Degradation-controlled tissue extracellular sponge for rapid hemostasis and wound repair after kidney injury.

Patients diagnosed with T1a cancer undergo partial nephrectomy to remove the tumors. In the process of removing the tumors, loss of kidney volume is inevitable, and current surgical methods focus solely on hemostasis and wound closure. Here, we developed an implantable form of decellularized extracellular matrix sponge to target both hemostasis and wound healing at the lesion site. A porous form of kidney decellularized matrix was achieved by fabricating a chemically cross-linked cryogel followed by lyophilization. The prepared kidney decellularized extracellular matrix sponge (kdES) was then characterized for features relevant to a hemostasis as well as a biocompatible and degradable biomaterial. Finally, histological evaluations were made after implantation in rat kidney incision model. Both gelatin sponge and kdES displayed excellent hemocompatibility and biocompatibility. However, after a 4-week observation period, kdES exhibited more favorable wound healing results at the lesion site. This suggests a promising potential for kdES as a supportive material in facilitating wound closure during partial nephrectomy surgery. KdES not only achieved rapid hemostasis for managing renal hemorrhage that is comparable to commercial hemostatic sponges, but also demonstrated superior wound healing outcomes.

Laminin-Augmented Decellularized Extracellular Matrix Ameliorating Neural Differentiation and Neuroinflammation in Human Mini-Brains.

Non-neural extracellular matrix (ECM) has limited application in humanized physiological neural modeling due to insufficient brain-specificity and safety concerns. Although brain-derived ECM contains enriched neural components, certain essential components are partially lost during the decellularization process, necessitating augmentation. Here, it is demonstrated that the laminin-augmented porcine brain-decellularized ECM (P-BdECM) is xenogeneic factor-depleted as well as favorable for the regulation of human neurons, astrocytes, and microglia. P-BdECM composition is comparable to human BdECM regarding brain-specificity through the matrisome and gene ontology-biological process analysis. As augmenting strategy, laminin 111 supplement promotes neural function by synergic effect with laminin 521 in P-BdECM. Annexin A1(ANXA1) and Peroxiredoxin(PRDX) in P-BdECM stabilized microglial and astrocytic behavior under normal while promoting active neuroinflammation in response to neuropathological factors. Further, supplementation of the brain-specific molecule to non-neural matrix also ameliorated glial cell inflammation as in P-BdECM. In conclusion, P-BdECM-augmentation strategy can be used to recapitulate humanized pathophysiological cerebral environments for neurological study.

3D bioprinted multilayered cerebrovascular conduits to study cancer extravasation mechanism related with vascular geometry.

Cerebral vessels are composed of highly complex structures that facilitate blood perfusion necessary for meeting the high energy demands of the brain. Their geometrical complexities alter the biophysical behavior of circulating tumor cells in the brain, thereby influencing brain metastasis. However, recapitulation of the native cerebrovascular microenvironment that shows continuities between vascular geometry and metastatic cancer development has not been accomplished. Here, we apply an in-bath 3D triaxial bioprinting technique and a brain-specific hybrid bioink containing an ionically crosslinkable hydrogel to generate a mature three-layered cerebrovascular conduit with varying curvatures to investigate the physical and molecular mechanisms of cancer extravasation in vitro. We show that more tumor cells adhere at larger vascular curvature regions, suggesting that prolongation of tumor residence time under low velocity and wall shear stress accelerates the molecular signatures of metastatic potential, including endothelial barrier disruption, epithelial-mesenchymal transition, inflammatory response, and tumorigenesis. These findings provide insights into the underlying mechanisms driving brain metastases and facilitate future advances in pharmaceutical and medical research.

Engineering of Uniform Epidermal Layers via Sacrificial Gelatin Bioink-Assisted 3D Extrusion Bioprinting of Skin.

