Computational Fluid Dynamics Analysis and Empirical Evaluation of Carboxymethylcellulose/Alginate 3D Bioprinting Inks for Screw-Based Microextrusion.

Three-dimensional microextrusion bioprinting technology uses pneumatics, pistons, or screws to transfer and extrude bioinks containing biomaterials and cells to print biological tissues and organs. Computational fluid dynamics (CFD) analysis can simulate the flow characteristics of bioinks in a control volume, and the effect on cell viability can be predicted by calculating the physical quantities. In this study, we developed an analysis system to predict the effect of a screw-based dispenser system (SDS) on cell viability in bioinks through rheological and CFD analyses. Furthermore, carboxymethylcellulose/alginate-based bioinks were used for the empirical evaluation of high-viscous bioinks. The viscosity of bioinks was determined by rheological measurement, and the viscosity coefficient for the CFD analysis was derived from a correlation equation by non-linear regression analysis. The mass flow rate derived from the analysis was successfully validated by comparison with that from the empirical evaluation. Finally, the cell viability was confirmed after bioprinting with bioinks containing C2C12 cells, suggesting that the developed SDS may be suitable for application in the field of bioengineering. Consequently, the developed bioink analysis system is applicable to a wide range of systems and materials, contributing to time and cost savings in the bioengineering industry.

Development of Silk Fibroin-Based Non-Crosslinking Thermosensitive Bioinks for 3D Bioprinting.

Three-dimensional (3D) bioprinting holds great promise for tissue engineering, allowing cells to thrive in a 3D environment. However, the applicability of natural polymers such as silk fibroin (SF) in 3D bioprinting faces hurdles due to limited mechanical strength and printability. SF, derived from the silkworm Bombyx mori, is emerging as a potential bioink due to its inherent physical gelling properties. However, research on inducing thermosensitive behavior in SF-based bioinks and tailoring their mechanical properties to specific tissue requirements is notably lacking. This study addresses these gaps through the development of silk fibroin-based thermosensitive bioinks (SF-TPBs). Precise modulation of gelation time and mechanical robustness is achieved by manipulating glycerol content without recourse to cross-linkers. Chemical analysis confirms ß-sheet conformation in SF-TPBs independent of glycerol concentration. Increased glycerol content improves gelation kinetics and results in rheological properties suitable for 3D printing. Overall, SF-TPBs offer promising prospects for realizing the potential of 3D bioprinting using natural polymers.

Development of 3D Printable Calcium Phosphate Cement Scaffolds with Cockle Shell Powders.

Three-dimensional (3D) printed calcium phosphate cement (CPC) scaffolds are increasingly being used for bone tissue repair. Traditional materials used for CPC scaffolds, such as bovine and porcine bone, generally contain low amounts of calcium phosphate compounds, resulting in reduced production rates of CPC scaffolds. On the other hand, cockle shells contain more than 99% CaCO3 in the form of amorphous aragonite with excellent biocompatibility, which is expected to increase the CPC production rate. In this study, 3D-printed cockle shell powder-based CPC (CSP-CPC) scaffolds were developed by the material extrusion method. Lactic acid and hyaluronic acid were used to promote the printability. The characterization of CSP-CPC scaffolds was performed using Fourier transform infrared spectra, X-ray diffraction patterns, and scanning electron microscopy. The biocompatibility of CSP-CPC scaffolds was evaluated using cell viability, Live/Dead, and alkaline phosphatase assays. In addition, CSP-CPC scaffolds were implanted into the mouse calvarial defect model to confirm bone regeneration. This study provides an opportunity to create high value added in fishing villages by recycling natural products from marine waste.

Development of a Scaffold-on-a-Chip Platform to Evaluate Cell Infiltration and Osteogenesis on the 3D-Printed Scaffold for Bone Regeneration.

Developing a scaffold for efficient and functional bone regeneration remains challenging. To accomplish this goal, a "scaffold-on-a-chip" device was developed as a platform to aid with the evaluation process. The device mimics a microenvironment experienced by a transplanted bone scaffold. The device contains a circular space at the center for scaffold insert and microfluidic channel that encloses the space. Such a design allows for monitoring of cell behavior at the blood-scaffold interphase. MC3T3-E1 cells were cultured with three different types of scaffold inserts to test its capability as an evaluation platform. Cellular behaviors, including migration, morphology, and osteogenesis with each scaffold, were analyzed through fluorescence images of live/dead assay and immunocytochemistry. Cellular behaviors, such as migration, morphology, and osteogenesis, were evaluated. The results revealed that our platform could effectively evaluate the osteoconductivity and osteoinductivity of scaffolds with various properties. In conclusion, our proposed platform is expected to replace current in vivo animal models as a highly relevant in vitro platform and can contribute to the fundamental study of bone regeneration.

