Cross-linked polymer blends from natural compounds, namely gelatin (Gel), chitosan (CS), and synthetic poly (vinyl alcohol) (PVA), have received increasing scrutiny because of their versatility, ...biocompatibility, and ease of use for tissue engineering. Previously, Gel/CS/PVA 1:1:1 hydrogel produced via the freeze-drying process presented enhanced mechanical properties. This study aimed to investigate the biocompatibility and chondrogenic potential of a steam-sterilized Gel/CS/PVA hydrogel using differentiation of human adipose-derived mesenchymal stromal cells (AD-hMSC) and cartilage marker expression. AD-hMSC displayed fibroblast-like morphology, 90% viability, and 69% proliferative potential. Mesenchymal profiles CD73 (98.3%), CD90 (98.6%), CD105 (97.0%), CD34 (1.11%), CD45 (0.27%), HLA-DR (0.24%); as well as multilineage potential, were confirmed. Chondrogenic differentiation of AD-hMSC in monolayer revealed the formation of cartilaginous nodules composed of glycosaminoglycans after 21 days. Compared to nonstimulated cells, hMSC-derived chondrocytes shifted the expression of CD49a from 2.82% to 40.6%, CD49e from 51.4% to 92.2%, CD54 from 9.66 to 37.2%, and CD151 from 45.1% to 75.8%. When cultured onto Gel/CS/PVA hydrogel during chondrogenic stimulation, AD-hMSC changed to polygonal morphology, and chondrogenic nodules increased by day 15, six days earlier than monolayer-differentiated cells. SEM analysis showed that hMSC-derived chondrocytes adhered to the surface with extended filopodia and abundant ECM formation. Chondrogenic nodules were positive for aggrecan and type II collagen, two of the most abundant components in cartilage. This study supports the biocompatibility of AD-hMSC onto steam-sterilized GE/CS/PVA hydrogels and its improved potential for chondrocyte differentiation. Hydrogel properties were not altered after steam sterilization, which is relevant for biosafety and biomedical purposes.
Three-dimensional (3D) hydrogels provide tissue-like complexities and allow for the spatial orientation of cells, leading to more realistic cellular responses in pathophysiological environments. ...There is a growing interest in developing multifunctional hydrogels using ternary mixtures for biomedical applications. This study examined the biocompatibility and suitability of human auricular chondrocytes from microtia cultured onto steam-sterilized 3D Chitosan/Gelatin/Poly(Vinyl Alcohol) (CS/Gel/PVA) hydrogels as scaffolds for tissue engineering applications. Hydrogels were prepared in a polymer ratio (1:1:1) through freezing/thawing and freeze-drying and were sterilized by autoclaving. The macrostructure of the resulting hydrogels was investigated by scanning electron microscopy (SEM), showing a heterogeneous macroporous structure with a pore size between 50 and 500 μm. Fourier-transform infrared (FTIR) spectra showed that the three polymers interacted through hydrogen bonding between the amino and hydroxyl moieties. The profile of amino acids present in the gelatin and the hydrogel was determined by ultra-performance liquid chromatography (UPLC), suggesting that the majority of amino acids interacted during the formation of the hydrogel. The cytocompatibility, viability, cell growth and formation of extracellular matrix (ECM) proteins were evaluated to demonstrate the suitability and functionality of the 3D hydrogels for the culture of auricular chondrocytes. The cytocompatibility of the 3D hydrogels was confirmed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, reaching 100% viability after 72 h. Chondrocyte viability showed a high affinity of chondrocytes for the hydrogel after 14 days, using the Live/Dead assay. The chondrocyte attachment onto the 3D hydrogels and the formation of an ECM were observed using SEM. Immunofluorescence confirmed the expression of elastin, aggrecan and type II collagen, three of the main components found in an elastic cartilage extracellular matrix. These results demonstrate the suitability and functionality of a CS/Gel/PVA hydrogel as a 3D support for the auricular chondrocytes culture, suggesting that these hydrogels are a potential biomaterial for cartilage tissue engineering applications, aimed at the regeneration of elastic cartilage.