To reconstruct an ideal full-thickness skin model, basal keratinocytes must be distributed as a confluent monolayer on the dermis. However, the currently available extrusion bioprinting method for the skin is limited when producing an air-exposed cellular monolayer because the cells are encapsulated within a bioink. This is the first study to use sacrificial gelatin-assisted extrusion bioprinting to reproduce a uniform and stratified epidermal layer. Experimental analyses of the rheological properties, printability, cell viability, and initial keratinocyte adhesion shows that the optimal gelatin bioink concentration is 4 wt.%. The appropriate thickness of the bioprinted gelatin structure for achieving a confluent keratinocyte layer is determined to be 400 µm. The suggested strategy generates a uniform keratinocyte monolayer with tight junctions throughout the central and peripheral regions, whereas manual seeding generates non-uniform cellular aggregates and vacancies. These results influence gene expression, exhibiting a propensity for epidermal differentiation. Finally, the gelatin-assisted keratinocytes are bioprinted onto a dermis composed of gelatin methacryloyl and dermis-derived decellularized extracellular matrix to establish a full-thickness skin model. Thus, this strategy leads to significant improvements in epidermal differentiation/stratification. The findings demonstrate that the gelatin-assisted approach is advantageous for recreating reliable full-thickness skin models with significant consistency for mass production.

3D Cell Printing of Advanced Vascularized Proximal Tubule-on-a-Chip for Drug Induced Nephrotoxicity Advancement.

Renal dysfunction due to drug-induced nephrotoxicity (DIN) affects >20% of the adult population worldwide. The vascularized proximal tubule is a complex structure that is often the primary site of drug-induced kidney injury. Herein, a vascularized proximal tubule-on-a-chip (Vas-POAC) was fabricated, demonstrating improved physiological emulation over earlier single-cell proximal tubule models. A perfusable model of vascularized proximal tubules permits the growth and proliferation of renal proximal tubule cells and adjacent endothelial cells under various conditions. An in vitro Vas-POAC showed mature expressions of the tubule and endothelial cell markers in the mature epithelium and endothelium lumens after 7 days of culture. Expression in the mature proximal tubule epithelium resembled the polarized expression of sodium-glucose cotransporter-2 and the de novo synthesis of ECM proteins. These perfusable Vas-POACs display significantly improved functional properties relative to the proximal tubules-on-a-chip (POAC), which lacks vascular components. Furthermore, the developed Vas-POAC model evaluated the cisplatin-induced nephrotoxicity and revealed enhanced drug receptivity compared to POAC. We further evaluated the capability of the developed proximal tubule model to act as a functional platform that targets screening drug doses that can cause renal proximal tubule injury in adults. Thus, our cell-printed models may prove valuable for screening, thoughtful mechanistic investigations of DIN, and discovery of drugs that interfere with tubule formation.

3D biofabrication of diseased human skin models in vitro.

Human skin is an organ located in the outermost part of the body; thus, it frequently exhibits visible signs of physiological health. Ethical concerns and genetic differences in conventional animal studies have increased the need for alternative in vitro platforms that mimic the structural and functional hallmarks of natural skin. Despite significant advances in in vitro skin modeling over the past few decades, different reproducible biofabrication strategies are required to reproduce the pathological features of diseased human skin compared to those used for healthy-skin models. To explain human skin modeling with pathological hallmarks, we first summarize the structural and functional characteristics of healthy human skin. We then provide an extensive overview of how to recreate diseased human skin models in vitro, including models for wounded, diabetic, skin-cancer, atopic, and other pathological skin types. We conclude with an outlook on diseased-skin modeling and its technical perspective for the further development of skin engineering.

The Correlation between Waist Circumference and the Pro-Inflammatory Adipokines in Diabetic Retinopathy of Type 2 Diabetes Patients.

Central obesity is one of the major risk factors for type 2 diabetes mellitus (DM), and the most common complication of DM is diabetic retinopathy. However, the exact relationship between obesity and DR remains unknown. In this study, we evaluate the effect of obesity on DR by comparing the aqueous humor-derived adipokines. For the analysis, 37 DR patients and 29 non-DR-patients participated. To evaluate the obesity of the patients, body mass index (BMI) and waist circumference (WC) were used. By comparing the concentrations of adipokines obtained from the aqueous humor of the two groups, the relationship between DR and adipokines was analyzed. In addition, by analyzing the correlation between obesity and adipokines in patients, the relationship between central obesity and DR was finally confirmed. The WC was significantly higher in patients than in the non-patient group. The concentrations of all adipokines compared in this study were significantly higher in the DR group than in the non-DM group (p < 0.05). Among them, adiponectin, leptin, TNF-α, Factor D (adipsin), lipocalin-2 (NGAL), Serpin E1 (PAI-1), and CXCL8 (IL-8) were confirmed to have a positive correlation with central obesity (defined as WC). These findings suggest that central obesity is strongly associated with the risk of DR.

Coaxial cell printing of a human glomerular model: anin vitroglomerular filtration barrier and its pathophysiology.

