Evaluation of functions and tissue compatibility of Poly (D,L-lactic-co-glycolic acid) (PLGA) for tissue engineering applications : 조직공학적 응용을 위한 Poly (D,L-lactic-co-glycolic acid) (PLGA)의 성능과 조직적합성 평가

양원선 The Graduate School, Yonsei University 2003 국내박사

본 연구는 두 가지로 나뉘어 진행되었다. Part I에서는 poly (D,L-lactic -co-glycolic acid) (PLGA)와 인체유래 진피 섬유아세포의 복합체의 성능과 조직적합성을 평가하였다. Part II에서는 조직공학에서의 응용을 위해 TiO_(2)를 코팅한 PLGA 필름을 개발하고 이를 평가하였다. Part I : 조직공학과 상처 치유에 이용되는 생체재료는 세포의 성장과 조직형성을 물리·기계적으로 지지해 주어야 한다. 일반적으로, 조직공학에 이용되는 생체재료는 콜라겐, 알지네이트와 같은 천연재료와 bladder submucosa와 small-intestinal submucosa와 같은 세포가 없는 매트릭스, 마지막으로 polyglycolic acid (PGA), polylactic acid (PLA), PLGA와 같은 합성 고분자로 나뉜다. 진피 대체물은 손상부위에 세포가 이동하도록 안내하는 역할을 해주어야 하고, 섬유아세포와 같은 진피조직의 세포에 대한 지지체로서의 기능을 해야 하며, 세포외기질들의 합성을 도울 수 있어야 한다. 손상부위에 잘 부착할 수 있어야 하고, 그 부위의 방어 기작과 상처치유에 도움을 주어야 하며, 탄력성과 항구성을 가져야 한다. 미적으로도 인간피부와 비슷한 형태를 나타낼 수 있는 성장력을 가져야 한다. 본 연구에서는 이전 연구에 근거하여, 3차원 다공성 PLGA 65/35를 선별하여 조직공학적 진피 대체물인 인체유래 진피 섬유아세포와 PLGA와의 복합체를 만든 후 이 복합체의 성능과 조직적합성을 평가하였다. 이를 위해, RT-PCR을 이용한 유전자 레벨에서의 제 1형 교원질을 확인하였고, 단백질 레벨에서의 교원질을 정량하였다. H & E 염색을 통하여 PLGA에서의 세포의 모양과 분포를 확인하였고, 면역조직화학 염색을 통하여 제 1형 교원질의 발현과 분포를 확인하였다. Part II : 플라즈마 기술은 재료의 표면을 원하는 대로 쉽게 변형 시킬 수 있어 생체재료의 세포 친수성을 증가시키기 위해 이용되고 있다. 또한, 플라즈마 처리를 통해 고분자 재료를 물리·기계적 특성의 변화 없이 대량으로 변형시킬 수 있다. 세포 친수성은 조직공학 분야에서 세포 지지체로서 이용되고 있는 PLGA와 같은 생분해성 고분자에 있어서 매우 중요한 요소이다. 본 연구에서는 PLGA 표면과 세포와의 상호작용을 증진시키기 위해 PLGA 표면에 magnetron sputtering 방법으로 TiO_(2)를 코팅하였다. PLGA 표면의 변화는 contact angle과 X-ray photoelectron spectroscopy (XPS)로 확인하였다. 세포 부착과 생장은 MTT와 scanning electron microsopy (SEM)으로 확인하였다. TiO_(2) 코팅된 PLGA 필름은 코팅이 안된 PLGA에 비해서 친수성으로 변하였고 세포 부착과 생장이 더 증가하였음을 확인 할 수 있었다. 또한, macrophage를 이용한 실험에서도 코팅되지 않은 PLGA와 비슷한 부착력을 확인 할 수 있었다. 따라서, TiO_(2) 코팅된 PLGA는 생체 적합한 세포 지지체로서의 응용 가능성이 있음을 확인 하였다. This study is divided into two categories. In part one, functions and tissue compatibility were evaluated in a composite of fibroblasts and poly (D,L-lactic-co-glycolic acid) (PLGA) scaffold. In part two, TiO_(2)-coated PLGA film was developed and evaluated for tissue engineering. Part I : In tissue engineering and wound-healing applications, scaffold materials are utilized to provide a mechanical support for cell growth and tissue formation. Generally, three classes of biomaterials have been used for engineering of genitourinary tissues: naturally derived materials (e.g., collagen and alginate), acellular tissue matrices (e.g. bladder submucosa and small-intestinal submucosa), and synthetic polymers [e.g., polyglycolic acid (PGA), polylactic acid (PLA), poly (lactic-co-glycolic acid) (PLGA)]. A dermal substitute should function as a guide for cells moving into the repair area, and serve as a scaffold for cells such as fibroblasts, and help synthesize extracellular matrix (ECM) components. It must adhere to the wound bed, support local defense mechanisms and wound healing. It must be elastic and have long-term durability and growth potential similar to human skin with good aesthetic quality. On the basis of previous study, we selected a three dimensional porous PLGA 65/35 and made composites of PLGA and human dermal fibroblast. The aim of this study is to develop the evaluation methods of function and tissue compatibility for tissue engineered dermal substitute. This study was focused on the functional analysis and tissue compatibility of artificial dermal substitute. The experiments were performed and confirmed at a level of gene expression (RT-PCR), protein expression (total collagen quantities) and histological analysis. Part II : Plasma technique can easily be used to introduce the desired functional groups or chains onto the surface of materials, so it has a special application to improve the cell affinity of scaffolds. Additionally, it has been demonstrated that plasma treatment is a unique and powerful method for modifying polymeric materials without altering their bulk properties. Cell affinity is the most important factor to be concerned with when biodegradable polymeric materials such as PLGA are utilized as a cell scaffold in tissue engineering. Therefore, in order to improve PLGA surface/cells interaction, we modified PLGA surface with TiO_(2) using magnetron sputtering method. The changes of their surface properties have been characterized by means of contact angle measurement and X-ray photoelectron spectroscopy (XPS). To confirm cell attachment and proliferation, MTT assay and scanning electron microscopy (SEM) were carried out. The results indicate TiO_(2)-coated PLGA film became hydrophilic and enhanced cell affinity and proliferation. We expect TiO_(2)-coated PLGA matrix can be a candidate for cell scaffolds in tissue engineering.