(1) Background: Currently, there are no pharmacological treatments that can modify the course of osteoarthritis (OA). For this reason, the present work is focused on generating knowledge for the ...development of new therapeutic alternatives for the treatment of OA. The objective of this work was to develop an articular hybrid implant with mesenchymal stem cells (MSCs) from sheep. The cells were differentiated into cartilage and bone using a bioabsorbable polymer with 3D printing Technology. (2) Methods: MSCs pre-differentiated to chondrocytes and osteoblasts were seeded on the 3D-printed scaffolds using polylactic acid (PLA). These were later implanted for 3 months in the thoracic ribs area and for 6 months inside the femoral head and outside of the joint capsule. After recovery, we analyzed the expressions of specific markers for bone and cartilage in the implants (3) Results: After 3 months, in lateral implants, the expression for bone markers (OPN, RUNX2) was similar to that of the control; at 6 months, we obtained a higher expression of bone markers in the implants with pre-differentiated MCS to osteoblasts outside and inside the joint. For cartilage markers, three months after the placement of the lateral implant, the expressions of Aggrecan and SOX9 COL2A1 were similar to those of the control, but the expression of COL2A1 was less; at 6 months, the three cartilage markers SOX9, Aggrecan, and COL2A1 showed significant expressions in the implant inside joint with pre-differentiated MCS to chondrocytes. (4) Conclusions: In this study, we demonstrated that the presence of pre-differentiated MSCs in the implants was a determinant factor for the expression of bone- and cartilage-specific markers at three and six months. We managed to generate a practical and easy-to-implement articular surface repair model.
Background
Complex meniscal lesions often require meniscectomy with favorable results in the short term but a high risk of early osteoarthritis subsequently. Partial meniscectomy treated with ...meniscal substitutes may delay articular cartilage degeneration.
Purpose
To evaluate the status of articular cartilage by T2 mapping after meniscal substitution with polyurethane scaffolds enriched with mesenchymal stem cells (MSC) and comparison with acellular scaffolds at 12 months.
Methods
Seventeen patients (18-50 years) with past meniscectomies were enrolled in 2 groups: (1) acellular polyurethane scaffold (APS) or (2) polyurethane scaffold enriched with MSC (MPS). Patients in the MPS group received filgrastim to stimulate MSC production, and CD90+ cells were obtained and cultured in the polyurethane scaffold. The scaffolds were implanted arthroscopically into partial meniscus defects. Concomitant injuries (articular cartilage lesions or cartilage lesions) were treated during the same procedure. Changes in the quality of articular cartilage were evaluated with T2 mapping in femur and tibia at 12 months.
Results
In tibial T2 mapping, values for the MPS group increased slightly at 9 months but returned to initial values at 12 months (P > 0.05). In the APS group, a clear decrease from 3 months to 12 months was observed (P > 0.05). This difference tended to be significantly lower in the APS group compared with the MPS group at the final time point (P = 0.18). In the femur, a slight increase in the MPS group (47.8 ± 3.4) compared with the APS group (45.3 ± 4.9) was observed (P > 0.05).
Conclusion
Meniscal substitution with polyurethane scaffold maintains normal T2 mapping values in adjacent cartilage at 12 months. The addition of MSC did not show any advantage in the protection of articular cartilage over acellular scaffolds (P > 0.05).
Objective. To evaluate minimum biosecurity parameters (MBP) for arthroscopic matrix-encapsulated autologous chondrocyte implantation (AMECI) based on patients’ clinical outcomes, magnetic resonance ...imaging (MRI) T2-mapping, Magnetic Resonance Observation of Cartilage Repair Tissue (MOCART) score, and International Cartilage Repair Society (ICRS) second-look arthroscopic evaluation, laying the basis for a future multicenter study. Design. Pilot clinical study. We analyzed the logistics to perform AMECI to treat focal chondral lesions in different hospitals following strict biosecurity parameters related to tissue and construct transportation, chondrocyte isolation, and cell expansion. Patient progress was analyzed with patient-reported outcome measures, MRI T2-mapping, MOCART, and ICRS arthroscopic second-look evaluation. Results. Thirty-five lesions in 30 patients treated in 7 different hospitals were evaluated. Cell viability before implantation was >90%. Cell viability in construct remnants was 87% ± 11% at 24 hours, 75% ± 17.1% at 48 hours, and 60% ± 8% at 72 hours after implantation. Mean final follow-up was 37 months (12-72 months). Patients showed statistically significant improvement in all clinical scores and MOCART evaluations. MRI T2-mapping evaluation showed significant decrease in relaxation time from 61.2 ± 14.3 to 42.9 ± 7.2 ms (P < 0.05). Arthroscopic second-look evaluation showed grade II “near normal” tissue in 83% of patients. Two treatment failures were documented. Conclusions. It was feasible to perform AMECI in 7 different institutions in a large metropolitan area following our biosecurity measures without any implant-related complication. Treated patients showed improvement in clinical, MRI T2-mapping, and MOCART scores, as well as a low failure rate and a favorable ICRS arthroscopic evaluation at a mid-term follow-up. Level of Evidence. 2b.