Much effort has been expended in emulating the kidney's glomerular unit because of its limitless potential in the field of drug screening and nephrotoxicity testing in clinics. Herein, we fabricate a functional bilayer glomerular microvessel-on-a-chip that recapitulates the specific arrangement of the glomerular endothelial cell, podocyte layers, and the intervening glomerular basement membrane (GBM) in a single step. Our perfusable chip allows for the co-culture of monolayer glomerular endothelium and podocyte epithelium, which display mature functional markers of glomerular cells, and their proper interactions produce GBM proteins, which are the major components of the GBMin vivo. Furthermore, we test the selective permeability capacity, a representative hallmark function of the glomerular filtration barrier. Lastly, we evaluate the response of our glomerular model to Adriamycin- and hyperglycemia-induced injury to evaluate its applicability for drug screening and glomerular disease modeling.

Extracellular matrix-based sticky sealants for scar-free corneal tissue reconstruction.

Regenerative medicine requires both tissue restoration and ease of compliance for clinical application. Considering this, sticky tissue sealants have been shown to have great potentials over surgical suturing and wound treatment. However, tissue sealants currently used pose challenges such as uncontrollable adhesion formation, mechanical mismatch, and lack of tissue restoration. A new sticky sealant based on gelatinized cornea-derived extracellular matrix (GelCodE) with a visible light-activating system is firstly being introduced in this study. De novo tissue regeneration relies on the matrisome in charge of tissue-organization and development within GelCodE while visible light-based photopolymerization with ruthenium/sodium persulfate rapidly induces covalent bonds with the adjacent tissues. The ease of not only in vivo application, biocompatibility, and biointegration, but also exceptional de novo tissue formation is demonstrated in this study. Interestingly, newly regenerated tissues were shown to have normal tissue-like matrices with little scar formation. Hence, this work presents a promising strategy to meet clinical demands for scar-free tissue recovery with superior ease of clinical application.

3D cell-printing of gradient multi-tissue interfaces for rotator cuff regeneration.

Owing to the prevalence of rotator cuff (RC) injuries and suboptimal healing outcome, rapid and functional regeneration of the tendon-bone interface (TBI) after RC repair continues to be a major clinical challenge. Given the essential role of the RC in shoulder movement, the engineering of biomimetic multi-tissue constructs presents an opportunity for complex TBI reconstruction after RC repair. Here, we propose a gradient cell-laden multi-tissue construct combined with compositional gradient TBI-specific bioinks via 3D cell-printing technology. In vitro studies demonstrated the capability of a gradient scaffold system in zone-specific inducibility and multi-tissue formation mimicking TBI. The regenerative performance of the gradient scaffold on RC regeneration was determined using a rat RC repair model. In particular, we adopted nondestructive, consecutive, and tissue-targeted near-infrared fluorescence imaging to visualize the direct anatomical change and the intricate RC regeneration progression in real time in vivo. Furthermore, the 3D cell-printed implant promotes effective restoration of shoulder locomotion function and accelerates TBI healing in vivo. In summary, this study identifies the therapeutic contribution of cell-printed constructs towards functional RC regeneration, demonstrating the translational potential of biomimetic gradient constructs for the clinical repair of multi-tissue interfaces.

Biomaterial-based 3D bioprinting strategy for orthopedic tissue engineering.

The advent of three-dimensional (3D) bioprinting has enabled impressive progress in the development of 3D cellular constructs to mimic the structural and functional characteristics of natural tissues. Bioprinting has considerable translational potential in tissue engineering and regenerative medicine. This review highlights the rational design and biofabrication strategies of diverse 3D bioprinted tissue constructs for orthopedic tissue engineering applications. First, we elucidate the fundamentals of 3D bioprinting techniques and biomaterial inks and discuss the basic design principles of bioprinted tissue constructs. Next, we describe the rationale and key considerations in 3D bioprinting of tissues in many different aspects. Thereafter, we outline the recent advances in 3D bioprinting technology for orthopedic tissue engineering applications, along with detailed strategies of the engineering methods and materials used, and discuss the possibilities and limitations of different 3D bioprinted tissue products. Finally, we summarize the current challenges and future directions of 3D bioprinting technology in orthopedic tissue engineering and regenerative medicine. This review not only delineates the representative 3D bioprinting strategies and their tissue engineering applications, but also provides new insights for the clinical translation of 3D bioprinted tissues to aid in prompting the future development of orthopedic implants. STATEMENT OF SIGNIFICANCE: 3D bioprinting has driven major innovations in the field of tissue engineering and regenerative medicine; aiming to develop a functional viable tissue construct that provides an alternative regenerative therapy for musculoskeletal tissue regeneration. 3D bioprinting-based biofabrication strategies could open new clinical possibilities for creating equivalent tissue substitutes with the ability to customize them to meet patient demands. In this review, we summarize the significance and recent advances in 3D bioprinting technology and advanced bioinks. We highlight the rationale for biofabrication strategies using 3D bioprinting for orthopedic tissue engineering applications. Furthermore, we offer ample perspective and new insights into the current challenges and future direction of orthopedic bioprinting translation research.

