimetic fabrication of scaffolds with specific functionality. In recent years, graphene oxide (GO) is considered as promising biomaterial due to its nontoxicity, high dispersity, and hydrophilic interaction, and these characteristics are key to stimulating the interactions between substrates and cells. METHOD: In this study, GO substrates were fabricated via chemically immobilizing GO at 1.0 mg/ml on glass slides. Furthermore, we examined the osteogenic responses of murine mesenchymal-like stem cells, C3H10T1/2 cells, on GO substrates. RESULTS: C3H10T1/2 cells on GO substrates resulted in increased cell surface area, enhanced cellular adhesions, and instigated osteogenic differentiation. Furthermore, priming of C3H10T1/2 cells with chondrocyte-conditioned medium (CM) could further induce a synergistic effect of osteogenesis on GO substrates. CONCLUSIONS: All of these data suggest that GO substrate along with CM is suitable for upregulating osteogenic responses of mesenchymal stem cells.

Bioglass-Incorporated Methacrylated Gelatin Cryogel for Regeneration of Bone Defects.

Cryogels have recently gained interest in the field of tissue engineering as they inherently possess an interconnected macroporous structure. Considered to be suitable for scaffold cryogel fabrication, methacrylated gelatin (GelMA) is a modified form of gelatin valued for its ability to retain cell adhesion site. Bioglass nanoparticles have also attracted attention in the field due to their osteoinductive and osteoconductive behavior. Here, we prepare methacrylated gelatin cryogel with varying concentration of bioglass nanoparticles to study its potential for bone regeneration. We demonstrate that an increase in bioglass concentration in cryogel leads to improved mechanical property and augmented osteogenic differentiation of mesenchymal cells during in vitro testing. Furthermore, in vivo testing in mice cranial defect model shows that highest concentration of bioglass nanoparticles (2.5 w/w %) incorporated in GelMA cryogel induces the most bone formation compared to the other tested groups, as studied by micro-CT and histology. The in vitro and in vivo results highlight the potential of bioglass nanoparticles incorporated in GelMA cryogel for bone regeneration.

Injectable in Situ Shape-Forming Osteogenic Nanocomposite Hydrogel for Regenerating Irregular Bone Defects.

The in situ forming injectable hydrogels are appealing for irregular bone defects because of the ease of administration and the addition of ceramics, molecules, and proteins into the hydrogel. We have developed in situ shape-forming hydrogel using oxidized alginate and gelatin as the hydrogel matrix. Whitlockite bioceramic nanoparticles (WH NPs) were incorporated, as they provide enhanced osteogenic stimulation compared to hydroxyapatite via providing higher local ion concentration. The drug simvastatin was also incorporated into the hydrogel system, as it increases the expression of BMP-2 thereby provide environment for bone regeneration. The presence of both WH nanoparticles and simvastatin would enhance bone regeneration potential. The whitlockite nanoparticles (80 ± 8 nm) were synthesized by precipitation method and were characterized. The nanocomposite hydrogel system was characterized by SEM, FTIR and rheologically. The gelation time of the in situ nanocomposite hydrogel was determined by rheological analysis as 28 s, whereas hydrogel alone showed 132 s. Addition of WH NPs not only shortened the gelation time but also increased the gel strength. The in vitro release of simvastatin from the nanocomposite hydrogel showed a release over a period of 28 days. The alkaline phosphatase (ALP) level also showed a significant increase. RUNX2 and BMP2 expressions showed that nanocomposite hydrogel enhanced the osteogenic differentiation. In vivo bone regeneration studies in mice cranial defect studies showed nanocomposite hydrogel was effective in regenerating the bone compared to controls. Thus, the simvastatin-incorporated oxidized alginate-gelatin/WH NPs hydrogel system has the potential to be used as a repairing and regenerative system in cranial bone defects.

Self-Healing and Adhesive Artificial Tissue Implant for Voice Recovery.

Loss of voice after vocal fold resection due to laryngeal cancer is a significant problem resulting in a low quality of life. Although there were many attempts to achieve a functional restoration of voice, challenges to regenerate vocal fold still remain due to its unique tissue mechanical characteristics such as pliability that produces phonation via vibration. In this study, we developed a mechanically compliant interpenetrating polymer network (IPN) hydrogel based on polyacrylamide (PAAM) and gelatin that matches physical and functional properties with native vocal fold tissue. The mechanical properties of this PAAM/gelatin (PG) hydrogel were optimized to have an elastic modulus of 5.4 kPa by adjusting the PAAM/gelatin ratio. In addition, the PG hydrogel demonstrated a minimal foreign body reaction upon implantation, and the hydrogel displayed a strong resistance to dehydration conditions that can last 40 days in the chamber with 60% humidity. Furthermore, the PG hydrogel demonstrated a self-healing ability that may allow ad-hoc implant augmentation. In addition, tough adhesion of the PG hydrogel resulted in stable attachment to vocal fold tissues. Finally, we demonstrated the functional restoration of voice on an ex vivo canine model by implanting the PG hydrogel as an artificial vocal fold tissue.