골조직공학을 위한 홍합접착단백질 기반의 다중복합적 인공세포외기질 : Multicomposite Artificial Extracellular Matrix Based on Mussel Adhesive Protein for Bone Tissue Engineering

최봉혁 포항공과대학교 일반대학원 2013 국내박사

The concept of artificial extracellular matrix (ECM) is very important approach in tissue engineering fields thanks to its biological activity. Artificial ECM can regulate diverse cellular behaviors including cell adhesion, proliferation, survival, morphology, and differentiation. Also, they are in charge of multiple functions, such as provision of 3-demensional structure and storage and presentation of growth factors. The first considering factor for design of artificial ECM is the type of desired cell/tissue because of difference of the macroscale, microscale, and nanoscale structures of ECM. Especially, in bone tissue engineering, organic protein, glycosaminoglycans, and inorganic calcium phosphate have been used to design bone-specific artificial ECM. Even though these molecules have superior biological activities, it is not easy to apply organic protein, glycosaminoglycans (GAGs), and inorganic calcium phosphate to scaffolds. For this reason, it is required that development of facile, efficient, and universal coating technique for multicomposite artificial ECM on surface of scaffold materials. Here, we proposed a novel functionalization material based on mussel adhesive protein (MAP). Adhesive property of MAP enabled efficient immobilization of ECM peptides without any protein and/or surface modifications, which significantly enhanced cellular behaviors including adhesion, spreading, proliferation, differentiation, and survival on each ECM mimics in 3 types of cells (pre-osteoblast, chondrocyte, and pre-adipocyte). Next, we performed the facile immobilization of glycosaminoglycan molecules, mainly hyaluronic acid (HA), using a MAP coating to improve the biocompatibility and biological activity of the titanium surface. Negatively charged HA was successfully immobilized onto the positively charged MAP-coated titanium surface by charge interactions without any chemical modifications. We found that pre-osteoblast behaviors, such as proliferation, spreading, and differentiation, were significantly increased on the MAP/HA-coated titanium compared to the bare, solely HA-treated, and solely MAP-layered surfaces. We also demonstrated that other negatively charged GAG molecules, including heparin sulfate (HS), chondroitin sulfate (CS), and dermatan sulfate (DS), could be successfully immobilized on a MAP-coated titanium surface. Moreover, we expanded the MAP-based coating method to fabricate a multilayer film of the two counterionic molecules of MAP and HA on to a titanium surface using the layer-by-layer technique. Finally, transitioning from general ECM peptides, we focused on the conjugated bioactive peptides related to bone-cell specific active peptides, such as RGD, basic fibroblast growth factor (bFGF), and human bone morphogenetic protein-2 (hBMP-2) which are relevant to cell adhesion, proliferation, and differentiation, respectively. Using these molecules, we constructed several chimeric MAPs using bone-cell specific active peptides and easily fabricated multicomposite artificial ECM environment via simple mixing with combination of chimeric MAPs. Multicomposite artificial ECM mimic mixtures were effective on diverse biological activities such as adhesion, proliferation, and differentiation in pre-osteoblast cell line. Although currently selected multicomposite artificial ECM mimic mixtures with calcium phosphate bone substitutes did not show satisfactory effects on in vivo bone regeneration, we expect positive effects from optimal multicomposite artificial ECM mimic combination with initial cell loading through further study. In addition, we also supplied GAG component on multicomposite artificial ECM to mimic natural ECM environment and GAG-applied multicomposite artificial ECM showed good biological performance in adhesion and proliferation of osteoblast cells. Collectively, our multicomposite artificial ECM mimics can be successfully applied in osteoblast culture and bone tissue engineering. Furthermore, MAP-based fusion technology and functionalization method can extend to other tissue and biomedical applications.

뼈와 망막색소상피의 재생을 위한 조직공학 및 세포치료에 관한 연구

박종호 전북대학교 일반대학원 2019 국내석사

Tissue engineering for tissue regeneration supports to overcome a shortage of cadaveric donor tissue for transplantation. The goal of this study was to fabricate an appropriate replacement for cadaveric tissue. In the study, alginate (AGT) hydrogel and Gallus gallus domesticus drived DBP (GDD) scaffold were utilized for tissue regeneration which has been applied to retinal and bone substitute respectively. Nature derived biomaterials have anti-inflammatory activity and biocompatibility. In the first study, we exploited the use of AGT incorporated with curcumin (CCI) forming a hydrogel based system CCI/AGT. The fabricated hydrogel could anchor RPE cell in it. In vitro cell analysis revealed that the CCI/AGT hydrogel shows good biocompatibility, enhanced cell growth ability and higher ECM formation compared to the pure AGT hydrogel. Hence, CCI/AGT hydrogel could be translated into a potential surgical graft for biological implantation of retinal tissue engineering. In the second study, GDD scaffolds were prepared by freeze-drying method from 1, 2 and 3% GDD solution and evaluated for osteogenic differentiation of BMSCs (bone marrow derived mesenchymal stem cells) on GDD scaffolds. we confirmed that cell viabillity, the gene expression and the quantity of ALP in 3% GDD scaffold superior to other sponges. As result, 3% GDD scaffold can be applied as potential biomaterial for bone regeneration.

Tissue Engineering of Temporomandibular Joint Disc Implants toward Clinical Translation

Donahue, Ryan Patrick University of California, Irvine ProQuest Disserta 2022 해외박사(DDOD)

