Tissue engineering. Part C, Methods最新文献

Stromal vascular fraction (SVF) is the primary isolate obtained after enzymatic digestion of adipose tissue that contains various cell types. Its successful application for cell-based construct preparation in an intra-operative setting for clinical bone augmentation and regeneration has been previously reported. However, the performance of SVF-based constructs compared with traditional ex vivo expanded adipose tissue-derived mesenchymal stromal cells (ATMSCs) remains unclear and direct comparative analyses are scarce. Consequently, we here aimed at comparing the in vitro osteogenic differentiation capacity of donor-matched SVF versus ATMSCs as well as their osteoinductive capacity. Human adipose tissue from nine different donors was used to isolate SVF, which was further purified via plastic-adherence to obtain donor-matched ATMSCs. Both cell populations were immunophenotypically characterized for mesenchymal stromal cell, endothelial, and hematopoietic markers after isolation and immunocytochemical staining was used to identify different cell types during prolonged cell culture. Based on normalization using plastic-adherence fraction determination, SVF and ATMSCs were seeded and cultured in osteogenic differentiation medium for 28 days. Further, SVF and ATMSCs were seeded onto devitalized bovine bone granules and subcutaneously implanted into nude mice. After 42 days of implantation, granules were retrieved, histologically processed, and stained with hematoxylin and eosin (HE) to assess ectopic bone formation. The ATMSCs were shown to be a homogenous cell population during cell culture, whereas SVF cultures consisted of multiple cell types. All donor-matched comparisons showed either accelerated or stronger mineralization for SVF cultures in vitro. However, neither SVF nor ATMSCs loaded on devitalized bone granules induced ectopic bone formation on subcutaneous implantation, as opposed to control granules loaded with bone morphogenetic protein-2 (BMP-2), which triggered ectopic bone formation with 100% incidence. Despite the observed lack of osteoinduction, our findings provide important in vitro evidence on the osteogenic superiority of intra-operatively available SVF as compared with donor-matched ATMSCs. Consequently, further studies should focus on optimizing the efficacy of these cell populations for implementation in orthotopic bone fracture or defect treatment.

Implant-supported dental prosthetics are widely used in dental practice. Sufficient peri-implant bone tissue is a crucial prerequisite for the long-term success of this treatment, as insufficient peri-implant bone volume hampers dental implant installation and negatively influences dental implant stability. However, due to tooth extraction, bone metabolism diseases, and trauma, bone defects in the jaw are common in patients, particularly in the elderly and those suffering from underlying conditions. If this is the case, the alveolar ridge has to be augmented for reliable implant placement. Various biomaterials, growth factors (GFs) or GF-based products, and trace elements have been tested and used for alveolar ridge augmentation. Among those biomaterials, calcium phosphates (CaPs) are the most popular due to their promising biocompatibility, great osteoconductivity, and distinguishing osteogenesis. Combining CaPs with GFs or trace elements can further favor bone defect repair. This review mainly focuses on applying artificial CaP biomaterials and their combination with bioactive agents to repair bone defects in implant dentistry.

Islet transplantation is a useful therapeutic choice for severe diabetes mellitus; however, limited donor supplies have interfered with the use of this treatment. Therefore, the establishment of alternative donor sources and engineered tissue, which enables to produce appropriate insulin for controlling blood glucose, is an important challenge. The adult pig is a promising and feasible donor source and materials for the engineered tissue for the clinical setting among various candidates. The recent progress of gene-editing technology contributes to possible clinical porcine xenotransplantation, including porcine islet xenotransplantation. For the success of future clinical porcine islet xenotransplantation, establishing an islet isolation technique for acquiring adequate, good-quality porcine islets is equally important to use a gene-edited pig. However, the characteristics of porcine islets are different from other species; therefore, establishing a suitable technique for porcine islets is challenging. Impact statement Recent technological progress promotes the feasibility of xenotransplantation, including islet xenotransplantation, for clinical setting. Adult pig is a promising and feasible donor source for islet xenotransplantation and engineered tissue, which enables to control blood glucose in recipients. It is important to acquire porcine islets in good qualities for the promotion, however, establishing a technique for adult porcine islet isolation is important but challenging because of the vulnerability of adult porcine islets. Deciding the proper timing of stopping pancreatic digestion is one of the important factors for obtaining adult porcine islets in good quality.