Interest in novel delivery systems that improve the cytoprotective and antioxidant effects of natural drugs has been explored recently due to the increase in the incidence of chronic diseases in ...which oxidation mechanisms are involved. Curcumin is a phenolic compound recently shown to be clinically significant due to its anti-inflammatory, anticancer, and antioxidant properties. However, this molecule possesses a low bioavailability and a high degradation rate in the presence of light. Therefore, we prepared nanoparticles of poly-ε-caprolactone and Pluronic® F-68 as a stabilizer and loaded these with curcumin (Cur–PCL nanoparticles) for antioxidant and cytoprotective applications. The nanoparticles did not induce cell death, but they did reduce cell proliferation without affecting cell migration and cell adhesion. Interestingly, Cur–PCL and poly-ε-caprolactone nanoparticles reduced the oxidative stress induced by hydrogen peroxide and presented a cytoprotective effect. Remarkably, poly-ε-caprolactone nanoparticles showed a decrement of 30% in reactive oxygen species presence compared to the positive control. The decrease of reactive oxygen species derived from the administration of poly-ε-caprolactone nanoparticles could be attributed to the presence of Pluronic® F-68. Taken together, these data indicated that these nanoparticles might have a clinical application in disorders related to reactive oxygen species formation.
Mobilized peripheral blood (MPB) bone marrow cells possess the potential to differentiate into a variety of mesenchymal tissue types and offer a source of easy access for obtaining stem cells for the ...development of experimental models with applications in tissue engineering. In the present work, we aimed to isolate by magnetic activated cell sorting CD90+ cells from MPB by means of the administration of Granulocyte-Colony Stimulating Factor and to evaluate cell proliferation capacity, after thawing of the in vitro culture of this population of mesenchymal stem cells (MSCs) in sheep. We obtained a median of 8.2 ± 0.6 million of CD90+ cells from the 20-mL MPB sample. After thawing, at day 15 under in vitro culture, the mean CD90+ cells determined by flow cytometry was 92.92 ± 1.29 % and cell duplication time determined by crystal violet staining was 47.59 h. This study describes for the first time the isolation, characterization, and post-in vitro culture thawing of CD90+ MSCs from mobilized peripheral blood in sheep. This population can be considered as a source of MSCs for experimental models in tissue engineering research.
Domingo de Ramos, or Palm Sunday, is a traditional Christian religious event where devotees use ramos, which are bouquets currently elaborated from palm leaves and other natural elements. In various ...countries, it is assumed this use of biodiversity leads to the depletion of the species involved. However, other important aspects must be considered, including the role of the people who produce and sell these ramos, the associated symbolism that has been overlooked, as well as commercial aspects that have barely been documented. This ethnobotanical study evaluates the regional-scale cultural, biological and socioeconomic aspects associated with Domingo de Ramos in central Mexico from an emic perspective.
Ethnographic and commercial information was obtained through interviews with ramos sellers in 28 municipalities in the state of Hidalgo, Mexico. We specifically sought sociodemographic data regarding the interviewees, as well as information pertaining to the ramos themselves and the palms. These aspects were explored with all of the sellers. The free list method was used to describe the uses and key elements associated with the ramos.
Although the ramos are used for religious purposes, they have eight different uses in the daily life of the sellers, the main one being "protection." They serve to protect families, crops and animals, as well as against several diseases. Likewise, they are considered valuable for diminishing strong storms. This belief in the protection conferred by the ramos preserves pre-Hispanic concepts and is combined with their use in blessing corresponding to Western beliefs. Ramos are made from 35 introduced and native plant species and comprise a base (made of palm, wheat or sotol), a "reliquia" (palm, rosemary, chamomile and laurel) and natural or artificial flowers. The ramos sellers are mostly adult women of indigenous origin and heads of family.
