Tissue engineering. Part C, Methods最新文献

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.

Primary human hepatocytes isolated from surgically resected liver tissue are an essential resource for pharmaceutical and toxicological studies. Patients undergoing partial liver resections have often received preoperative chemotherapy. The aim of our study was to investigate whether preoperative chemotherapy has effects on the outcome of cell isolation or the metabolic function of cultured hepatocytes. Liver specimens from 48 patients were used for hepatocyte isolation. Out of these, 21 patients had prior chemotherapy, with fluoropyrimidine-based regimen in 14 patients. Viability and cell yield as parameter for the outcome of isolation, as well as transaminase levels, urea or albumin secretion to the culture medium were not different between hepatocytes from pretreated and untreated donor. Furthermore, the transcription levels of cytochrome P450 (CYP) 1A2, CYP 2B6, and CYP 3A4 of cultured hepatocytes were not affected by prior chemotherapy of the tissue donors. In conclusion, hepatocytes from tissue donors that underwent fluoropyrimidine-based chemotherapy regimens before isolation seem to perform as well as hepatocytes without preoperative chemotherapy exposure. Our results suggest that hepatocytes from patients who received combination chemotherapy before liver resection are an uncompromised resource for pharmacological and toxicological studies. Impact statement Isolated primary human hepatocytes are an essential resource for pharmacological and toxicological studies. Our results present further evidence that isolated hepatocytes from patients who received combination chemotherapy before liver resection are an uncompromised resource for pharmacological and toxicological studies-especially when fluoropyrimidine-based regimens are used.

Mesenchymal stem cells (MSCs) stimulate nerve and tissue regeneration and are primed for clinical translation. Application of autologous MSCs is limited by requirements for expedient harvesting procedures, proliferative expansion to increase number of cells, and reduced regenerative potential due to aging or pathological conditions. Because MSCs are immune privileged, allogeneic MSCs may serve as "off-the-shelf" cell-based reconstructive treatments to support nerve repair. Therefore, we examined the safety and immune response parameters of allogeneic MSCs seeded on NeuraGen^® Nerve Guides (NNGs) in a rabbit model. NNGs with or without allogeneic rabbit MSCs were applied to rabbit sciatic nerves. Randomly assigned treatment included group I (no surgery control, n = 3) or groups II and III (sciatic nerve wrapped with unseeded or allogeneic MSC-seeded NNGs; n = 5/group). Rabbits were euthanized after 2 weeks to monitor functional recovery by histological evaluation of sciatic nerves and tibialis anterior (TA) muscle. Host reactions to allogeneic MSCs were analyzed by assessment of body and tissue weight, temperature, as well as hematological parameters, including white blood cell count (WBC), spleen histology, and CD4⁺ and CD8⁺ T lymphocytes. Histological analyses of nerves and spleen were all unremarkable, consistent with absence of overt systemic and local immune responses upon allogeneic MSC administration. No significant differences were observed in WBC or CD4⁺ and CD8⁺ T lymphocytes across unseeded and seeded treatment groups. Thus, allogenic MSCs are safe for use and may be considered in lieu of autologous MSCs in translational animal studies as the basis for future clinical nerve repair strategies. Impact statement Autologous mesenchymal stem cells (MSC) have been reported to enhance nerve regeneration when used in conjunction with nerve graft substitutes. However, autologous stem cell sources delay treatment and may be susceptible to age- or disease-related dysfunctions. In this study, we investigated the safety of allogeneic MSCs and the optimal number of cells for nerve conduit delivery in a rabbit model. When compared with unseeded nerve conduits, allogeneic MSC-seeded conduits did not induce a systemic or local immune response. The findings of this study will ultimately facilitate the clinical translation of a universal donor cell-based treatment option for nerve defects.

Chronic kidney disease (CKD) is the irreversible loss of nephron function, leading to a build-up of toxins, prolonged inflammation, and ultimately fibrosis. Currently, no effective therapies exist to treat CKD due to its complex pathophysiology. Mesenchymal stem/stromal cell (MSC) transplantation is a promising strategy to treat kidney diseases, and multiple clinical trials are currently ongoing. We previously demonstrated that rat bone marrow-derived MSC (BMSC) sheets transplanted onto surgically decapsulated kidney exert therapeutic effects that suppressed renal fibrosis progression based on enhanced vascularization. However, there are clinical concerns about kidney decapsulation such as impaired glomerular filtration rate and Na⁺ ion and H₂O excretion, leading to kidney dysfunction. Therefore, for transitioning from basic research to translational research using cell sheet therapy for kidney disease, it is essential to develop a new cell sheet transplantation strategy without kidney decapsulation. Significantly, we employed cell sheets engineered from clinical-grade human clonal BMSC (cBMSC) and transplanted these onto intact renal capsule to evaluate their therapeutic ability in the rat ischemia-reperfusion injury (IRI) model. Histological analysis 1-day postsurgery showed that cBMSC sheets engrafted well onto intact renal capsule. Interestingly, some grafted cBMSCs migrated into the renal parenchyma. At 1-3 days postsurgery (acute stage), grafted cBMSC sheets prevented tubular epithelial cell injury. At 28 days postsurgery (chronic phase), we observed that grafted cBMSC sheets suppressed renal fibrosis in the rat IRI model. Taken together, engineered cBMSC sheet transplantation onto intact renal capsule suppresses tubular epithelial cell injury and renal fibrosis, supporting further development as a possible clinically relevant strategy. Impact statement Chronic kidney disease (CKD) produces irreversible loss of nephron function, leading to toxemia, prolonged inflammation, and ultimately kidney fibrosis. Currently, no therapies exist to effectively treat CKD due to its complex pathophysiology. Mesenchymal stem/stromal cells (MSCs) are widely known to secret therapeutic paracrine factors, which is expected to provide a new effective therapy for unmet medical needs. However, unsatisfied MSC quality and administration methods to patients limit their therapeutic effects. In this study, we engineered clonal bone marrow-derived MSC sheets and established clinically relevant cell sheet transplantation strategy to treat renal fibrosis, which would improve MSC treatment for kidney disease.