Abstract Engineered adipose tissue constructs that are capable of reconstructing soft tissue with adequate volume would be worthwhile in plastic and reconstructive surgery. Tissue printing offers the ...possibility of fabricating anatomically relevant tissue constructs by delivering suitable matrix materials and living cells. Here, we devise a biomimetic approach for printing adipose tissue constructs employing decellularized adipose tissue (DAT) matrix bioink encapsulating human adipose tissue-derived mesenchymal stem cells (hASCs). We designed and printed precisely-defined and flexible dome-shaped structures with engineered porosity using DAT bioink that facilitated high cell viability over 2 weeks and induced expression of standard adipogenic genes without any supplemented adipogenic factors. The printed DAT constructs expressed adipogenic genes more intensely than did non-printed DAT gel. To evaluate the efficacy of our printed tissue constructs for adipose tissue regeneration, we implanted them subcutaneously in mice. The constructs did not induce chronic inflammation or cytotoxicity postimplantation, but supported positive tissue infiltration, constructive tissue remodeling, and adipose tissue formation. This study demonstrates that direct printing of spatially on-demand customized tissue analogs is a promising approach to soft tissue regeneration.
The ability to print and pattern all the components that make up a tissue (cells and matrix materials) in three dimensions to generate structures similar to tissues is an exciting prospect of ...bioprinting. However, the majority of the matrix materials used so far for bioprinting cannot represent the complexity of natural extracellular matrix (ECM) and thus are unable to reconstitute the intrinsic cellular morphologies and functions. Here, we develop a method for the bioprinting of cell-laden constructs with novel decellularized extracellular matrix (dECM) bioink capable of providing an optimized microenvironment conducive to the growth of three-dimensional structured tissue. We show the versatility and flexibility of the developed bioprinting process using tissue-specific dECM bioinks, including adipose, cartilage and heart tissues, capable of providing crucial cues for cells engraftment, survival and long-term function. We achieve high cell viability and functionality of the printed dECM structures using our bioprinting method.
It is essential to characterize the cellular properties of mesenchymal stem cell populations to maintain quality specifications and control in regenerative medicine. Biofunctional materials have been ...designed as artificial matrices for the stimulation of cell adhesion and specific cellular functions. We have developed recombinant maltose‐binding protein (MBP)‐fused proteins as artificial adhesion matrices to control human mesenchymal stem cell (hMSC) fate by using an integrin‐independent heparin sulfate proteoglycans‐mediated cell adhesion. In this study, we characterize cell adhesion‐dependent cellular behaviors of human adipose‐derived stem cells (hASCs) and human bone marrow stem cells (hBMSCs). We used an MBP‐fused basic fibroblast growth factor (MF)‐coated surface and fibronectin (FN)‐coated surface to restrict and support, respectively, integrin‐mediated adhesion. The cells adhered to MF exhibited restricted actin cytoskeleton organization and focal adhesion kinase phosphorylation. The hASCs and hBMSCs exhibited different cytoplasmic projection morphologies on MF. Both hASCs and hBMSCs differentiated more dominantly into osteogenic cells on FN than on MF. In contrast, hASCs differentiated more dominantly into adipogenic cells on MF than on FN, whereas hBMSCs differentiated predominantly into adipogenic cells on FN. The results indicate that hASCs exhibit a competitive differentiation potential (osteogenesis vs. adipogenesis) that depends on the cell adhesion matrix, whereas hBMSCs exhibit both adipogenesis and osteogenesis in integrin‐mediated adhesion and thus hBMSCs have noncompetitive differentiation potential. We suggest that comparing differentiation behaviors of hMSCs with the diversity of cell adhesion is an important way to characterize hMSCs for regenerative medicine.
A recombinant protein was designed to generate a novel cell adhesion mechanism. We found marked differences between human adipose‐derived stem cells and human bone marrow stem cells in cellular behavior and differentiation, depending on the cell adhesion mechanism.
A new concept, assembling cell‐laden tissue modules, is for the first time proposed for soft tissue engineering. Adipose‐vascular tissue modules composed of a synthetic polymer‐based substructure and ...customized bioinks using planar 3D cell printing are engineered. Such tissue modules are systematically assembled into a synthetic polymer‐based module holder fabricated with rotational 3D printing, resulting in the development of a flexible and volumetric tissue assembly. Whereas most of the previous studies about the construction of adipose tissue are limited to hypoxia, poor vascularization, rapid resorption, and mismatch in mechanical properties, it is aimed to realize the construction of nonhypoxic, flexible, and volume‐stable tissue assembly in this study. The significance of engineered tissue assembly is proven through various in vitro and in vivo evaluations. In particular, stable volume and remarkable neovascularization/adipogenesis are observed in the implanted assembly over four weeks. Interestingly, the size of newly formed lipid droplets and the remodeled morphology in the assembly are comparable to those in native adipose tissue. As far as it is known, this work is a first report suggesting a cell printing‐based tissue assembly for functional reconstruction of soft tissue.
