Tissue Engineering Part C: Methods

Transforming growth factor-beta 1 (TGF-β1) has been reported to promote chondrogenic differentiation and proliferation in the multipotent stromal cell (MSCs), and the transforming growth factor-beta 3 (TGF-β3) tends to be exclusively in promoting cell differentiation alone. The objective of this study was to determine the effect of TGF-β1 and -β3 on the MSCs chondrogenic differentiation on the poly (vinyl alcohol)-chitosan-poly (ethylene glycol) (PVA-NOCC-PEG) scaffold, compared with that of monolayer and pellet cultures. In this study, P2 rabbit bone marrow-derived MSCs were seeded either on the untreated six-well plate (for monolayer culture) or onto the PVA-NOCC-PEG scaffold or cultured as a pellet culture. The cultures were maintained in a chemically defined serum-free medium supplemented with 10 ng/mL of either TGF-β1 or TGF-β3. Cell viability assay, biochemical assay, and real-time polymerase chain reaction were performed to determine the net effect of cell proliferation and chondrogenic differentiation of each of the growth factors. The results showed that the PVA-NOCC-PEG scaffold enhanced MSCs cell proliferation from day 12 to 30 (p < 0.05); however, no significant differences were observed in the cell proliferation between the cultures supplemented with or without TGF-β1 and TGF-β3 (p > 0.05). In terms of chondrogenic differentiation, the PVA-NOCC-PEG scaffold augmented the GAGs secretion in MSCs and the mRNA expression levels of Sox9, Col2a1, Acan, and Comp were elevated (p < 0.05). However, there was no significant difference between both the TGF-β1 and TGF-β3-treated groups (p > 0.05). In conclusion, TGF-β1 and TGF-β3 enhanced the chondrogenic differentiation of MSCs seeded on the PVA-NOCC-PEG scaffold; however, there was no significant difference between the effect of TGF-β1 and TGF-β3.

The state of host cells is reflected in the cargo carried by their extracellular vesicles (EVs). This makes EV a potential source of biomarkers for human diseases. Piwi-interacting RNA (piRNA) regulates gene expression through epigenetic regulation and post-transcriptional gene silencing. Thus, piRNA profiling in EVs derived from human clinical samples could identify markers that characterize disease stages and unveil their roles in disease pathology. This review aimed to report the expression profiles of EV-derived piRNA (EV-piRNA) in various human samples, as well as their role in each pathology. A systematic review was conducted to collate the findings of human EV-piRNA from original research articles published in indexed scientific journals up to February 16, 2022. Article searches were performed in PubMed, Web of Science, and Scopus databases, using a combination of keywords, including “EV” and “piRNA.” A total of 775 nonredundant original articles were identified. After subjecting articles to inclusion and exclusion criteria, 34 articles were accepted for this review. The piRNA expression levels among the small RNA profiles of human-derived EVs range from 0.09% to 43.84%, with the lowest expression level reported in urine-derived EVs and the highest percentage in plasma-derived EVs. Differentially expressed EV-piRNAs have been identified in patients with specific disease conditions compared to their counterparts (healthy control), suggesting an association between piRNA and progression in various diseases. Seven articles identified piRNA putative target genes and/or the pathway enrichment of piRNA target genes, and one study demonstrated a direct role of piRNA candidates in disease pathology. In conclusion, EV-piRNA has been isolated successfully from various human body fluids. EV-piRNA is a new research niche in human disease pathology. The expression profiles of EV-piRNA in various tissue types and disease conditions remain largely unexplored. Furthermore, there is currently a lack of guidelines on piRNA bioinformatics analysis, which could lead to inconsistent results and thus hinder the progression of piRNA discoveries. Finally, the lack of published scientific evidence on the role of EV-piRNA supports the need for future research to focus on the functional analysis of EV-piRNA as part of the route in piRNA discoveries.

Human hair is a potential biomaterial for biomedical applications. Improper disposal of human hair may pose various adverse effects on the environment and human health. Therefore, proper management of human hair waste is pivotal. Human hair fiber and its derivatives offer various advantages as biomaterials such as biocompatibility, biodegradability, low toxicity, radical scavenging, electroconductivity, and intrinsic biological activity. Therefore, the favorable characteristics of human hair have rendered its usage in tissue engineering (TE) applications including skin, cardiac, nerve, bone, ocular, and periodontal. Moreover, the strategies by utilizing human hair as a biomaterial for TE applications may reduce the accumulation of human hair. Thus, it also improves human hair waste management while promoting natural, environmental-friendly, and nontoxic materials. Furthermore, promoting sustainable materials production will benefit human health and well-being. Hence, this article reviews and discusses human hair characteristics as sustainable biomaterials and their recent application in TE applications.

