Search Results (135)

Titanium–aluminum–vanadium (Ti6Al4V) is a material chosen for spine, orthopedic, and dental implants due to its combination of desirable mechanical and biological properties. Lasers have been used to modify metal surfaces, enabling the generation of a surface on Ti6Al4V with distinct micro- and nano-scale structures. Studies indicate that topography with micro/nano features of osteoclast resorption pits causes bone marrow stromal cells (MSCs) and osteoprogenitor cells to favor differentiation into an osteoblastic phenotype. This study examined whether the biological response of human MSCs to Ti6Al4V surfaces is sensitive to laser treatment-controlled micro/nano-topography. First, 15 mm diameter Ti6Al4V discs (Spine Wave Inc., Shelton, CT, USA) were either machined (M) or additively manufactured (AM). Surface treatments included no laser treatment (NT), nanosecond laser (Ns), femtosecond laser (Fs), or nanosecond followed by femtosecond laser (Ns+Fs). Surface wettability, roughness, and surface chemistry were determined using sessile drop contact angle, laser confocal microscopy, X-ray photoelectron spectroscopy (XPS), and scanning electron microscopy (SEM). Human MSCs were cultured in growth media on tissue culture polystyrene (TCPS) or test surfaces. On day 7, the levels of osteocalcin (OCN), osteopontin (OPN), osteoprotegerin (OPG), and vascular endothelial growth factor 165 (VEGF) in the conditioned media were measured. M NT, Fs, and Ns+Fs surfaces were hydrophilic; Ns was hydrophobic. AM NT and Fs surfaces were hydrophilic; AM Ns and Ns+Fs were hydrophobic. Roughness (Sa and Sz) increased after Ns and Ns+Fs treatment for both M and AM disks. All surfaces primarily consisted of oxygen, titanium, and carbon; Fs had increased levels of aluminum for both M and AM. SEM images showed that M NT discs had a smooth surface, whereas AM surfaces appeared rough at a higher magnification. Fs surfaces had a similar morphology to their respective NT disc at low magnification, but higher magnification revealed nano-scale bumps not seen on NT surfaces. AM Fs surfaces also had regular interval ridges that were not seen on non-femto laser-ablated surfaces. Surface roughness was increased on M and AM Ns and Ns+Fs disks compared to NT and Fs disks. OCN was enhanced, and DNA was reduced on Ns and Ns+Fs, with no difference between them. OPN, OPG, and VEGF levels for laser-treated M surfaces were unchanged compared to NT, apart from an increase in OPG on Fs. MSCs grown on AM Ns and Ns+Fs surfaces had increased levels of OCN per DNA. These results indicate that MSCs cultured on AM Ns and AM Ns+Fs surfaces, which exhibited unique roughness at the microscale and nanoscale, had enhanced differentiation to an osteoblastic phenotype. The laser treatments of the surface mediated this enhancement of MSC differentiation and warrant further clinical investigation. Full article

Interest in Current Good Manufacturing Practices (cGMP)-grade extracellular vesicles (EVs) is expanding. Some obstacles in this new but rapidly growing field include a lack of standardization and scalability. This review focuses on automated biomanufacturing of EVs in conditioned media collected from cultured mesenchymal stromal cells (MSCs). Different automated cell culture systems are discussed, including factors affecting EV quantity and quality, isolating EVs manufactured in an automated system, and validations needed. The ultimate goal when manufacturing cGMP-grade EVs is to identify a specific application and characterize the EV population in detail. This is achieved by validating every step of the process, choosing appropriate release criteria, and assuring batch-to-batch consistency. Due to the lack of standards in the field, it is critical to ensure that the cGMP-grade EVs meet FDA standards pertaining to identity, reproducibility, sterility, safety, purity, and potency. A closed-system automated bioreactor can be a valuable tool to generate cGMP-EVs in a scalable, economical, and reproducible manner. Full article