A new concept, assembling cell‐laden tissue modules, is for the first time proposed for engineering a flexible, volumetric, and functional adipose construct through combinational 3D printing.
Volumetric muscle loss (VML) is an irrecoverable injury associated with muscle loss greater than 20%. Although hydrogel-based 3D engineered muscles and the decellularized extracellular matrix (dECM) ...have been considered for VML treatment, they have shown limited efficacy. We established a novel VML treatment with dECM bioink using 3D cell printing technology. Volumetric muscle constructs composed of cell-laden dECM bioinks were generated with a granule-based printing reservoir. The 3D cell printed muscle constructs exhibited high cell viability without generating hypoxia and enhanced de novo muscle formation in a VML rat model. To improve functional recovery, prevascularized muscle constructs that mimic the hierarchical architecture of vascularized muscles were fabricated through coaxial nozzle printing with muscle and vascular dECM bioinks. Spatially printing tissue-specific dECM bioinks offers organized microenvironmental cues for the differentiation of each cell and improves vascularization, innervation, and functional recovery. Our present results suggest that a 3D cell printing and tissue-derived bioink-based approach could effectively generate biomimetic engineered muscles to improve the treatment of VML injuries.
Abstract Stem cells offer significant therapeutic promise for the treatment of ischemic disease. However, stem cells transplanted into ischemic tissue exhibit limited therapeutic efficacy due to poor ...engraftment in vivo . Several strategies for improving the survival and engraftment of stem cells in ischemic tissue have been developed including transplantation in combination with growth factor delivery, genetic modification of stem cells, and the use of cell-transplantation scaffolds. Here, we demonstrate that human adipose-derived stromal cells (hADSCs) cultured and grafted as spheroids exhibit improved therapeutic efficacy for ischemia treatment. hADSCs were cultured in monolayer or spheroids. Spheroid cultures were more effective in preconditioning hADSCs to a hypoxic environment, upregulating hypoxia-adaptive signals (i.e., stromal cell-derived factor-1α and hypoxia-inducible factor-1α), inhibiting apoptosis, and enhancing secretion of both angiogenic and anti-apoptotic factors (i.e., hepatocyte growth factor, vascular endothelial growth factor, and fibroblast growth factor 2) compared to monolayer cultures. Moreover, cell harvesting following spheroid cultures avoided damage to extracellular matrices due to harsh proteolytic enzyme treatment, thereby preventing anoikis (apoptosis induced by a lack of cell–matrix interaction). Following intramuscular transplantation to ischemic hindlimbs of athymic mice, hADSC spheroids showed improved cell survival, angiogenic factor secretion, neovascularization, and limb survival as compared to hADSCs grafted as dissociated cells. Taken together, spheroid cultures precondition hADSCs to a hypoxic environment, and grafting hADSCs as spheroids to ischemic limbs improves therapeutic efficacy for ischemia treatment due to enhanced cell survival and paracrine effects. Spheroid-based cell delivery could be a simple and effective strategy for improving stem cell therapy for ischemic diseases, eliminating the need for growth factor delivery, biomaterial scaffolds or genetic modification.