Temporomandibular joint (TMJ) disorders (TMDs) are a group of painful and debilitating conditions, affecting 5–25% of the general US population. While generally an outdated umbrella term, recent work has delineated TMDs into those of myogenous (associated with the muscles) and arthrogenous (associated with the joint) etiologies. Specifically, in the arthrogenous category, the fibrocartilaginous TMJ disc, situated between the temporal bone of the skull and the mandible, is central to TMDs; up to 70% of all TMD cases include a pathology called disc displacement, which is an abnormal positioning of the disc. As a result of this, a condition known as disc perforation can also develop. Current treatments for discal TMDs quickly progress to end-stage surgical techniques because non-surgical approaches are only palliative and do not induce reparative effects. Recently, tissue engineering has been proposed as an intermediate solution that may be able to regenerate TMJ disc defects. However, prior to translation of tissue-engineered therapeutics, 1) tissue engineering methodologies must be optimized to create mechanically robust constructs for implantation in the orthotopic environment, 2) given that neocartilage surgical implantation causes an immune response, immune challenge of tissue-engineered implants must be investigated in vitro toward implant survivability in vivo, and 3) implants must be tested for safety and efficacy in a suitable large animal model. Toward overcoming these three hurdles, the global objectives of this dissertation are 1) to engineer neocartilage implants that can withstand the demanding environment of the TMJ disc, both mechanically and immunogenically, and 2) to expand treatable indications of tissue-engineered TMJ disc implants to perforation defects via preclinical investigations in a suitable large animal model. Toward expanding indications for discal TMDs, this work first examined focal perforation defects in the Yucatan minipig TMJ disc in tandem with allogeneic, self-assembled implants derived from costal chondrocytes. Across 24 weeks, implant treatment was safe and efficacious in healing focal (i.e., 3 mm diameter) perforation TMJ disc defects. For safety, full body necropsy, blood work, and local joint responses indicated that implants were well-tolerated immunogenically. In terms of efficacy, repair tissues of implant-treated discs were 6.2-times tougher, 8.9-times more resilient, 3.4-times stronger, and had a 2.5-times higher strain at failure, compared to fill tissues of empty defect controls. This represented significantly improved healing of TMJ disc perforation defects in the Yucatan minipig. Prior to scaling-up to larger defects, the tissue engineering process and immune response to constructs were examined. Across three studies examining the tissue engineering processes, 1) juvenile costal chondrocytes from the minipig were selected as the ideal tissue donor source, 2) 56 days of culture in the self-assembling process, which mimics native porcine knee cartilage development, resulted in the greatest tensile properties, and 3) large (i.e., 11 x 17 mm) implants derived from highly passaged (i.e., passage 6) cells were mechanically robust and flat. In another two studies assessing the immune response to implants, 1) minipig macrophages from the blood and bone marrow were harvested and characterized, and 2) macrophage co-culture revealed constructs were protected from macrophage inflammatory challenge and resulting degradation via their robust matrix content and bioactive factor application during the self-assembling process.Using the information generated from the in vitro studies described, a second in vivo study was performed examining regeneration of large (i.e., 6 mm diameter) TMJ disc perforation defects when treated with self-assembled implants. Implant-treated discs exhibited complete closure of defects with regenerated tissue after only 8 weeks, recapitulating between 64.4% and 81.2% of native disc tensile properties. Controls remained perforated after 8 weeks. Ultimately, this study further bolstered the safety and efficacy of self-assembled implants toward future use in human discal TMDs, such as disc displacement and perforation. This dissertation establishes the translational pathway for tissue-engineered implants to clinical use in humans, potentially providing long-term relief of pain and improved function for the millions of people suffering from discal TMDs.

Development of collagen-based foam bioink utilizing the meringue principle and its application in tissue engineering

Koo, Youngwon Sungkyunkwan University 2025 국내박사

This dissertation investigates the development of a collagen-based foam bioink for fabricating three-dimensional porous constructs embedded with cells, specifically tailored for tissue regeneration in bone and muscle. Bioprinting, a leading technology in tissue engineering, requires bioinks optimized for biocompatibility, ensuring safe cell transport while minimizing adverse effects in vivo. Natural polymer-based bioinks, particularly those using collagen, are widely studied for their ability to support cellular activity. However, hydrogel-based bioinks often face challenges in nutrient diffusion, necessitating the inclusion of porous structures to sustain cell viability in three-dimensional constructs. To address these limitations, this study developed a foam bioink utilizing the amphiphilic properties of collagen, employing a whipping process inspired by the principles of meringue formation. The foam bioink's physical properties, foam formation efficiency, and biological performance were thoroughly evaluated. Furthermore, hydroxyapatite and silk fibroin were incorporated to tailor the foam bioinks for bone and muscle regeneration, respectively. Their efficacy in promoting tissue-specific differentiation was validated through in vitro and in vivo experiments. A centrifugation process was also introduced to enhance the mechanical properties and structural stability of the foam bioink, facilitating the fabrication of hierarchical porous architectures mimicking the microstructure of bone tissue. This novel approach offers a robust platform to overcome the limitations of conventional hydrogel-based bioinks. In conclusion, this work demonstrates the potential of collagen-based foam bioink technology as a versatile tool in bioprinting applications, paving the way for advanced tissue engineering and the regeneration of complex tissue structures. 본 연구는 콜라겐을 기반으로 하는 거품형 바이오잉크를 개발하여 세포가 포함된 3차원 공극 구조를 제작하고 뼈 및 근육 등 조직 재생에 특화된 세포구조체를 개발하는 것을 목표로 한다. 특정한 조직의 구조와 기능을 재생시키고자 하는 조직 재생 공학 분야에서, 맞춤형 제작에 특화되어 있는 바이오프린팅 기술은 가장 널리 활용되고 개발되어 왔으며, 이에 최적화된 바이오잉크의 개발도 덩달아 활발히 연구되어 왔다. 바이오잉크를 개발하는 데 있어 가장 중요한 특성은 생체적합성이며, 이는 세포를 운반하며 체내에서 부작용 및 염증 반응을 최소화하는 데 필수적이다. 이 때문에 대부분의 바이오잉크는 하이드로겔 기반이며, 특히 세포의 활성을 돕는 콜라겐과 같은 천연 고분자 기반 바이오잉크가 각광받고 있다. 그러나 하이드로겔 기반 바이오잉크는 양분 전달의 한계 때문에 3차원 세포 구조체 제작 시 세포에 양분을 전달할 수 있는 공극 구조가 필수적으로 포함되어야 한다. 기존에는 하이드로겔 기반 바이오잉크를 격자 구조로 프린팅하거나, 프린팅 후 제거되며 공극 구조를 형성하는 희생물질이 포함된 바이오잉크를 사용하는 방법 등이 사용되었으나, 본 연구에서는 콜라겐이라는 단백질의 특성을 활용하여 휘핑whipping 공정을 통해 공극 구조가 내재되어 있는 거품형 바이오잉크를 개발하고 뼈와 근육 각 조직 특성에 맞춰 거품형 바이오잉크를 개선하는 연구를 진행하였다. 본 연구에서 콜라겐 기반 거품형 바이오잉크는 기본적으로 단백질의 양친성amphiphilicity을 이용하여 거품을 만들어내는 머랭meringue의 원리를 활용하여 개발하였으며, 거품 형성 정도와 바이오잉크의 물성 및 생물학적 효능을 평가하였다. 더 나아가 뼈와 근육 재생을 위해 각각 하이드록시아파타이트와 실크 피브로인이 혼합된 콜라겐 기반 거품형 바이오잉크를 개발하고, 각 조직으로의 분화를 세포 및 동물 모델을 사용한 실험을 통해 검증하였다. 또한 원심분리 공정을 거쳐 물성과 형상 가공성 및 유지력이 개선된 콜라겐 거품형 바이오잉크를 개발하여 계층적인 공극 구조를 갖는 뼈 조직의 미세 구조를 더욱 유사하게 재현할 수 있는 바이오프린팅 기술을 개발하였다. 결론적으로 콜라겐을 이용한 거품형 바이오잉크를 만드는 기술은 기존의 하이드로겔 기반 바이오잉크의 한계를 극복하고 바이오프린팅 기술 기반 조직 재생 공학 분야에서 다양한 조직들을 재생하고 재현하는 데 활용될 수 있는 가능성을 제시한다.