This study of Domingo de Ramos, carried out at a regional scale, highlights a syncretism that is reflected in both the symbolic importance of ramos palm and in the species used, as well as socioeconomic aspects that had not previously been identified in the study area and reflect the occurrence of complex relationships in non-timber forest products that remain little addressed.
Radiosterilized pig skin (RPS) has been used as a dressing for burns since the 1980s. Its similarity to human skin in terms of the extracellular matrix (ECM) allows the attachment of mesenchymal stem ...cells, making it ideal as a scaffold to create cellularized constructs. The use of silver nanoparticles (AgNPs) has been proven to be an appropriate alternative to the use of antibiotics and a potential solution against multidrug-resistant bacteria. RPS can be impregnated with AgNPs to develop nanomaterials capable of preventing wound infections. The main goal of this study was to assess the use of RPS as a scaffold for autologous fibroblasts (Fb), keratinocytes (Kc), and mesenchymal stem cells (MSC) in the treatment of second-degree burns (SDB). Additionally, independent RPS samples were impregnated with AgNPs to enhance their properties and further develop an antibacterial dressing that was initially tested using a burn mouse model. This protocol was approved by the Research and Ethics Committee of the INRLGII (INR 20/19 AC). Transmission electron microscopy (TEM) and dynamic light scattering (DLS) analysis of the synthesized AgNPs showed an average size of 10 nm and rounded morphology. Minimum inhibitory concentrations (MIC) and Kirby–Bauer assays indicated that AgNPs (in solution at a concentration of 125 ppm) exhibit antimicrobial activity against the planktonic form of S. aureus isolated from burned patients; moreover, a log reduction of 1.74 ± 0.24 was achieved against biofilm formation. The nanomaterial developed with RPS impregnated with AgNPs solution at 125 ppm (RPS-AgNPs125) facilitated wound healing in a burn mouse model and enhanced extracellular matrix (ECM) deposition, as analyzed by Masson’s staining in histological samples. No silver was detected by energy-dispersive X-ray spectroscopy (EDS) in the skin, and neither by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) in different organs of the mouse burn model. Calcein/ethidium homodimer (EthD-1), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and scanning electron microscopy (SEM) analysis demonstrated that Fb, Kc, and MSC could attach to RPS with over 95% cell viability. Kc were capable of releasing FGF at 0.5 pg above control levels, as analyzed by ELISA assays. An autologous RPS-Fb-Kc construct was implanted in a patient with SDB and compared to an autologous skin graft. The patient recovery was assessed seven days post-implantation, and the patient was followed up at one, two, and three months after the implantation, exhibiting favorable recovery compared to the gold standard, as measured by the cutometer. In conclusion, RPS effectively can be used as a scaffold for the culture of Fb, Kc, and MSC, facilitating the development of a cellularized construct that enhances wound healing in burn patients.
Psoriasis and atopic dermatitis (AD) are characterized by enhanced skin inflammation, which results in hyperproliferation and the recruitment of immune cells into the skin. For that reason, it is ...needed a chemical capable to reduce cell proliferation and the recruitment of cells. The search for new molecules for therapeutic skin treatment mainly focuses on the antioxidant and anti-inflammatory properties, highlighting the rheological properties of polymeric polypeptides. We studied L-arginine (L-Arg) grafted (-g-) to enzymatic poly(gallic acid) (PGAL). The latter is a multiradical antioxidant with greater properties and thermal stability. The derivative was enzymatically polymerized in an innocuous procedure. The poly(gallic acid)-g-L-Arg molecule (PGAL-g-L-Arg) inhibits bacterial strains which also have been involved in the progression of psoriasis and AD. However, it is important to analyze their biological effect on skin cells. The cell viability was analyzed by calcein/ethidium homodimer assays and crystal violet. The proliferation and cell attachment were determined by a curve of time and quantitation of the optical density of crystal violet. To analyze the cell migration a wound-healing assay was performed. This synthesis demonstrates that it is not cytotoxic at high concentrations (250 μg/mL). We observed a decrease in the proliferation, migration, and adhesion of dermal fibroblasts in vitro but the compound could not avoid the increase of reactive oxygen species in the cell. Based on our findings, PGAL-g-L-Arg is a promising candidate for treating skin diseases such as psoriasis and AD where decreasing the proliferation and cell migration could help to avoid inflammation.
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