An Mg-based alloy device manufactured via a superplastic forming process (Mg-AZ31+SPF) and coated using a hydrothermal method (Mg AZ31+SPF+HT) was investigated as a method to increase mechanical and osteointegration capability. The cell viability and osteointegrative properties of alloy-derived Mg AZ31+SPF and Mg AZ31+SPF+HT extracts were investigated regarding their effect on human mesenchymal stem cells (hMSCs) (maintained in basal (BM) and osteogenic medium (OM)) after 7 and 14 days of treatment. The viability was analyzed through metabolic activity and double-strand DNA quantification, while the osteoinductive effects were evaluated through qRT-PCR, osteoimage, and BioPlex investigations. Finally, a preliminary liquid mass spectrometry analysis was conducted on the secretome of hMSCs. Biocompatibility analysis revealed no toxic effect on cells’ viability or proliferation during the experimental period. A modulation effect was observed on the osteoblast pre-commitment genes of hMSCs treated with Mg-AZ31+SPF+HT in OM, which was supported by mineralization nodule analysis. A preliminary mass spectrometry investigation highlighted the modulation of protein clusters involved in extracellular exosomes, Hippo, and the lipid metabolism process. In conclusion, our results revealed that the Mg AZ31+SPF+HT extracts can modulate the canonical and non-canonical osteogenic process in vitro, suggesting their possible application in bone tissue engineering. Full article

Mesenchymal stem cells (MSCs) hold significant potential in regenerative medicine, tissue engineering, and cultivated meat production. However, large-scale MSC production is limited by their need for surface adherence during growth. This study evaluates two biocompatible materials—collagen-coated alginate microcarriers and polylactic-co-glycolic acid (PLGA) nanofibers—as novel growth substrates to enhance MSC proliferation. Physicochemical characterization confirmed successful collagen integration on both materials. In vitro, bone marrow-derived MSCs (bmMSCs) cultured on collagen-coated alginate microcarriers exhibited significantly enhanced growth compared to commercial microcarriers, while PLGA nanofibers supported bmMSC growth comparable to traditional growth surfaces. Scanning Electron Microscopy (SEM) revealed that bmMSCs adhered not only to the surface but also grew within the porous structure of the alginate microcarriers. Mycoplasma testing confirmed that the bmMSCs were free from contamination. Both materials were assessed for biocompatibility using ISO-10993 guidelines, demonstrating no skin or ocular irritation, supporting their potential for in situ applications in clinical and therapeutic settings. This study highlights the promise of collagen-coated alginate microcarriers and PLGA nanofibers for scalable MSC production, offering efficient, biocompatible alternatives to traditional growth surfaces in regenerative medicine and cultivated meat manufacturing. Future research should focus on optimizing these materials for larger-scale production and exploring specific applications in therapeutic and food sectors. Full article

Knee osteoarthritis (OA) is a chronic, progressive, inflammatory, and degenerative whole-joint disease. Early-stage OA treatments typically include physiotherapy, weight-loss, pain relief medications, and intra-articular knee injections, such as corticosteroids, hyaluronic acid, or platelet-rich plasma. These treatments primarily provide symptomatic relief rather than reversing or halting disease progression. Recently, mesenchymal stromal cell (MSC) injections have garnered attention due to their immunomodulatory and regenerative capacities. MSCs, which can be derived from sources such as bone marrow, umbilical cord, or adipose tissue, and can be allogeneic or autologous, have demonstrated promising results in both animal models and several human studies. However, different protocols have been employed, presenting challenges for comparing outcomes. In this review, we address these variable settings, evaluate current practices, and identify key factors critical in optimizing MSC-based therapies by critically reviewing clinical trials of ex vivo expanded MSC therapies for OA undertaken between 2008 and 2023. Specific attention was given to two key aspects: (1) the cell culture process employed in manufacturing of autologous or allogeneic MSC products, and (2) the post-culture methods employed in storage, reconstitution and administration of the MSCs. Our findings suggest that standardizing MSC production for clinical applications remains a significant challenge, primarily due to variations in tissue sources, harvesting techniques, and manufacturing protocols, and due to broad discrepancies in reporting. Thus, we propose a set of minimal reporting criteria to guide future clinical trials. A common reporting guideline is a critical step towards a more standardized MSC production across different laboratories and clinical settings, thereby enhancing reproducibility and advancing the field of regenerative medicine for knee OA, as well as other disease settings. Full article