Enhancing Functional Properties of Self-Assembled Neomenisci and Neocartilage toward Their Implantation in a Suitable Preclinical Large Animal Model

Gonzalez-Leon, Erik University of California, Irvine ProQuest Disserta 2022 해외박사(DDOD)

Knee meniscus injury is frequent, resulting in over 1 million surgeries annually in the United States and Europe. Loss of meniscus tissue has been associated with early onset knee osteoarthritis due to an increase in joint contact pressures in meniscectomized knees; thus, meniscal injury also leads to damage on articular cartilage surfaces within the knee joint. Clinically available replacement strategies range from allograft transplantation to synthetic implants. Although short-term efficacy has been demonstrated with some of these treatments, factors such as long-term durability and chondroprotective efficacy remain unpredictable. Because of the near-avascularity of this fibrocartilaginous tissue and its intrinsic lack of healing, tissue engineering has been proposed as a solution for meniscus repair and replacement. In particular, bioactive and mechanical stimulation during culture can be used to enhance mechanical properties and drive extracellular matrix content toward native tissue levels. Before an effective tissue-engineering strategy for treating meniscal lesions can be translated to the clinic, the United States Food and Drug Administration (FDA) requires rigorous preclinical testing of the safety and efficacy of these technologies in a large animal model. However, guidance documents for meniscus repair technologies are nonexistent and no gold-standard animal model has been established for preclinical meniscus research. Thus, toward translating tissue engineering technologies to clinical applications, the global objectives of this research are: 1) to enhance self-assembled neomeniscus and neocartilage mechanical and biochemical properties through application of bioactive or mechanical stimuli, and 2) to identify appropriate implantation and integration methods in a large animal model to validate the repair capacity of the tissue-engineering strategies developed in vitro. To address these objectives, this research: 1) enhanced the mechanical and extracellular matrix properties of neomenisci using bioactive factors TGF-β1, chondroitinase ABC, and lysyl oxidase-like 2 (collectively termed “TCL”), in addition to lysophosphatidic acid (LPA); 2) improved neocartilage mechanical and biochemical properties through sequential application of two forms of mechanical stimuli (uniaxial tension and fluid-induced shear); 3) established the Yucatan minipig as a suitable preclinical animal model for meniscus research by showing that several gross morphological, mechanical, and biochemical properties were within ranges of values reported in human menisci; and 4) evaluated the efficacy of neocartilage implanted in the medial meniscus of Yucatan minipigs toward repairing meniscal lesions. An approach employing bioactive stimuli to enhance both extracellular matrix content and organization of neomenisci toward augmenting their mechanical properties was investigated. Specifically, self-assembled neomenisci were treated with TCL+LPA. Supporting our hypothesis, TCL+LPA treatment synergistically improved circumferential tensile stiffness and strength, significantly enhanced collagen and pyridinoline crosslink contents per dry weight by 61% and 81% over controls, respectively, and achieved tensile anisotropy (circumferential/radial) values of neomenisci close to 4. This study utilized a combination of bioactive stimuli for use in neomeniscus tissue engineering studies that improved functional properties to achieve anisotropic tensile properties, which is a crucial mechanical aspect of the native meniscus, providing a promising path toward deploying these neomenisci as functional repair and replacement tissues. To investigate whether a hyperelastic model could capture changes to native and engineered meniscus functional properties to better inform meniscus tissue engineering strategies, three different hyperelastic models were applied to mechanical and biochemical data from native tissue treated with bioactive treatments, namely collagenase. Experimental data from neomenisci treated with bioactive factors in a previously published study, specifically TCL+LPA treated neomenisci, were also examined using hyperelastic analysis. Small-strain analysis, which is largely phenomenological, is typically used to model the meniscus; however, the meniscus experiences large strains (~40%) under normal loading conditions. Collagenase treatment on native meniscus samples led to significant decreases in tensile properties and collagen content compared to untreated controls. The three hyperelasticity models examined were Neo-Hookean, Yeoh, and fiber-reinforced neo-Hookean models; it was hypothesized that a microstructural, hyperelastic model would best describe the experimental data and provide model parameters that would correlate with the biochemical content of both engineered and native tissues. Out of the three, the fiber-reinforced Neo-Hookean model, which incorporates tissue microstructural properties, was found to be the best model based on goodness-of-fit. Positive correlations between both collagen content (ρ = 0.81) and pyridinoline crosslinking (ρ = 0.69) and the fiber modulus (γ), which is a stress-like material property determined from mechanical tests of the tissue, were identified. Interestingly, the strongest correlation existed between the collagen to GAG ratio (ρ = 0.84) and the nonlinearity parameter (α). Together, these data provide a hyperelastic model that allows for deeper understanding of meniscal function with regard to its structural properties, and aids tissue engineers in the design of functional neomenisci toward their use in repair and replacement technologies. The manipulation of neocartilage construct mechanical properties toward native tissue values can. (Abstract shortened by ProQuest).