Development of Plum Seed-Derived Carboxymethylcellulose Bioink for 3D Bioprinting.

Three-dimensional bioprinting represents an innovative platform for fabricating intricate, three-dimensional (3D) tissue structures that closely resemble natural tissues. The development of hybrid bioinks is an actionable strategy for integrating desirable characteristics of components. In this study, cellulose recovered from plum seed was processed to synthesize carboxymethyl cellulose (CMC) for 3D bioprinting. The plum seeds were initially subjected to α-cellulose recovery, followed by the synthesis and characterization of plum seed-derived carboxymethyl cellulose (PCMC). Then, hybrid bioinks composed of PCMC and sodium alginate were fabricated, and their suitability for extrusion-based bioprinting was explored. The PCMC bioinks exhibit a remarkable shear-thinning property, enabling effortless extrusion through the nozzle and maintaining excellent initial shape fidelity. This bioink was then used to print muscle-mimetic 3D structures containing C2C12 cells. Subsequently, the cytotoxicity of PCMC was evaluated at different concentrations to determine the maximum acceptable concentration. As a result, cytotoxicity was not observed in hydrogels containing a suitable concentration of PCMC. Cell viability was also evaluated after printing PCMC-containing bioinks, and it was observed that the bioprinting process caused minimal damage to the cells. This suggests that PCMC/alginate hybrid bioink can be used as a very attractive material for bioprinting applications.

Reduced graphene oxide-incorporated calcium phosphate cements with pulsed electromagnetic fields for bone regeneration.

Natural calcium phosphate cements (CPCs) derived from sintered animal bone have been investigated to treat bone defects, but their low mechanical strength remains a critical limitation. Graphene improves the mechanical properties of scaffolds and promotes higher osteoinduction. To this end, reduced graphene oxide-incorporated natural calcium phosphate cements (RGO-CPCs) are fabricated for reinforcement of CPCs' characteristics. Pulsed electromagnetic fields (PEMFs) were additionally applied to RGO-CPCs to promote osteogenic differentiation ability. The fabricated RGO-CPCs show distinct surface properties and chemical properties according to the RGO concentration. The RGO-CPCs' mechanical properties are significantly increased compared to CPCs owing to chemical bonding between RGO and CPCs. In in vitro studies using a mouse osteoblast cell line and rat-derived adipose stem cells, RGO-CPCs are not severely toxic to either cell type. Cell migration study, western blotting, immunocytochemistry, and alizarin red staining assay reveal that osteoinductivity as well as osteoconductivity of RGO-CPCs was highly increased. In in vivo study, RGO-CPCs not only promoted bone ingrowth but also enhanced osteogenic differentiation of stem cells. Application of PEMFs enhanced the osteogenic differentiation of stem cells. RGO-CPCs with PEMFs can overcome the flaws of previously developed natural CPCs and are anticipated to open the gate to clinical application for bone repair and regeneration.

Enhanced Osteogenesis of Dental Pulp Stem Cells In Vitro Induced by Chitosan-PEG-Incorporated Calcium Phosphate Cement.