A male Mongolian child with a complete congenital absence of both nose and nasal passage had a poor survival prognosis due to respiratory distress. To enable his survival, a new nose capable of ...conferring respiratory function was constructed. Following reconstructive surgery, an absence of mucoepithelium in the nasal passage can lead to rhinostenosis. To avoid this complication, a custom‐made nasal silicone stent was created using three‐dimensional (3D) printing technology in conjunction with the patient's computed tomography data. The stent was implanted for 2 months to maintain the shape and size of the nasal passage. At 2 months after stent implantation, the mucoepithelium tissue in the passage had successfully regenerated with no immune reaction. Three years after stent removal, respiratory function, nasal passage structure, and external nose shape were maintained without additional medical care. These results indicate the successful nasal reconstruction in an arhinia patient using a customized, 3D‐printed nasal stent. Laryngoscope, 129:582–585, 2019
Autologous cartilages or synthetic nasal implants have been utilized in augmentative rhinoplasty to reconstruct the nasal shape for therapeutic and cosmetic purposes. Autologous cartilage is ...considered to be an ideal graft, but has drawbacks, such as limited cartilage source, requirements of additional surgery for obtaining autologous cartilage, and donor site morbidity. In contrast, synthetic nasal implants are abundantly available but have low biocompatibility than the autologous cartilages. Moreover, the currently used nasal cartilage grafts involve additional reshaping processes, by meticulous manual carving during surgery to fit the diverse nose shape of each patient. The final shapes of the manually tailored implants are highly dependent on the surgeons’ proficiency and often result in patient dissatisfaction and even undesired separation of the implant. This study describes a new process of rhinoplasty, which integrates three-dimensional printing and tissue engineering approaches. We established a serial procedure based on computer-aided design to generate a three-dimensional model of customized nasal implant, and the model was fabricated through three-dimensional printing. An engineered nasal cartilage implant was generated by injecting cartilage-derived hydrogel containing human adipose-derived stem cells into the implant containing the octahedral interior architecture. We observed remarkable expression levels of chondrogenic markers from the human adipose-derived stem cells grown in the engineered nasal cartilage with the cartilage-derived hydrogel. In addition, the engineered nasal cartilage, which was implanted into mouse subcutaneous region, exhibited maintenance of the exquisite shape and structure, and striking formation of the cartilaginous tissues for 12 weeks. We expect that the developed process, which combines computer-aided design, three-dimensional printing, and tissue-derived hydrogel, would be beneficial in generating implants of other types of tissue.
Vulvovaginal reconstruction is challenging. In this study, we evaluated the outcomes of vulvovaginal reconstruction with “perineal perforator switch flap” (PPSF) and compared it with other ...conventional flaps. In addition, the long-term esthetic results were compared with the perineal perforator propeller flap (PPPF), which we previously used.
We retrospectively reviewed the clinical data of 16 patients (27 flaps) who underwent vulvovaginal reconstruction with PPSF. After tumor resection, perineal perforators close to the genitofemoral sulcus were identified. The flap was designed as an island with the perforator at the center. The flap was elevated while preserving the soft tissue surrounding the pedicle, and transferred to the defect via the subcutaneous tunnel without pedicle skeletonization.
All flaps survived and no major surgical complications were observed. The total follow-up period was 16.13±3.38 months. The mean operation time was 79.38±19.65 min, and the initiation of walking and the length of hospitalization were 1.69 ± 0.79 and 5.69 ± 0.79 days, respectively. Perineal function was well preserved. Comparison of esthetic results with PPPF showed that PPSF showed better results in symmetrical and labial shape (2.29 ± 0.73 vs. 3.13 ± 0.81; p=0.015, 2.43 ± 1.02 vs. 3.25 ± 0.68; p=0.031, respectively), and in total score. (10.29 ± 2.16 vs. 12.31 ± 1.82; p=0.017).
PPSF was technically simple and significantly reduced the duration of operation and the overall recovery time. PPSF also prevented delay in radiation. Therefore, PPSF is a promising method for vulvovaginal reconstruction.
Silica particles (SPs) induce cell proliferation and osteogenic differentiation. We reported that SPs in the scaffold induced early stage osteogenic differentiation.
A polycaprolactone (PCL) scaffold ...was fabricated with a 10 wt% SPs. The surface of PCL scaffold was coated with a 10 µg/mL collagen solution. Next, the scaffold was conjugated with 2 μM SPs, 2 μg/mL bone morphogenetic protein 2 (BMP2), or 2 μM BMP2-conjugated SPs (BCSPs). Green fluorescent protein-coupled BMP2 was applied to fabricate the scaffold. The fluorescence intensity was analyzed by confocal microscopy. The mRNA levels of the early osteogenic differentiation marker, alkaline phosphatase (ALP), were analyzed by real-time quantitative polymerase chain reaction. Levels of BMP2, RUNX2, ERK1/2, and AKT were assessed by western blotting.
ALP mRNA levels were significantly higher in the BCSP-conjugated scaffold than in the other scaffolds. In the early stage of osteogenic differentiation, the protein levels of BMP2, RUNX2, ERK1/2, and AKT in cells were significantly higher in the BCSP-conjugated scaffold than in other scaffolds. Thus, the BCSP composite scaffold induced rapid osteogenic differentiation.
These results suggest that BCSP composite can be used to promote early stage osteogenic differentiation and show promise as a material for use in scaffolds for bone regeneration.
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