TiO2 기반 나노구조의 표면 기능화된 세포함유 계면의 골형성 유도 효과

루피스칸델 전북대학교 일반대학원 2022 국내박사

Bone tissue engineering using titanium-based material with their modification is strongly motivated in the potential applications for orthopedic dental and hard tissue engineering. The porous three-dimensional constructs based on TiO2 have been proposed as scaffolding material along with high biocompatibility and osteoconductivity in large bone defects. However, using bare titanium (Ti) substrate as bio-implants could failure to interact between a cell-to-implant interface that can lead to loosening and dislocating away from the implant site. Consequently, implant-associated complications/infections and need multiple surgeries rise biomedical and clinical burdens. It still requires modification to achieve robust osteointegration between the Ti implant and surrounding bone. To overcome these challenges, numerous methods have been introduced to fabricate porous implant surfaces with a variety of coating materials on Ti implant. In our first work, we engineered osteoconductive and osteoinductive bio-substrate of chitosan (CS) crosslinked polyaniline (PANI) nanonets coated on titanium nanotube (TiO2NTs), to mimic bone tissue’s major components via electrophoretic deposition using cyclic voltammetry. Inspired by the architectural and tunable mechanical properties of such tissue, the TiO2NTs-PANI@CS-based biofilm conferred robust anticorrosion, mechanical integrity and excellent bioavailability to enhance biological functions for the host tissue response. The light weight and high surface area of the material (TiO2NTs-PANI@CS) exhibited almost twofold increase in anti-corrosion behavior compared to TiO2NTs can be assigned to the chemical stability of PANI. PANI@CS layer can interact with osteoconductive hydroxyapatite through chelating or chemical bonding for deposition of bone ceramic at surface promoting bone tissue regeneration and osteoblastic differentiation of the human bone marrow-derived mesenchymal stem (hBM-MSCs) cells. The in vitro bone-implant bio interface demonstrates active cellular activity including cell adhesion, proliferation, and migration of the cells. The secretion of protein (collagen type-I) and phosphatase activities indicate the characteristic of TiO2NTs-PANI@CS that resemble the bone cell-extracellular matrix (ECM). Notably, the bone-related genes (collagen-I, OPN, OCN, and RUNX 2) were highly expressed within the TiO2NTs-PANI@CS over the period of 14 days, indicating greater bone cell differentiation. These findings demonstrate that the in vitro functionality of the cells on the osteoinductive-like platform of TiO2NTs-PANI@CS improves the efficiency for osteoblastic cell regeneration and that the substrate potentially has utility in bone tissue engineering applications. Furthermore, Bioactive mesoporous binary metal oxide nanoparticles allied with polymeric scaffolds can mimic natural extracellular matrix because of their self-mineralized functional matrix are highly demanded in bone tissue engineering. However, the synthesis of nanostructured metal oxide nanoparticles to incorporate in polymeric tissue scaffolds are still challenging. To address this issue, we developed fibrous scaffolds of polycaprolactone (PCL) integrating well-dispersed TiO2@ZrO2 nanoparticles (NPs) via electrospinning for a tissue engineering approach. The scaffold with 0.1 wt.% of bioceramic (TiO2@ZrO2) shows synergistic effects on physicochemical and bioactivity suited to stem cell attachment/proliferation. The bioceramic-based scaffold shows excellent antibacterial activity that can prevent implant-associated infections. The scaffolds show higher porosity, higher specific surface area, and improved thermal stability which enhanced quick electrolytic movement for cellular communication through a bio-interface to endorse the regenerative effect on pre-osteoblast (MC3T3-E1) cells. In addition, the TiO2@ZrO2 in scaffold serves as a stem cell microenvironment to accelerate cell-to-cell interactions, including cell growth, morphology/orientation, differentiation, and regeneration. The NPS in PCL exerts superior biocompatibility on MC3T3-E1 cells inducing osteogenic differentiation. The ALP activity and ARS staining confirm the upregulation of bone-related proteins and minerals suggesting the scaffolds exhibit osteoinductive abilities and contribute to bone cell regeneration. Based on this result, the bimetallic oxide could become a novel bone ceramic tailor TiO2@ZrO2 composite tissue-construct and keep potential nanomaterials-based scaffold for bone tissue engineering strategy. Keywords: Titanium nanotube (TiO2NTs), TiO2@ZrO2 NPs, Bioceramics, antibacterial, tissue engineering, osteoconductive, Immunocytochemistry 티타늄 기반 재료를 활용하는 뼈조직 공학 연구는 매우 큰 잠재적 응용 가능성으로 정형외과, 치과 및 경조직공학 연구분야에 다양하게 응용되어 왔다. TiO2을 기반으로 한 다공성 3차원 구조는 높은 생체적합성 및 골전도성을 갖는 지지체 재료로 제안되고 있다. 하지만, 순수 티타늄(Ti)을 바이오 임플란트 재료로 사용할 경우, 세포와 임플란트 계면 사이의 상호작용이 어려워 임플란트 삽입 부위에서 탈구 현상이 발생할 수 있다. 이는 결과적으로 임플란트 관련 합병증/감염 및 재수술이 요구되며, 이로 인해 임상적 부담이 증가한다. Ti 임플란트와 주변 뼈 사이의 강한 골유착을 형성하기 위해서는 다양한 재료적 변형이 요구된다. 이러한 문제를 해결하기 위해 Ti 임플란트에 다양한 코팅 재료를 사용하여 다공성 임플란트 표면을 제작하는 많은 연구 및 방법들이 개발되고 있다. 첫 번째 연구에서는 전기영동증착을 위해 순환전압전류법을 사용하여 키토산(CS)이 코팅된 티타늄나노튜브(TiO2NTs)에 폴리아닐린(PANI)을 가교하여 나노네트구조의 생체기판을 제작하였다. 이는 뼈조직의 주요 구성 요소를 모방하며 골전도성(osteoconduction) 및 골유도성(osteoinduction)의 특징을 갖고 있다. 생체조직의 구조적∙기계적 특성을 모방하여 제작된 TiO2NTs-PANI@CS 기반의 바이오필름은 강력한 부식방지능력과 기계적 무결성 및 우수한 생체활성을 가짐으로써, 조직반응에 대한 생물학적 기능을 향상시켰다. 합성된 재료(TiO2NTs-PANI@CS)는 TiO2NT보다 가볍고, 높은 표면적을 갖으며 PAN의 화학적 안정성으로 내부식성이 약 2배 이상 향상되었다. 또한, PANI@CS표면에는 킬레이트화(chelating) 또는 화학적 결합 방식을 통해 골세라믹 침착 및 골전도성 수산화인회석(hydroxyapatite)을 결합하였다. 이러한 특징은 인간 골수 유래 중간엽 줄기세포(hBM-MSCs)의 골조직 재생 및 조골 분화를 촉진한다. In vitro 실험을 통해 세포 부착, 증식 및 세포 이동을 포함한 세포활성 효과를 확인하였다. PANI@CS에서 분비되는Collagen type I과 Phosphatase의 활성은 골세포의 세포외기질과 유사한 수준을 나타내었다. 특히, 골분화 관련 유전자(collagen I, OPN, OCN, RUNX 2)는 TiO2NTs-PANI@CS 내에서 14일 동안 높게 발현되어 골세포 분화가 유도되었음을 알 수 있다. 이러한 결과를 토대로 TiO2NTs-PANI@CS의 골유도성 효과는 조골세포 재생을 향상시킴으로써, 개발된 본 기판은 뼈 조직공학 응용분야에서 잠재적인 유용성을 가지고 있다. 고분자 스캐폴드와 결합된 나노다공성 금속 산화물 나노입자는 자가 광물화 기능으로 세포외기질(ECM)을 모방할 수 있으며, 이는 뼈 조직공학 연구에서 요구되는 기능이다. 하지만, 고분자 조직 스캐폴드와 결합할 수 있는 나노구조의 금속 산화물질의 나노입자 합성은 여전히 도전적인 과제이다. 이를 해결할 수 있는 방법으로 전기방사법을 활용해 TiO2@ZrO2 나노입자가 분산된 PCL 섬유질 스캐폴드를 제작하였다. 0.1 wt%의 바이오세라믹(TiO2@ZrO2)이 함유된 섬유질 스캐폴드는 줄기세포의 부착 및 증식에 적합한 물리∙화학적 특성 및 생리활성 능력에서 시너지 효과를 나타냈다. 바이오세라믹 기반의 스캐폴드는 우수한 항균 특성을 보임으로써 임플란트 관련 감염을 예방할 수 있다. 증가된 비표면적과 다공성, 열 안정성을 보여주는 섬유질 스캐폴드는 전 조골세포(MC3T3-E1)에 대한 재생효과를 보여주었다. 또한, 바이오인터페이스(bio-interface)를 통한 세포간 신호체계를 빠르게 전달할 수 있도록 향상시켰다. 스캐폴드에 함유된 TiO2@ZrO2는 줄기세포의 생장, 형태/배향, 분화 및 재생을 포함한 세포 간 상호작용을 가속화하는 역할을 하였다. PCL섬유상에 존재하는 나노파티클은 골 형성 분화를 유도하는 MC3T3-E1세포에서 우수한 생체적합성을 나타냈다. ALP활성 및 ARS 염색을 통해 뼈 관련 단백질 및 미네랄 형성이 증가되었음을 확인하였고, 이를 통해 제작된 섬유질 스캐폴드는 골유도 및 뼈 세포 재생에 효과적임을 확인하였다. 이러한 결과를 바탕으로 두가지 금속(bimetallic)의 산화물은 새로운 TiO2@ZrO2복합 조직 구조가 될 수 있으며, 뼈 조직 공학분야에서 잠재적인 나노 물질 기반의 스캐폴드로 제안될 수 있다. 키워드: 티타늄나노튜브(TiO2NTs); TiO2@ZrO2 나노파티클; 바이오세라믹; 항균성; 조직공학; 골전도성; 면역세포화학