The use of bone graft materials is required for the treatment of bone defects damaged beyond the critical defect; therefore, injectable calcium phosphate cement (CPC) is actively used after surgery. The application of various polymers to improve injectability, mechanical strength, and biological function of injection-type CPC is encouraged. We previously developed a chitosan-PEG conjugate (CS/PEG) by a sulfur (VI) fluoride exchange reaction, and the resulting chitosan derivative showed high solubility at a neutral pH. We have demonstrated the CPC incorporated with a poly (ethylene glycol) (PEG)-grafted chitosan (CS/PEG) and developed CS/PEG CPC. The characterization of CS/PEG CPC was conducted using Fourier transform infrared spectroscopy (FT-IR) and X-ray diffraction (XRD). The initial properties of CS/PEG CPCs, such as the pH, porosity, mechanical strength, zeta potential, and in vitro biocompatibility using the WST-1 assay, were also investigated. Moreover, osteocompatibility of CS/PEG CPCs was carried out via Alizarin Red S staining, immunocytochemistry, and Western blot analysis. CS/PEG CPC has enhanced mechanical strength compared to CPC, and the cohesion test also demonstrated in vivo stability. Furthermore, we determined whether CS/PEG CPC is a suitable candidate for promoting the osteogenic ability of Dental Pulp Stem Cells (DPSC). The elution of CS/PEG CPC entraps more calcium ion than CPC, as confirmed through the zeta potential test. Accordingly, the ion trapping effect of CS/PEG is considered to have played a role in promoting osteogenic differentiation of DPSCs. The results strongly suggested that CS/PEG could be used as suitable additives for improving osteogenic induction of bone substitute materials.

Enhanced Osteogenic Differentiation of Periodontal Ligament Stem Cells Using a Graphene Oxide-Coated Poly(ε-caprolactone) Scaffold.

Periodontal diseases occur through bacterial infection in the oral cavity, which can cause alveolar bone loss. Several efforts have been made to reconstruct alveolar bone, such as grafting bone substitutes and 3D-printed scaffolds. Poly(ε-caprolactone) (PCL) is biocompatible and biodegradable, thus demonstrating its potential as a biomaterial substitute; however, it is difficult for cells to adhere to PCL because of its strong hydrophobicity. Therefore, its use as a biomaterial has limitations. In this study, we used graphene oxide (GO) as a coating material to promote the osteogenic differentiation ability of PCL scaffolds. First, 3D-printed PCL scaffolds were fabricated, and the oxygen plasma treatment and coating conditions were established according to the concentration of GO. The physical and chemical properties of the prepared scaffolds were evaluated through water contact angle analysis, Raman spectroscopy, and image analysis. In addition, the adhesion and proliferation of periodontal ligament stem cells (PDLSCs) on the GO scaffolds were assessed via the water-soluble tetrazolium salt-1 (WST-1) assay, and the osteogenic differentiation ability was evaluated through alizarin red S staining. The results confirmed that the cell proliferation and osteogenic differentiation of the PDLSCs were enhanced in the scaffolds coated with oxygen plasma and GO. In conclusion, the plasma-treated GO-coating method that we developed can be used to promote the cell proliferation and osteogenic differentiation of the scaffolds.

Development of novel gene carrier using modified nano hydroxyapatite derived from equine bone for osteogenic differentiation of dental pulp stem cells.

Hydroxyapatite (HA) is a representative substance that induces bone regeneration. Our research team extracted nanohydroxyapatite (EH) from natural resources, especially equine bones, and developed it as a molecular biological tool. Polyethylenimine (PEI) was used to coat the EH to develop a gene carrier. To verify that PEI is well coated in the EH, we first observed the morphology and dispersity of PEI-coated EH (pEH) by electron microscopy. The pEH particles were well distributed, while only the EH particles were not distributed and aggregated. Then, the existence of nitrogen elements of PEI on the surface of the pEH was confirmed by EDS, calcium concentration measurement and fourier transform infrared spectroscopy (FT-IR). Additionally, the pEH was confirmed to have a more positive charge than the 25 kD PEI by comparing the zeta potentials. As a result of pGL3 transfection, pEH was better able to transport genes to cells than 25 kD PEI. After verification as a gene carrier for pEH, we induced osteogenic differentiation of DPSCs by loading the BMP-2 gene in pEH (BMP-2/pEH) and delivering it to the cells. As a result, it was confirmed that osteogenic differentiation was promoted by showing that the expression of osteopontin (OPN), osteocalcin (OCN), and runt-related transcription factor 2 (RUNX2) was significantly increased in the group treated with BMP-2/pEH. In conclusion, we have not only developed a novel nonviral gene carrier that is better performing and less toxic than 25 kD PEI by modifying natural HA (the agricultural byproduct) but also proved that bone differentiation can be effectively promoted by delivering BMP-2 with pEH to stem cells.

3D-Printed Poly(ε-Caprolactone)/Hydroxyapatite Scaffolds Modified with Alkaline Hydrolysis Enhance Osteogenesis In Vitro.