Engineering clinically relevant musculoskeletal tissues at a human scale is a considerable challenge. Developmentally inspired scaffold-free approaches for engineering cartilage tissues have shown great promise in recent years, enabling the generation of highly biomimetic tissues. Despite the relative success of these approaches, the absence of a supporting scaffold or hydrogel creates challenges in the development of large-scale tissues. Combining numerous scaled-down tissue units (herein termed microtissues) into a larger macrotissue represents a promising strategy to address this challenge. The overall success of such approaches, however, relies on the development of strategies which support the robust and consistent chondrogenic differentiation of clinically relevant cell sources such as mesenchymal stem/stromal cells (MSCs) within microwell arrays to biofabricate numerous microtissues rich in cartilage-specific extracellular matrix components. In this article, we first describe a simple method to manufacture cartilage microtissues at various scales using novel microwell array stamps. This system allows the rapid and reliable generation of cartilage microtissues and can be used as a platform to study microtissue phenotype and development. Based on the unexpected discovery that Endothelial Growth Medium (EGM) enhanced MSC aggregation and chondrogenic capacity within the microwell arrays, this work also sought to identify soluble factors within the media capable of supporting robust differentiation using heterogeneous MSC populations. Hydrocortisone was found to be the key factor within EGM that enhanced the chondrogenic capacity of MSCs within these microwell arrays. This strategy represents a promising means of generating large numbers of high-quality, scaffold-free cartilage microtissues for diverse biofabrication applications. Impact statement This study addresses a key challenge facing emerging modular biofabrication strategies that use microtissues as biological building blocks. Namely, achieving the necessary robust and consistent differentiation of clinically relevant cell sources, for example, mesenchymal stem/stromal cells (MSCs), and the accumulation of sufficient tissue-specific extracellular matrix (ECM) to engineer tissue of scale. We achieved this by establishing hydrocortisone as a simple and potent method for improving MSC chondrogenesis, resulting in the biofabrication of high-quality (ECM rich) cartilage microtissues. These findings could enable the generation of more scalable engineered cartilage by ensuring the formation of high-quality microtissue building blocks generated using heterogeneous MSC populations.

In the peripheral nervous system, Schwann cells (SCs) play a crucial role in axonal growth, metabolic support of neurons, and the production of myelin sheaths. Expansion of SCs after extraction from human or animal nerves is a long and often low-yielding process. We established a rapid cell culture method using a defined serum-free medium to differentiate human induced pluripotent stem cells (iPSCs) into SCs in only 21 days. The SC identity was characterized by expression of SRY-Box Transcription factor 10 (SOX10), S100b, glial fibrillary acidic protein (GFAP), P75, growth-associated protein 43 (GAP43), and early growth response 2 (EGR2) markers. The SC purity reached 87% as assessed by flow cytometry using the specific SOX10 marker, and 69% based on S100b expression. When SCs were cocultured with iPSC-derived motor neurons two-dimensionally or three-dimensionally (3D), they also expressed the markers of myelin MBP, MPZ, and gliomedin. Likewise, when they were seeded on the opposite side of a porous collagen sponge from motor neurons in the 3D model, they were able to migrate through it and colocalize with motor axons after 8 weeks of maturation. Moreover, they were shown by transmission electron microscopy to form myelin sheaths around motor axons. These results suggest that the use of autologous iPSC-derived SCs for clinical applications such as the repair of peripheral nerve damage, the treatment of spinal cord injuries, or for demyelinating diseases could be a valuable option. Impact Statement Peripheral nerve injuries can cause the complete paralysis of the upper or lower limbs, which considerably reduces the quality of life of patients. To repair this injury, many approaches have been developed by tissue engineering. Combining biomaterials with Schwann cells (SCs) has been shown to be an effective solution for stimulating nerve regeneration. However, the challenge faced concerns the strategy for obtaining autologous SCs to treat patients. A promising approach is to differentiate them from the patient's own cells, previously induced into pluripotent stem cells. We propose a fast culture method to generate functional SCs differentiated from induced pluripotent stem cells.