Over 4500 mm3 Engineered Liver Scaffold for Implantable Artificial Liver

웬 찬 충 울산대학교 일반대학원 2024 국내박사

Liver diseases, encompassing conditions from hepatitis to cirrhosis, present a global health challenge affecting millions and straining healthcare systems. These diseases are often debilitating and life-threatening. Liver transplantation, long an essential life-saving procedure for patients with end- stage liver disease, is hindered by a constant shortage of suitable donor organs and the need for lifelong immunosuppression to prevent graft rejection. A promising solution arises in liver tissue engineering, which aims to create functional liver tissue using methodologies like decellularized extracellular matrix scaffolds and 3D bioprinting. Liver tissue engineering aims to diminish reliance on organ donors by using a patient's own cells to create bio-engineered liver tissue. In doing so, this field promises to substantially reduce the challenges associated with organ shortages and the need for lifelong immunosuppression, effectively minimizing complications from immune-related responses. Part 1 of this dissertation comprises two pivotal chapters. Chapter 1 provides information on the liver anatomy. We delve into the liver's components, from the diversity of cell types to the complexities of the extracellular matrix. The chapter also dissects the essential role of growth factors in orchestrating the liver's functions. The liver regeneration process, a unique attribute of the liver, is unveiled. It also addresses the sobering reality of liver diseases, explaining various afflictions, their causes, and their profound implications for liver health. Chapter 2 ventures into liver tissue engineering, introducing the utilization of various cells and biomaterials for creating artificial liver tissues. Here, we explore the techniques and methodologies that underpin this field. Liver tissue engineering presents a promising avenue, blending advanced science and technology, to craft synthetic liver tissues that might approach the functionality of a healthy liver. These two chapters collectively lay the foundation for an in-depth exploration of the liver's intricacies, from its structural anatomy to the exciting prospects of engineering liver tissues. Part 2 details our artificial liver project, focusing on developing a large-volume functional liver scaffold comprising three distinct models for two different culture conditions. These models are meticulously designed to explore the influence of cell culture conditions, including both static and dynamic flow conditions. Inspired by previous research, we employed 3D extrusion bioprinting with an inverse-gravity technique and a laminar-flow device to generate these large-volume constructs. To address the challenge of nutrient and oxygen diffusion within large scaffolds, we implemented a pumping system that directly delivers cell culture medium through a hollow channel embedded within the scaffold. Additionally, we conducted investigations into the appropriate material matrix for cell maturation, aiming to mimic the biological environment of native liver tissue and sustain long-term culture. A rigorous biological analysis process was employed to evaluate our models. This process included assessment of cell proliferation, cell viability, liver function through albumin and urea secretion, and examination of cell morphology. This chapter marks significant progress in advancing the field of artificial liver research.