The 3D-printed bioactive ceramic incorporated Poly(ε-caprolactone) (PCL) scaffolds show great promise as synthetic bone graft substitutes. However, 3D-printed scaffolds still lack adequate surface properties for cells to be attached to them. In this study, we modified the surface characteristics of 3D-printed poly(ε-caprolactone)/hydroxyapatite scaffolds using O2 plasma and sodium hydroxide. The surface property of the alkaline hydrolyzed and O2 plasma-treated PCL/HA scaffolds were evaluated using field-emission scanning microscopy (FE-SEM), Alizarin Red S (ARS) staining, and water contact angle analysis, respectively. The in vitro behavior of the scaffolds was investigated using human dental pulp-derived stem cells (hDPSCs). Cell proliferation of hDPSCs on the scaffolds was evaluated via immunocytochemistry (ICC) and water-soluble tetrazolium salt (WST-1) assay. Osteogenic differentiation of hDPSCs on the scaffolds was further investigated using ARS staining and Western blot analysis. The result of this study shows that alkaline treatment is beneficial for exposing hydroxyapatite particles embedded in the scaffolds compared to O2 plasma treatment, which promotes cell proliferation and differentiation of hDPSCs.

Development and characterization of waste equine bone-derived calcium phosphate cements with human alveolar bone-derived mesenchymal stem cells.

Calcium phosphate cements (CPCs) are regarded as promising graft substitutes for bone tissue engineering. However, their wide use is limited by the high cost associated with the complex synthetic processes involved in their fabrication. Cheaper xenogeneic calcium phosphate (CaP) materials derived from waste animal bone may solve this problem. Moreover, the surface topography, mechanical strength, and cellular function of CPCs are influenced by the ratio of micro- to nano-sized CaP (M/NCaP) particles. In this study, we developed waste equine bone (EB)-derived CPCs with various M/NCaP particle ratios to examine the potential capacity of EB-CPCs for bone grafting materials. Our study showed that increasing the number of NCaP particles resulted in reductions in roughness and porosity while promoting smoother surfaces of EB-CPCs. Changes in the chemical properties of EB-CPCs by NCaP particles were observed using X-ray diffractometry. The mechanical properties and cohesiveness of the EB-CPCs improved as the NCaP particle content increased. In an in vitro study, EB-CPCs with a greater proportion of MCaP particles showed higher cell adhesion. Alkaline phosphatase activity indicated that osteogenic differentiation by EB-CPCs was promoted with increased NCaP particle content. These results could provide a design criterion for bone substitutes for orthopedic disease, including periodontal bone defects.

Aligned Nanofiber-Guided Bone Regeneration Barrier Incorporated with Equine Bone-Derived Hydroxyapatite for Alveolar Bone Regeneration.

Post-surgery failure of dental implants due to alveolar bone loss is currently critical, disturbing the quality of life of senior dental patients. To overcome this problem, bioceramic or bone graft material is loaded into the defect. However, connective tissue invasion instead of osteogenic tissue limits bone tissue regeneration. The guided bone regeneration concept was adapted to solve this problem and still has room for improvements, such as biochemical similarity or oriented structure. In this article, an aligned electrospun-guided bone regeneration barrier with xenograft equine bone-derived nano hydroxyapatite (EBNH-RB) was fabricated by electrospinning EBNH/PCL solution on high-speed rotating drum collector and fiber characterization, viability and differentiation enhancing properties of mesenchymal dental pulp stem cell on the barrier was determined. EBNH-RB showed biochemical and structural similarity to natural bone tissue electron microscopy image analysis and x-ray diffractometer analysis, and had a significantly better effect in promoting osteogenesis based on the increased bioceramic content by promoting cell viability, calcium deposition and osteogenic marker expression, suggesting that they can be successfully applied to regenerate alveolar bone as a guided bone regeneration barrier.

Evaluation of the Osteogenic Potential of Stem Cells in the Presence of Growth Hormone under Magnetic Field Stimulation.