Predicting multipotency of human adult stem cells derived from various donors through deep learning.

Adult stem cell-based therapeutic approaches have great potential in regenerative medicine because of their immunoregulatory properties and multidifferentiation capacity. Nevertheless, the outcomes of stem cell­based therapies to date have shown inconsistent efficacy owing to donor variation, thwarting the expectation of clinical effects. However, such donor dependency has been elucidated by biological consequences that current research could not predict. Here, we introduce cellular morphology-based prediction to determine the multipotency rate of human nasal turbinate stem cells (hNTSCs), aiming to predict the differentiation rate of keratocyte progenitors. We characterized the overall genes and morphologies of hNTSCs from five donors and compared stemness-related properties, including multipotency and specific lineages, using mRNA sequencing. It was demonstrated that transformation factors affecting the principal components were highly related to cell morphology. We then performed a convolutional neural network-based analysis, which enabled us to assess the multipotency level of each cell group based on their morphologies with 85.98% accuracy. Surprisingly, the trend in expression levels after ex vivo differentiation matched well with the deep learning prediction. These results suggest that AI­assisted cellular behavioral prediction can be utilized to perform quantitative, non-invasive, single-cell, and multimarker characterizations of live stem cells for improved quality control in clinical cell therapies.

Feasibility and safety of a novel 3D-printed biodegradable biliary stent in an in vivo porcine model: a preliminary study.

To assess the feasibility and safety of a novel 3D-printed biodegradable biliary stent using polycaprolactone (PCL) in an in vivo porcine model. In this animal study using domestic pigs, biodegradable radiopaque biliary stents made of polycaprolactone (PCL) and barium sulfate were produced using 3D printing and surgically inserted into the common bile duct (CBD) of pigs (stent group, n = 12). Another five pigs were allocated to the control group that only underwent resection and anastomosis of the CBD without stent insertion. To check the position and status of the stents and stent-related complications, follow-up computed tomography (CT) was performed every month. The pigs were sacrificed 1 or 3 months after surgery, and their excised CBD specimens were examined at both the macroscopic and microscopic levels. Three pigs (one in the stent group and two in the control group) died within one day after surgery and were excluded from further analysis; the remaining 11 in the stent group and 3 in the control group survived the scheduled follow-up period (1 month, 5 and 1; and 3 months, 6 and 2 in stent and control groups, respectively). In all pigs, no clinical symptoms or radiologic evidence of biliary complications was observed. In the stent group (n = 11), stent migration (n = 1 at 3 months; n = 2 at 1 month) and stent fracture (n = 3 at 2 months) were detected on CT scans. Macroscopic evaluation of the stent indicated no significant change at 1 month (n = 3) or fragmentation with discoloration at 3 months (n = 5). On microscopic examination of CBD specimens, the tissue inflammation score was significantly higher in the stent group than in the control group (mean ± standard deviation (SD), 5.63 ± 2.07 vs. 2.00 ± 1.73; P = 0.039) and thickness of fibrosis of the CBD wall was significantly higher than that of the control group (0.46 ± 0.12 mm vs. 0.21 ± 0.05 mm; P = 0.012). Despite mild bile duct inflammation and fibrosis, 3D-printed biodegradable biliary stents showed good feasibility and safety in porcine bile ducts, suggesting their potential for use in the prevention of postoperative biliary strictures.

Blood-Lymphatic Integrated System with Heterogeneous Melanoma Spheroids via In-Bath Three-Dimensional Bioprinting for Modelling of Combinational Targeted Therapy.