환경제어가 가능한 인베스 3D 세포 프린팅 기술을 통한 지방세포로 조밀하게 채워진 지방 구조체의 개발

안민준 포항공과대학교 일반대학원 2022 국내박사

Adipose tissue is a representative loose connective tissue composed mostly of lipid-accumulating adipocytes, which differentiate from preadipocytes. Adipose tissue is densely packed with mature adipocytes, and this morphology is associated with its functionality. Conventionally, adipose tissue plays a role in fat deposition, protection of neighboring organs, and insulation. However, adipose tissue has recently been identified as a major endocrine organ. The adipose tissue coordinates with several organs through numerous adipokines secreted from mature adipocytes and is responsible for overall health. Consequently, morphological and functional recapitulation of adipose constructs is becoming gradually important in biological and pathological studies. Although direct use of adipocytes results in immediate recapitulation of native functions, their fragile properties result in many dead cells during fabrication. Moreover, it remains challenging limitations to recapitulate the morphology of native adipose tissue with fully populated adipogenic lipid droplets, mainly due to poor adipogenesis resulted from the low cell amount in a unit area of current adipose constructs. This thesis suggests the use of environmentally controlled in-bath three-dimensional (3D) cell printing to engineer morphologically and functionally biomimetic densely packed adipose tissues in vitro. To achieve this, a hybrid bioink containing alginate was first developed for bath suspension, because alginate is a well-known biomaterial that lacks cell-binding motifs. It was hypothesized that alginate in the hybrid bioink would provide in-bath printed cells with a cell-unfriendly environment, which regulates cell migratory behavior to allow dominant proliferation in the printed region. In addition, we hypothesized that selectively proliferated preadipocytes would form a densely packed adipose tissue construct after preadipocyte differentiation. The optimal concentration of the hybrid bath bioink (1% alginate + 1.5% adipose-derived decellularized matrix) was determined by rheological assessments, shape maintenance ability, and selective cell proliferation capability. In the hybrid bath bioink, preadipocytes (printed at >107 cells/ml) proliferated without cell migration towards the bath, forming a densely populated cellular constucts. After adipogenesis, the morphological and quantitative results demonstrated that the selectively proliferated preadipocytes could differentiate into mature adipocytes. The resulting in vivo-like densely packed adipose tissue was successfully engineered in vitro when compared with conventional approaches. The levels of representative hormones secreted by the mature adipocytes were measured. The results showed that the densely packed adipose tissue construct secreted the highest levels of adipokines among the experimental groups, indicating that organotypic cell-to-cell interactions enhance adipose tissue maturation. The densely packed adipose tissue construct also showed pathological changes associated with obesity under relevant conditions found in obese patients. The adipose tissue shows obesity characteristics under a prolonged hyperglycemic environment, leading to an increase in adipocyte size (adipocyte hypertrophy). We applied the obesity-inducing condition to the densely packed adipose tissue construct to determine if the phenomenon could be embodied. Apparent adipocyte hypertrophy was observed in our construct compared with the healthy adipose tissue construct cultured under normal glucose conditions. Most obesity-associated complications result from insulin resistance in hypertrophic adipocytes. The staining results showed few activated insulin receptors in the obese adipose tissue construct. Glucose uptake tests showed no notable changes upon insulin treatment of the obese adipose tissue construct. These outcomes revealed that the hallmarks of obese adipose tissue were successfully induced in the adipose tissue construct under obesity-inducing conditions. Furthermore, we recapitulated obesity-induced adipose tissue inflammation in vitro through coculture with monocytes. The interaction between hypertrophic adipocytes and monocytes significantly increased pro-inflammatory cytokine secretion in the obese adipose tissue construct with monocytes. We expected that this pro-inflammatory response would induce monocytes to differentiate into M1 macrophages as an adapted phenotype to the physiological state. To confirm this, an immunostaining was performed using CD206 and CD68 antibodies against M2 and M1 macrophages, respectively. The results demonstrated the predominant differentiation of monocytes to M1 macrophages in the obese adipose tissue construct. Interestingly, crown-like structures, the representative histological hallmark of obesity-associated inflamed adipose tissue, were discovered in the horizontal cross-sectional areas of the obese adipose tissue construct cocultured with monocytes. In summary, this thesis suggests an environmentally controlled in-bath three-dimensional (3D) cell-printing technique to create native-mimetic adipose tissues in vitro. To this end, a hybrid bath bioink developed for inducing selective cell proliferation was first developed. The proposed technique enabled preadipocytes to be located in a predesigned restricted region, and they became a densely packed adipose tissue construct consisting of native mimetic adipocytes. The achieved construct could recapitulate the pathological changes associated with obesity under prolonged nutrient availability. Monocytes cocultured with the obese adipose tissue construct differentiated into M1 macrophages, resulting in chronic inflammation in the adipose tissue. When cocultured with monocytes, they differentiated predominantly into M1 or M2 macrophages as per their resident states. The crown-like structure demonstrated the successful recapitulation of chronically inflamed adipose tissue in vitro. Overall, the proposed cell printing strategy shows potential as a reliable engineering platform not only for deepening the biological understanding of adipose tissue, but also for addressing several diseases related to adipose tissues, such as obesity.

Three-Dimensional (3D) Cell Printing of Engineered Patches Using Tissue-Specific Bioinks for Uterine Regeneration and Fertility Restoration : Three-Dimensional (3D) Cell Printing of Engineered Patches Using Tissue-Specific Bioinks for Uterine Regeneration and Fertility Restoration