The human alveolar bone-derived mesenchymal stem cells (hABMSCs) are considered an attractive source for the development of bone tissues. However, their mechanism of action is still unclear. This work aimed to investigate the potential of the natural human growth hormone (NHGH) derived from stem cells under magnetic field (MF) stimulation for tissue engineering by exploring the paracrine or autocrine effects of hABMSCs in vitro. The secretion of anti-inflammatory cytokines and growth factors from hABMSCs was profoundly affected by the intensity of the applied MFs. The effects of stimulated MFs on vascular endothelial growth factor (VEGF) and bone morphogenetic protein-2 (BMP-2) production were quantified by an ELISA kit. Notably, higher cell metabolic activity was observed in MF stimulation compared to the control, and this was more prominent in 130 mT strength of MF. An enhancement in the production of VEGF and BMP-2 was noted in MFs compared to the control. Moreover, higher accumulation of osteogenesis-related genes has occurred in MFs than the control. Furthermore, a significant enhancement in cell metabolic activity and mineralized nodule formations was spotted in the presence of NHGH via MF stimulation; vis-à-vis, MF stimulation only through autocrine and paracrine effects demonstrated the better osteogenic potential of NHGH in the presence of MFs for tissue engineering applications.

Enhanced Osteogenesis of Human Mesenchymal Stem Cells in Presence of Single-Walled Carbon Nanotubes.

Human mesenchymal stem cells (hMSCs) have attracted significant attention for tissue engineering because of their ability to differentiate into bone cells, chondrocytes, adipocytes, and muscle cells. Single-walled carbon nanotubes (SWCNTs) have been considered as a potential material for tissue engineering applications due to their unique properties, such as high aspect ratio, excellent electrocatalytic activity, and biocompatibility. Here we prepared exfoliated SWCNTs layers through an ultra-sonication process in the acidic medium and evaluated their cytotoxicity using hMSCs. Improved viability and osteogenesis of hMSCs were observed in the presence of exfoliated SWCNTs. Besides, the higher expression of osteogenic differentiation-related genes in the presence of exfoliated SWCNTs further confirmed their enhanced osteogenic nature. These results indicated the potential of SWCNTs as a biomaterial for tissue engineering applications.

Evaluation of Bone Regeneration Potential of Long-Term Soaked Natural Hydroxyapatite.

Natural hydroxyapatite (HA) was derived from pig bones (PBs) for tissue engineering applications through heat treatment. Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), X-ray fluorescence (XRF), energy-dispersive X-ray spectroscopy (EDX), and field emission scanning electron microscopy (FE-SEM) were employed for the analysis of heat-treated PB powder. In addition, inductively coupled plasma (ICP) mass spectroscopy was applied to examine the phase organizations and chemical composition of PB powder. The Ca/P ratio in bone powder was between 1.5 and 1.6. Moreover, in vitro and in vivo studies were performed to assess the biocompatibility of long-term soaked bone powder. These results can be used to (i) establish chemical characterization by analyzing SEM microstructures and undertaking XRD, XRF, EDX, FTIR, and ICP atomic emission spectrometry; (ii) perform cytotoxicity evaluations using human mesenchymal stem cells; and (iii) conduct assessments using an in vivo animal model and histological observations. No significant changes in chemical composition were observed in long-term soaked samples with respect to the original HA. Cells properly adhered to the surface of soaked samples, and rapid hilling of the defected skull was observed in the presence of the soaked sample, indicating its potential for use as a tissue engineering biomaterial. These results suggested that heat treatment at 1200 °C could produce natural bioceramics based on calcium phosphate, which retained their osteogenic potential after 36 months of soaking.

Epidermal Growth Factor-Releasing Radially Aligned Electrospun Nanofibrous Patches for the Regeneration of Chronic Tympanic Membrane Perforations.

Chronic tympanic membrane (TM) perforations can cause otorrhea. To date, various types of tissue engineering techniques have been applied for the regeneration of chronic TM perforations. However, the application of nanofibers with radially aligned nanostructures and the simultaneous release of growth factors have never been applied in the regeneration of chronic TM perforations. Here, epidermal growth factor (EGF)-releasing radially aligned nanofibrous patches (ERA-NFPs) are developed and applied for the regeneration of chronic perforated TMs. First, radial alignments and the presence of EGF in the ERA-NFPs are analyzed. EGF is confirmed to be released from the ERA-NFPs until 8 weeks. In an in vitro study, cell viability assay, immunocytochemistry, and wound-healing assay indicate rational enhancement of healing by the combination of radial alignments and EGF release. The effect of ERA-NFPs on TM cells is revealed by quantitative real-time polymerase chain reaction. An in vivo animal study shows that the ERA-NFPs effectively stimulates the healing of the chronic TM perforations. The TMs healed by ERA-NFPs show histological properties similar to those of normal TMs. These results indicate that ERA-NFPs may be an efficient platform for the regeneration of chronic TM perforations, laying the foundation for nonsurgical treatments of chronic otitis media.