Although metastatic melanoma can be managed with chemotherapy, its heterogeneity and resistance to therapy remain poorly understood. In addition to the spread of melanoma in the bloodstream, melanoma-stroma interaction and the lymphatic system play active roles in said heterogeneity and resistance, leading to its progression and metastasis. Reproducing the complexities of the melanoma microenvironment in vitro will help understanding its progression and enhance the translatability of potential cancer therapeutics. A blood-lymphatic integrated system with heterogeneous melanoma spheroids (BLISH) using the in-bath bioprinting process is developed. The process uniformly prints size-controllable metastatic melanoma spheroids along with biomimetic blood and lymphatic vessels (LVs). The system reproduces hallmark events of metastatic melanoma, such as tumor stroma interaction, melanoma invasion, and intravasation. The application of the system to investigate the anticancer effect of combinational targeted therapy suggests that it can be used to study the pathophysiology of melanoma and improve the accuracy of drug response monitoring in skin cancer.

Enhancement strategy for effective vascular regeneration following myocardial infarction through a dual stem cell approach.

Since an impaired coronary blood supply following myocardial infarction (MI) negatively affects heart function, therapeutic neovascularization is considered one of the major therapeutic strategies for cell-based cardiac repair. Here, to more effectively achieve therapeutic neovascularization in ischemic hearts, we developed a dual stem cell approach for effective vascular regeneration by utilizing two distinct types of stem cells, CD31+-endothelial cells derived from human induced pluripotent stem cells (hiPSC-ECs) and engineered human mesenchymal stem cells that continuously secrete stromal derived factor-1α (SDF-eMSCs), to simultaneously promote natal vasculogenesis and angiogenesis, two core mechanisms of neovascularization. To induce more comprehensive vascular regeneration, we intramyocardially injected hiPSC-ECs to produce de novo vessels, possibly via vasculogenesis, and a 3D cardiac patch encapsulating SDF-eMSCs (SDF-eMSC-PA) to enhance angiogenesis through prolonged secretion of paracrine factors, including SDF-1α, was implanted into the epicardium of ischemic hearts. We verified that hiPSC-ECs directly contribute to de novo vessel formation in ischemic hearts, resulting in enhanced cardiac function. In addition, the concomitant implantation of SDF1α-eMSC-PAs substantially improved the survival, retention, and vasculogenic potential of hiPSC-ECs, ultimately achieving more comprehensive neovascularization in the MI hearts. Of note, the newly formed vessels through the dual stem cell approach were significantly larger and more functional than those formed by hiPSC-ECs alone. In conclusion, these results provide compelling evidence that our strategy for effective vascular regeneration can be an effective means to treat ischemic heart disease.

A Bioprinted Bruch's Membrane for Modeling Smoke-Induced Retinal Pigment Epithelium Degeneration via Hybrid Membrane Printing Technology.

The retinal pigment epithelium (RPE) not only forms the outer blood-retinal barrier (oBRB) but also plays a multifunctional role in the ocular system. The loss of this epithelium leads to serious diseases resulting in vision impairment. No effective treatment is available for the repair of RPE damage. A functional in vitro RPE model that allows the recapitulation of oBRB-related pathophysiological responses is lacking. Here, a hybrid membrane printing technology is developed to fabricate cellular monolayers on the basement membrane to mimic human Bruch's membrane (BM). Using this technology, in vitro oBRB model containing the RPE monolayer on the printed BM with stable mechanical properties and fibril diameter similar to that of natural BM is developed. Compared to traditional collagen bioink, BM-based bioink significantly promotes RPE functions in vitro. Finally, smoking-like conditions are exposed to the model to recapitulate the absorption of mainstream cigarette smoke which is known as one of the risk factors for the disease progression. RPE function is damaged due to oxidative stress. Furthermore, the versatility of the model as a drug-testing platform is confirmed by the suppression of oxidative stress via antioxidants. This technology shows potential for fabricating a functional oBRB model that reflects patient conditions.

Kidney Decellularized Extracellular Matrix Enhanced the Vascularization and Maturation of Human Kidney Organoids.