AdipoRon is an adiponectin receptor 1, 2 (ADIPOR1 and ADIPOR2) agonist with numerous reported physiological benefits in murine models of human disease, including a proposed reduction in fibrosis. However, AdipoRon has never been investigated in rabbits, which provide a robust model for orthopedic conditions. We examined the safety of intravenous (IV) AdipoRon in New Zealand White (NZW) female rabbits surgically stressed by a procedure that mimics human arthrofibrosis. Fifteen female NZW rabbits were prospectively studied using increasing AdipoRon doses based on established literature. AdipoRon was dissolved in dimethyl sulfoxide (DMSO), diluted in normal saline, and administered IV preoperatively and for 5 subsequent days postoperatively. The primary outcome was overall toxicity to rabbits, whereas secondary outcomes were change in rabbit weights and hemodynamics and defining acid-base characteristics of the drug formulation. Two rabbits expired during preoperative drug administration at 25 mg/kg. Remaining rabbits received preoperative doses of DMSO (vehicle), 2.5, 5, or 10 mg/kg of AdipoRon without complications. On postoperative day 1, one rabbit sustained a tonic-clonic seizure after their second dose of 10 mg/kg AdipoRon. The remaining 12 rabbits (4 in each group) received six serial doses of vehicle, 2.5, or 5 mg/kg of AdipoRon without adverse effects. All formulations of AdipoRon were within safe physiological pH ranges (4-5). We are the first to report the use of IV AdipoRon in a surgically stressed rabbit model of orthopedic disease. AdipoRon doses of 5 mg/kg or less appear to be well-tolerated in female NZW rabbits. Impact statement We provided the first in vivo toxicity assessment and dose optimization of a new antifibrotic experimental medication, AdipoRon, in a surgically stressed rabbit model of knee arthrofibrosis.

Purpose: There is still a lack of effective treatments for cartilage damage. Cartilage tissue engineering could be a promising treatment method. Human umbilical cord Wharton's jelly (HUCWJ) and hydrogels have received wide attention as a scaffold for tissue engineering. They have not been widely used in clinical studies as their effectiveness and safety are still controversial. This study systematically compared the ability of these two biological tissue engineering materials to carry chondrocytes to repair cartilage injury in vivo. Methods: Chondrocytes were cocultured with HUCWJ or hydrogel for in vivo transplantation. The treatments comprised the HUCWJ+cell, hydrogel+cell, and blank groups. A rabbit model with articular cartilage defect in the knee joint area was established. The defective knee cartilage of different rabbit groups was treated for 3 and 6 months. The efficacy of the various treatments on articular cartilage injury was evaluated by immunohistochemistry and biochemical indices. Results: We found that the HUCWJ+cell and hydrogel+cell groups promoted cartilage repair compared with the blank group, which had no repair effect. The treatment efficacy of each group at 6 months was significantly better than that at 3 months. HUCWJ showed accelerated cartilage repair ability than the hydrogel. Conclusion: This study showed that HUCWJ is useful in cartilage tissue engineering to enhance the efficacy of chondrocyte-based cartilage repair, providing new insights for regenerative medicine. Impact statement Human umbilical cord Wharton's jelly (HUCWJ) and hydrogel are the suitable extracellular matrix for cartilage tissue engineering. This study assessed the capacity of HUCWJ- and hydrogel-loaded chondrocytes to repair cartilage injury in vivo. The data demonstrate that both HUCWJ and hydrogel effectively facilitated cartilage repair, and the repair effects of HUCWJ were significantly better compared with hydrogel, therefore providing a potential candidate for clinical practice of cartilage regeneration therapy.