Neurogenic Differentiation of Human Dental Pulp Stem Cells on Graphene-Polycaprolactone Hybrid Nanofibers.

Stem cells derived from dental tissues-dental stem cells-are favored due to their easy acquisition. Among them, dental pulp stem cells (DPSCs) extracted from the dental pulp have many advantages, such as high proliferation and a highly purified population. Although their ability for neurogenic differentiation has been highlighted and neurogenic differentiation using electrospun nanofibers (NFs) has been performed, graphene-incorporated NFs have never been applied for DPSC neurogenic differentiation. Here, reduced graphene oxide (RGO)-polycaprolactone (PCL) hybrid electrospun NFs were developed and applied for enhanced neurogenesis of DPSCs. First, RGO-PCL NFs were fabricated by electrospinning with incorporation of RGO and alignments, and their chemical and morphological characteristics were evaluated. Furthermore, in vitro NF properties, such as influence on the cellular alignments and cell viability of DPSCs, were also analyzed. The influences of NFs on DPSCs neurogenesis were also analyzed. The results confirmed that an appropriate concentration of RGO promoted better DPSC neurogenesis. Furthermore, the use of random NFs facilitated contiguous junctions of differentiated cells, whereas the use of aligned NFs facilitated an aligned junction of differentiated cells along the direction of NF alignments. Our findings showed that RGO-PCL NFs can be a useful tool for DPSC neurogenesis, which will help regeneration in neurodegenerative and neurodefective diseases.

Effects of pulsing of light on the dentinogenesis of dental pulp stem cells in vitro.

Low power light (LPL) treatment has been widely used in various clinical trials, which has been known to reduce pain and inflammation and to promote wound healing. LPL was also shown to enhance differentiation of stem cells into specific lineages. However, most studies have used high power light in mW order, and there was lack of studies about the effects of very low power light in µW. In this study, we applied 810 nm LPL of 128 µW/cm2 energy density in vitro. Upon this value, continuous wave (CW) irradiation did not induce any significant changes for differentiation of human dental pulp stem cells (hDPSCs). However, the membrane hyperpolarization, alkaline phosphatase activity, and intracellular oxidative stress were largely enhanced in the pulsed wave (PW) with 30% of duty cycle and 300-3000 Hz frequencies-LPL in which LED driver work in the form of square wave. After 21 days of daily LPL treatment, Western blot revealed the dentinogenesis in this condition in vitro. This study demonstrates that the very low power light at 810 nm enhanced significant differentiation of hDPSCs in the PW mode and there were duty cycle dependency as well as pulsing frequency dependency in the efficiency.

Chitosan/PEI patch releasing EGF and the EGFR gene for the regeneration of the tympanic membrane after perforation.

Damage to the eardrum causes acute pain and can lead to chronic otitis media if it develops into chronic tympanic membrane (TM) perforations. Chronic TM perforations are usually treated with surgical methods such as tympanoplasty and myringoplasty. However, these surgeries are not only complicated and difficult but also cost a lot of money. Our research team developed chitosan patches (E-CPs) that release epidermal growth factor (EGF) as a patch therapy to replace surgical methods. However, there was a limitation in the healing ratio of the treatment compared to the surgical methods. In this study, we developed EGF and epidermal growth factor receptor (EGFR) gene-releasing polyethyleneimine (PEI)/chitosan patches (EErP-CPs) to increase the regeneration of TM perforations. The addition of PEI increased the adhesion and migration ability of TM cells on the patches. The simultaneous release of the EGF and the EGFR gene further enhanced TM cell proliferation, adhesion and migratory ability. It was confirmed that the EGF protein and EGFR gene were released for 30 days; however, EGF was released and increased TM cell viability almost immediately after treatment and EGFR took a minimum of 3 days before showing its effect on improved cell viability. It was also shown that EErP-CPs are more hydrophilic and have more positive charge than E-CP because of added amine groups from PEI. In conclusion, the developed EErP-CPs resulted in the improved healing of TM perforations and can potentially be applied to the regeneration of both chronic and acute tympanic membrane perforations.