Kidney organoids derived from human pluripotent stem cells (hPSCs) have extensive potential for disease modelling and regenerative medicine. However, the limited vascularization and immaturity of kidney organoids have been still remained to overcome. Extracellular matrix (ECM) can provide mechanical support and a biochemical microenvironment for cell growth and differentiation. Here in vitro methods using a kidney decellularized extracellular matrix (dECM) hydrogel to culture hPSC-derived kidney organoids, which have extensive vascular network and their own endothelial cells, are reported. Single-cell transcriptomics reveal that the vascularized kidney organoids cultured using the kidney dECM have more mature patterns of glomerular development and higher similarity to human kidney than those cultured without the kidney dECM. Differentiation of α-galactosidase A (GLA)-knock-out hPSCs generated using CRISPR/Cas9 into kidney organoids by the culture method using kidney dECM efficiently recapitulate Fabry nephropathy with vasculopathy. Transplantation of kidney organoids with kidney dECM into kidney of mouse accelerates the recruitment of endothelial cells from the host mouse kidney and maintains vascular integrity with the more organized slit diaphragm-like structures than those without kidney dECM. The kidney dECM methodology for inducing extensive vascularization and maturation of kidney organoids can be applied to studies for kidney development, disease modeling, and regenerative medicine.

Three-Dimensional Microfilament Printing of a Decellularized Extracellular Matrix (dECM) Bioink Using a Microgel Printing Bath for Nerve Graft Fabrication and the Effectiveness of dECM Graft Combined with a Polycaprolactone Conduit.

Various synthetic and decellularized materials are being used to reconstruct peripheral nerve defects and replace autologous nerve grafts. In this study, we developed a microgel printing bath to three-dimensionally (3D) print a peripheral nervous system decellularized extracellular matrix nerve graft reinforced by a polycaprolactone (PCL) conduit. The straightforward fabrication method of an alginate microgel-supplemented printing bath allows a 30 µm filament resolution of a low viscous decellularized extracellular matrix hydrogel with neutral pH. When applied to a sciatic nerve defect model of rats, the total number of regenerated axons and relative gastrocnemius muscle weight ratio were comparable to those of the autologous nerve graft group. Meanwhile, the results were superior to those of the porcine decellularized nerve graft group or the 3D printed decellularized extracellular matrix graft group. This study will be the first step demonstrating that the 3D printed decellularized extracellular matrix (dECM) graft with a PCL conduit is an effective and reliable choice to replace an autologous nerve graft in the near future.

3D Bioprinting of an In Vitro Model of a Biomimetic Urinary Bladder with a Contract-Release System.

The development of curative therapy for bladder dysfunction is usually hampered owing to the lack of reliable ex vivo human models that can mimic the complexity of the human bladder. To overcome this issue, 3D in vitro model systems offering unique opportunities to engineer realistic human tissues/organs have been developed. However, existing in vitro models still cannot entirely reflect the key structural and physiological characteristics of the native human bladder. In this study, we propose an in vitro model of the urinary bladder that can create 3D biomimetic tissue structures and dynamic microenvironments to replicate the smooth muscle functions of an actual human urinary bladder. In other words, the proposed biomimetic model system, developed using a 3D bioprinting approach, can recreate the physiological motion of the urinary bladder by incorporating decellularized extracellular matrix from the bladder tissue and introducing cyclic mechanical stimuli. The results showed that the developed bladder tissue models exhibited high cell viability and proliferation rate and promoted myogenic differentiation potential given dynamic mechanical cues. We envision the developed in vitro bladder mimicry model can serve as a research platform for fundamental studies on human disease modeling and pharmaceutical testing.

Mechanically and biologically promoted cell-laden constructs generated using tissue-specific bioinks for tendon/ligament tissue engineering applications.

Tendon and ligament tissues provide stability and mobility crucial for musculoskeletal function, but are particularly prone to injury. Owing to poor innate healing capacity, the regeneration of mature and functional tendon/ligament (T/L) poses a formidable clinical challenge. Advanced bioengineering strategies to develop biomimetic tissue implants are highly desired for the treatment of T/L injuries. Here, we presented a cell-based tissue engineering strategy to generate cell-laden tissue constructs comprising stem cells and tissue-specific bioinks using 3D cell-printing technology. We implemented anin vitropreconditioning approach to guide semi-organized T/L-like formation before thein vivoapplication of cell-printed implants. Duringin vitromaturation, tissue-specific decellularized extracellular matrix-based cellular constructs facilitated long-termin vitroculture with high cell viability and promoted tenogenesis with enhanced cellular/structural anisotropy. Moreover, we demonstrated improved cell survival/retention uponin vivoimplantation of pre-matured constructs in nude mice with de novo tendon formation and improved mechanical strength. Althoughin vivomechanical properties of the cell-printed implants were lower than those of human T/L tissues, the results of this study may have significant implications for future cell-based therapies in tendon and ligament regeneration and translational medicine.