Three-dimensional (3D) bioprinting demonstrates technology that is capable of producing structures comparable to native tissues in the human body. The freeform reversible embedding of suspended hydrogels (FRESH) technique involves hydrogel-based bio-inks printed within a thermo-reversible support bath to provide mechanical strength to the printed construct. Smaller and more uniform microsphere sizes of FRESH were reported to aid in enhancing printing resolution and construct accuracy. Therefore, we sought to optimize the FRESH generation protocol, particularly by varying stir speed and stir duration, in hopes to further improve microsphere size and uniformity. We observed optimal conditions at a stir speed of 600 rpm and stir duration for 20 h that generated the smallest microspheres with the best uniformity. Comparison of using the optimized FRESH to the commercial FRESH LifeSupport to bioprint single filament and geometrical constructs revealed reduced single filament diameters and higher angular precision in the optimized FRESH bio-printed constructs compared with those printed in the commercial FRESH. Overall, our refinement of the FRESH manufacturing protocol represents an important step toward enhancing 3D bioprinting resolution and construct fidelity. Improving such technologies allows for the fabrication of highly accurate constructs with anatomical properties similar to native counterparts. Such work has significant implications in the field of tissue engineering for producing accurate human organ model systems. Impact statement Freeform reversible embedding of suspended hydrogels (FRESH) is a method of sacrificial three-dimensional (3D) bioprinting that offers support to reinforce bio-ink extrusion during printing. During FRESH generation, the stir speed and stir duration of the mixture can significantly impact FRESH microsphere characteristics. In this study, we optimized FRESH microspheres to significantly improve resolution and accuracy in bioprinting. This advancement in FRESH-based 3D bioprinting technologies allows for the fabrication of highly accurate constructs with anatomical properties similar to native counterparts and has significant implications in the field of tissue engineering and translational medicine.

The respiratory tract is one of the frontline barriers for biological defense. Lung epithelial intercellular adhesions provide protection from bacterial and viral infections and prevent invasion into deep tissues by pathogens. Dysfunction of lung epithelial intercellular adhesion caused by pathogens is associated with development of several diseases, such as acute respiratory distress syndrome, pneumonia, and asthma. To elucidate the pathological mechanism of respiratory infections, two-dimensional cell cultures and animal models are commonly used, although are not useful for evaluating host specificity or human biological response. With the rapid progression and worldwide spread of severe acute respiratory syndrome coronavirus-2, there is increasing interest in the development of a three-dimensional (3D) in vitro lung model for analyzing interactions between pathogens and hosts. However, some models possess unclear epithelial polarity or insufficient barrier functions and need the use of complex technologies, have high cost, and long cultivation terms. We previously reported about the fabrication of 3D cellular multilayers using a layer-by-layer (LbL) cell coating technique with extracellular matrix protein, fibronectin (FN), and gelatin (G). In the present study, such a LbL cell coating technique was utilized to construct a human 3D lung model in which a monolayer of the human lower airway epithelial adenocarcinoma cell line Calu-3 cells was placed on 3D-cellular multilayers composed of FN-G-coated human primary pulmonary fibroblast cells. The 3D lung model thus constructed demonstrated an epithelial-fibroblast layer that maintained uniform thickness until 7 days of incubation. Moreover, expressions of E-cadherin, ZO-1, and mucin in the epithelial layer were observed by immunohistochemical staining. Epithelial barrier integrity was evaluated using transepithelial electrical resistance values. The results indicate that the present constructed human 3D lung model is similar to human lung tissues and also features epithelial polarity and a barrier function, thus is considered useful for evaluating infection and pathological mechanisms related to pneumonia and several pathogens. Impact statement A novel in vitro model of lung tissue was established. Using a layer-by-layer cell coating technique, a three-dimensional cultured lung model was constructed. The present novel model was shown to have epithelial polarity and chemical barrier functions. This model may be useful for investigating interaction pathogens and human biology.

Local tumor treatment is a feasible measure for patients with glioblastoma (GBM) who are unsuitable for surgical resection. Interferon-elastin-like polypeptide [IFN-ELP(V)] is a slow-release, biodegradable, thermosensitive fusion protein with antitumor immunity, and resveratrol (Res) is a polyphenolic compound with an antitumor effect. In this study, we found that intratumor injection of IFN-ELP(V) combined with intraperitoneal injection of Res is more effective in delaying GBM growth in mice. Specifically, in an orthotopic GBM model, we found a significant improvement in the median survival with this strategy. Our results suggested that the combined use of IFN-ELP(V) and Res has a dramatic synergistic effect on GBM, thus providing a novel and effective therapeutic strategy for tumors. Impact statement We report a novel and effective strategy in which the combined use of interferon-elastin-like polypeptide [IFN-ELP(V)] and Res effectively inhibits glioblastoma growth. IFN-ELP(V) can create a reservoir in the tumor and continuously release IFN to produce a powerful in situ antitumor immune response; furthermore, the combination of IFN-ELP(V) and Res is more effective in inhibiting tumor growth.