Background: The supply chain refers to the full process of creating and providing a good or service, starting with the raw materials and ending with the final customer. It requires cooperation and coordination between many parties, including the suppliers, manufacturers, distributors, retailers, and customers. Methods: In the medicinal supply chain (MSC), the critical nature of these processes becomes more complicated. It requires strict regulation, quality control, and traceability to ensure patient safety and compliance with regulatory standards. This study is conducted to suggest a smooth channel to deal with the challenges and adaptability of the MSC. Different MSC challenges are considered as criteria which deal with various adaptation plans. Multi-criteria decision-making (MCDM) methodologies are taken as optimization tools and probabilistic linguistic term sets (PLTSs) are considered for express uncertainty. Results: The subscript degree function (SDF) and deviation degree function (DDF) are introduced to evaluate the crisp value of the PLTSs. An MSC model is constructed to optimize the sustainable medicinal supply chain and overcome various barriers to MSC problems. Conclusions: Additionally, sensitivity analysis and comparative analysis were conducted to check the robustness and flexibility of the system. Finally, the conclusion section determines the optimal weighted criteria for the MSC problem and identifies the best possible solutions for MSC using PLTS-based MCDM methodologies. Full article

Batch-to-batch reproducibility and robust quality assessment are crucial for producing cell-free biologics, such as conditioned medium (CM) derived from mesenchymal stem/stromal cells (MSCs). This study investigated the effects of freezing CM at −80 °C prior to concentration, a step that could occur in large scale pipelines, compared to freshly processed CM. Quality assessment included total protein quantification; extracellular vesicle evaluation using nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and cytofluorimetry; and biochemical analysis using Raman spectroscopy. The freezing process resulted in a 34% reduction in total protein content, as confirmed for selected bioactive mediators, and significant depletion of specific particle types, particularly larger ones. Interestingly, the total particle concentration and polydispersity remained stable. Alterations in Raman spectra highlighted changes in protein, lipid, and nucleic acid content. These findings demonstrate that even routine steps like freezing can alter CM composition, likely due to temperature-induced structural changes in biological molecules. Careful consideration of pre- and intra-processing handling temperatures is critical for preserving the integrity of CM and ensuring consistent quality. This study emphasizes the importance of refining manufacturing protocols in the production of cell-free biologics. Full article

Metal 3D printing is increasingly being used to manufacture titanium–aluminum–vanadium (Ti6Al4V) implants. In vitro studies using 2D substrates demonstrate that the osteoblastic differentiation of bone marrow stromal cells (MSCs) on Ti6Al4V surfaces, with a microscale/nanoscale surface topography that mimics an osteoclast resorption pit, involves non-canonical Wnt signaling; Wnt3a is downregulated and Wnt5a is upregulated, leading to the local production of BMP2 and semaphorin 3A (sema3A). In this study, it was examined whether the regulation of MSCs in a 3D environment occurs by a similar mechanism. Human MSCs from two different donors were cultured for 7, 14, or 21 days on porous (3D) or solid (2D) constructs fabricated by powder-bed laser fusion. mRNA and secretion of osteoblast markers, as well as factors that enhance peri-implant osteogenesis, were analyzed, with a primary focus on the Wnt family, sema3A, and microRNA-145 (miR-145) signaling pathways. MSCs exhibited greater production of osteocalcin, latent and active TGFβ1, sema3A, and Wnt16 on the 3D constructs compared to 2D, both of which had similar microscale/nanoscale surface modifications. Wnt3a was reduced on 2D constructs as a function of time; Wnt11 and Wnt5a remained elevated in the 3D and 2D cultures. To better understand the role of Wnt16, cultures were treated with rhWnt16; endogenous Wnt16 was blocked using an antibody. Wnt16 promoted proliferation and inhibited osteoblast differentiation, potentially by reducing production of BMP2 and BMP4. Wnt16 expression was reduced by exogenous Wnt16 in 3D cells. Addition of the anti-Wnt16 antibody to the cultures reversed the effects of exogenous Wnt16, indicating an autocrine mechanism. Wnt16 increased miR-145-5p, suggesting a potential feedback mechanism. The miR-145-5p mimic increased Wnt16 production and inhibited sema3A in a 3D porous substrate-specific manner. Wnt16 did not affect sema3A production, but it was reduced by miR-145-5p mimic on the 3D constructs and stimulated by miR-145-5p inhibitor. Media from 7-, 14-, and 21-day cultures of MSCs grown on 3D constructs inhibited osteoclast activity to a greater extent than media from the 2D cultures. The findings present a significant step towards understanding the complex molecular interplay that occurs in 3D Ti6Al4V constructs fabricated by additive manufacturing. In addition to enhancing osteogenesis, the 3D porous biomimetic structure inhibits osteoclast activities, indicating its role in modulating bone remodeling processes. Our data suggest that the pathway mediated by sema3A/Wnt16/miR145-5p was enhanced by the 3D surface and contributes to bone regeneration in the 3D implants. This comprehensive exploration contributes valuable insights to guide future strategies in implant design, customization, and ultimately aims at improving clinical outcomes and successful osseointegration. Full article