SEN, TUGCE 포항공과대학교 일반대학원 2026 국내박사

여성 생식계에서 자궁은 여성의 건강과 성공적인 임신에 핵심적인 역할을 수행하는 기관이다. 자궁 요인 불임(uterine factor infertility)은 불임의 주요 원인 중 하나로, 자궁벽의 손상을 유발하는 질환이나 외과적 수술로 인해 자궁의 형태 이상 및 기능 상실이 발생하면서 나타난다. 현재 임상적으로 적용되고 있는 호르몬 치료나 외과적 재건술은 일시적인 증상 완화 효과를 보일 수 있으나, 자궁의 구조적·기능적 완전 회복을 달성하기에는 한계가 있다. 자궁은 자궁내막(endometrium)과 자궁근층(myometrium)으로 구성되어 있으며, 이 두 층은 에스트로겐과 프로게스테론과 같은 스테로이드 호르몬의 조절 하에 상호 협력적으로 작용하여 배아 착상과 임신을 지지한다. 이 중 어느 한 층이라도 손상될 경우, 자궁벽의 기능적 조화가 붕괴되어 생식 능력 저하로 이어질 수 있다. 이와 같은 기존 치료법의 한계를 극복하기 위한 대안으로 조직공학 기반 자궁 재생 연구가 주목받고 있다. 그러나 다른 조직공학 장기들과 비교할 때, 자궁 조직공학은 아직 초기 단계에 머물러 있으며, 자궁내막과 자궁근층을 동시에 기능적으로 회복시키는 것은 여전히 도전적인 과제로 남아 있다. 자궁벽의 구조적 복잡성을 고려할 때, 다중 조직 계면을 재현하고 세포 간 상호작용을 촉진하며 구조적·기능적 회복을 동시에 유도할 수 있는 생체모사형 지지체의 개발이 필수적이다. 이를 위해서는 자궁의 고유한 미세환경을 정밀하게 모사할 수 있는 새로운 바이오소재 및 다층 재생이 가능한 바이오제조 기술이 요구된다. 본 학위논문에서는 조직 특이적 바이오잉크와 3D 바이오프린팅 기술을 활용하여 자궁벽의 기능적 재생을 위한 생체모사 패치형 플랫폼을 개발하는 것을 목표로 하였다. 제2장에서는 돼지 자궁 조직을 탈세포화하여 자궁 층별 특성을 반영하는 바이오잉크를 제조하였으며, 각 바이오잉크는 해당 자궁 조직의 생화학적·구조적 특성을 효과적으로 보존하였다. 개발된 바이오잉크는 주요 세포외기질 단백질과 생물학적 신호를 유지하면서도 압출 기반 3D 프린팅에 적합한 유변학적 특성과 우수한 세포 적합성을 나타냈다. 또한, 자궁내막 기질세포의 호르몬 반응성과 얇은 자궁내막 마우스 모델에서의 향상된 재생 효과를 통해, 해당 바이오잉크가 자궁 재생을 위한 조직 특이적이고 생물학적으로 활성화된 미세환경을 제공함을 확인하였다. 제3장에서는 자궁내막 특이적 바이오잉크를 활용하여 자궁내막의 층상 구조를 모사한 3D 바이오프린팅 기반 자궁내막 패치(EndoPatch)를 제작하였다. 해당 패치는 상피 오가노이드, 기질세포, 자궁 자연살해세포(uNK 세포), 혈관내피세포를 조직유사 배열로 통합함으로써, 상피–기질 간 생체모사 계면을 성공적으로 구현하였다. 아셔만 증후군 동물 모델에 EndoPatch를 이식한 결과, 비처치군 대비 자궁내막 두께 증가, 샘 구조 재생, 혈관화가 유의미하게 향상되었으며, 생식 기능 또한 효과적으로 회복되었다. 이러한 결과는 EndoPatch가 자궁내막 재건을 위한 기능적 바이오소재 플랫폼으로서 높은 잠재력을 지님을 시사한다. 제4장에서는 자궁내막 및 자궁근층 특이적 바이오잉크를 통합한 3D 바이오프린팅 공정을 통해 자궁 전층(full-thickness) 재생을 위한 자궁벽 패치(UteroPatch)를 제작하였다. UteroPatch는 자궁의 구조적 계층성을 성공적으로 재현하였으며, 각 층은 자궁내막 상피세포, 기질세포, 혈관내피세포 및 평활근세포에 적합한 미세환경을 제공하였다. 전층 자궁 손상 랫드 모델에 이식한 결과, 이식체는 숙주 조직과 원활하게 통합되었으며, 정돈된 콜라겐 재형성과 함께 자궁내막 및 자궁근층의 구조적 회복이 관찰되었다. 더 나아가, UteroPatch 이식군에서는 정상 대조군과 유사한 배아 착상 및 태아 성장 결과가 확인되어, 본 패치가 임신 과정에 부정적인 영향을 미치지 않으면서 기능적 회복을 효과적으로 지원함을 입증하였다. 종합적으로, 본 학위논문은 탈세포화 자궁 유래 바이오잉크와 고도화된 3D 바이오프린팅 기술을 융합하여 자궁 조직 재생을 위한 통합적인 바이오공학 전략을 제시한다. 조직 특이적 바이오잉크 개발과 이를 기반으로 한 자궁내막 및 자궁벽 패치 시스템의 구축을 통해, 구조적·호르몬적·기능적 재생이 가능한 플랫폼을 확립하였다. 본 연구 결과는 자궁 모사형 지지체 설계에 대한 이해를 심화시키는 동시에, 자궁 요인 불임 치료 및 생식 기능 회복을 위한 향후 임상 전환 연구의 토대를 제공할 것으로 기대된다. The female reproductive system, particularly the uterus, plays a vital role in women’s health and successful pregnancy. Uterine factor infertility is one of the primary causes of infertility, often resulting from diseases or surgical procedures that damage the uterine wall and lead to malformation and loss of function. Although current clinical approaches, such as hormonal therapies and surgical reconstruction, can offer temporary relief, they remain insufficient for achieving complete and functional regeneration. The uterus consists of two major layers, the endometrium and myometrium, that work in concert under the influence of steroid hormones such as estrogen and progesterone to support embryo implantation and pregnancy. Damage to either of these layers disrupts these layers functional harmony, resulting in impaired fertility. Given the limitations of current treatments, tissue engineering has emerged as a promising strategy for uterine repair and regeneration. However, compared with other tissue-engineered organs, uterine tissue engineering remains at an early stage, and achieving functional recovery of both the endometrium and myometrium remains a significant challenge. Considering the complexity of the uterine wall, it is critically important to develop biomimetic scaffolds that can regenerate multiple tissue interfaces, promote cellular communication, and restore both structure and function. For this purpose, novel biomaterials and biofabrication strategies are needed to enable multilayered regeneration that closely replicates the native uterine environment. In this dissertation, we aimed to develop biomimetic, patch-type platforms using tissue-specific bioinks and 3D bioprinting technology for functional regeneration of the uterine wall. In Chapter 2, uterine layer-specific bioinks were obtained via decellularization of porcine uterus, each reflecting the biochemical and structural features of the corresponding uterine tissue. These bioinks retained key extracellular matrix proteins and bioactive cues, exhibiting suitable rheological properties for extrusion-based 3D printing and excellent cytocompatibility with uterine cell types. The hormone-responsive behavior of encapsulated endometrial stromal cells and the enhanced regenerative effect in a thin-endometrium mouse model confirmed that the developed bioinks provide a biologically active and tissue-specific microenvironment for uterine repair. In Chapter 3, the endometrium-specific bioink was further utilized to fabricate a 3D-bioprinted endometrial patch (EndoPatch) designed to recapitulate the stratified structure of the endometrium. The patch incorporated epithelial organoids, stromal cells, uterine natural killer (uNK) cells, and endothelial cells in an organotypic arrangement, achieving a biomimetic interface between the epithelial and stromal compartments. In vivo transplantation of the EndoPatch into an Asherman’s syndrome model demonstrated significant improvement in endometrial thickness, glandular regeneration, and vascularization, as well as restoration of fertility compared to untreated groups. These findings highlight the EndoPatch’s potential as a functional biomaterial platform for endometrial reconstruction. In Chapter 4, a full-thickness uterine wall patch (UteroPatch) was fabricated using a 3D bioprinting approach integrating both endometrium-specific and myometrium-specific bioinks. The UteroPatch successfully replicated the structural hierarchy of the uterus, with each layer providing a distinct microenvironment for endometrial epithelial, stromal, endothelial, and smooth muscle cells. In vivo implantation into a rat full-thickness uterine injury model demonstrated seamless integration with host tissue, organized collagen remodeling, and restoration of both endometrial and myometrial layers. Moreover, the UteroPatch-treated group showed normal embryo implantation and fetal growth comparable to the healthy control group, demonstrating that the bioprinted patch supported functional recovery without adverse effects on pregnancy. Collectively, this dissertation presents a comprehensive bioengineering strategy for uterine tissue regeneration by combining decellularized uterus-derived bioinks with advanced 3D bioprinting techniques. The development of tissue-specific bioinks, followed by fabrication of endometrial and uterine patch systems, establishes a platform capable of structural, hormonal, and functional regeneration. These findings not only deepen the understanding of uterus-mimetic scaffold design but also provide a foundation for future translational applications in reproductive medicine, particularly for treating uterine factor infertility and enhancing fertility restoration.