Caffeine is therapeutically effective for treating apnea, cellulite formation, and pain management. It also exhibits neuroprotective and antioxidant activities in different models of Parkinson's disease and Alzheimer's disease. However, caffeine administration in a minimally invasive and sustainable manner through the transdermal route is challenging owing to its hydrophilic nature. Therefore, this study demonstrated a transdermal delivery approach for caffeine by utilizing hydrogel microneedle (MN) as a permeation enhancer. The influence of formulation parameters such as molecular weight (MW) of PMVE/MA (polymethyl vinyl ether/maleic anhydride) copolymer and sodium bicarbonate (NaHCO₃) concentration on the swelling kinetics and mechanical integrity of the hydrogel MNs was investigated. In addition, the effect of different MN application methods and needle densities of hydrogel MN on the skin insertion efficiency and penetration depth was also evaluated. The swelling degree at equilibrium percentage (% S_eq) recorded for hydrogels fabricated with Gantrez S-97 (MW = 1,500,000 Da) was significantly higher than formulation with Gantrez AN-139 (MW = 1,080,000 Da). Increasing the concentration of NaHCO₃ also significantly increased the % S_eq. Moreover, a 100% penetration was recorded for both the applicator and combination of applicator and thumb pressure compared with only 11% for thumb pressure alone. The average diameter of micropores created by the applicator method was 62.94 μm, which was significantly lower than the combination of both applicator and thumb pressure MN application (100.53 μm). Based on histological imaging, the penetration depth of hydrogel MN increased as the MN density per array decreased. The hydrogel MN with the optimized formulation and skin insertion parameters was tested for caffeine delivery in an in vitro Franz diffusion cell setup. Approximately 2.9 mg of caffeine was delivered within 24 h, and the drug release profile was best fitted to the Korsmeyer–Peppas model, displaying Super Case II kinetics. In conclusion, a combination of thumb and impact application methods and reduced needle density improved the skin penetration efficiency of hydrogel MNs. The results also show that hydrogel MNs fabricated from 3% w/w NaHCO₃ and high MW of copolymer exhibit optimum physical and swelling properties for enhanced transdermal delivery.

The use of gelatin microspheres (GMs) as a cell carrier has been extensively researched. One of its limitations is that it dissolves rapidly in aqueous settings, precluding its use for long-term cell propagation. This circumstance necessitates the use of crosslinking agents to circumvent the constraint. Thus, this study examines two different methods of crosslinking and their effect on the microsphere's physicochemical and cartilage tissue regeneration capacity. Crosslinking was accomplished by physical (dehydrothermal [DHT]) and natural (genipin) crosslinking of the three-dimensional (3D) GM. We begin by comparing the microstructures of the scaffolds and their long-term resistance to degradation under physiological conditions (in an isotonic solution, at 37°C, pH = 7.4). Infrared spectroscopy indicated that the gelatin structure was preserved after the crosslinking treatments. The crosslinked GM demonstrated good cell adhesion, viability, proliferation, and widespread 3D scaffold colonization when seeded with human bone marrow mesenchymal stem cells. In addition, the crosslinked microspheres enhanced chondrogenesis, as demonstrated by the data. It was discovered that crosslinked GM increased the expression of cartilage-related genes and the biosynthesis of a glycosaminoglycan-positive matrix as compared with non-crosslinked GM. In comparison, DHT-crosslinked results were significantly enhanced. To summarize, DHT treatment was found to be a superior approach for crosslinking the GM to promote better cartilage tissue regeneration.

Transforming growth factor-beta (TGF-β) is an important regulator of joint homeostasis, of which dysregulation is closely associated with the development of osteoarthritis (OA). In normal conditions, its biological functions in a joint environment are joint protective, but it can be dramatically altered in different contexts, making its therapeutic application a challenge. However, with the deeper insights into the TGF-β functions, it has been proven that TGF-β augments cartilage regeneration by chondrocytes, and differentiates both the precursor cells of chondrocytes and stem cells into cartilage-generating chondrocytes. Following documentation of the therapeutic efficacy of chondrocytes augmented by TGF-β in the last decade, there is an ongoing phase III clinical trial examining the therapeutic efficacy of a mixture of allogeneic chondrocytes and TGF-β-overexpressing cells. To prepare cartilage-restoring chondrocytes from induced pluripotent stem cells (iPSCs), the stem cells are differentiated mainly using TGF-β with some other growth factors. Of note, clinical trials evaluating the therapeutic efficacy of iPSCs for OA are scheduled this year. Mesenchymal stromal stem cells (MSCs) have inherent limitations in that they differentiate into the osteochondral pathway, resulting in the production of poor-quality cartilage. Despite the established essential role of TGF-β in chondrogenic differentiation of MSCs, whether the coordinated use of TGF-β in MSC-based therapy for degenerated cartilage is effective is unknown. We herein reviewed the general characteristics and mechanism of action of TGF-β in a joint environment. Furthermore, we discussed the core interaction of TGF-β with principal cells of OA cell-based therapies, the chondrocytes, MSCs, and iPSCs.