Spheroids, as three-dimensional (3D) cell aggregates, can be prepared using various methods, including hanging drops, microwells, microfluidics, magnetic manipulation, and bioreactors. However, current spheroid manufacturing techniques face challenges such as complex workflows, the need for specialized personnel, and poor batch reproducibility. In this study, we designed a support-free, 3D-printed microwell chip and developed a compatible low-cell-adhesion process. Through simulation and experimental validation, we rapidly optimized microwell size and the coating process. We successfully formed three types of spheroids—human immortalized epidermal cells (HaCaTs), umbilical cord mesenchymal stem cells (UC-MSCs), and human osteosarcoma cells (MG63s)—on the chip. Fluorescent viability staining confirmed the biocompatibility and reliability of the chip. Finally, drug response experiments were conducted using the chip. Compared to traditional methods, our proposed strategy enables high-throughput production of size-controlled spheroids with excellent shape retention, while enhanced gas exchange during culture improves differentiation marker expression. This platform provides an efficient and cost-effective solution for biosensing applications, such as drug screening, disease modeling, and personalized therapy monitoring. Furthermore, the chip shows significant potential for real-time in vitro monitoring of cellular viability, reaction kinetics, and drug sensitivity, offering valuable advancements in biosensor technology for life sciences and medical applications. Full article

This study investigates the residual stresses arising from welding and machining processes, recognizing their adverse implications in manufacturing. Employing experimental analysis and simulation techniques, the research scrutinizes residual stress alterations resulting from sequential butt welding and subsequent machining. Utilizing MSC Marc Mentat software(version 2016), three-dimensional models are developed to simulate these processes. The finite element model from welding simulation seamlessly integrates into cutting simulations via the pre-state option. The experimental procedures involve 100 × 100 × 10 mm AISI 304 steel plates subjected to sequential welding and machining, with residual stresses measured at each stage. A comparative analysis between experimental and simulation results elucidates variations in residual stresses induced by sequential processes. The study focuses on examining the initial stress state post-welding and numerically assessing stress modifications due to milling. The results suggest minimal material removal insignificantly affects stress distribution and magnitude at the weld centerline. However, increased material removal leads to noticeable changes in through-thickness transverse stress within the weld zone, contrasting with marginal alterations in through-thickness longitudinal stress. Regions distanced from the weld seam show substantial increases in through-thickness longitudinal stress compared to marginal changes in through-thickness transverse stress. Full article

Background/Objectives: Cartilage injuries and osteoarthritis are prevalent public health problems, due to their disabling nature and economic impact. Mesenchymal stromal cells (MSCs) isolated from different tissues have the immunomodulatory capacity to regulate local joint environment. This translational study aims to compare cartilage restoration from MSCs from the synovial membrane (SM) and dental pulp (DP) by a tissue-engineered construct with Good Manufacturing Practices. Methods: A controlled experimental study was conducted on fourteen miniature pigs, using scaffold-free Tissue Engineering Constructs (TECs) from DP and SM MSCs, with a 6-month follow-up. Total thickness cartilage defects were created in both hind knees; one side was left untreated and the other received a TEC from either DP (n = 7) or SM (n = 7). An MRI assessed the morphology using the MOCART scoring system, T2 mapping evaluated water, and collagen fiber composition, and histological analysis was performed using the ICRS-2 score. Results: The untreated group had a mean MOCART value of 46.2 ± 13.4, while the SM-treated group was 65.7 ± 15.5 (p < 0.05) and the DP-treated group was 59.0 ± 7.9 (n.s.). The T2 mapping indicated a mean value of T2 of 54.9 ± 1.9 for native cartilage, with the untreated group at 50.9 ± 2.4 (p < 0.05). No difference was found between the T2 value of native cartilage and the treated groups. The ICRS-2 mean values were 42.1 ± 14.8 for the untreated group, 64.3 ± 19.0 for SM (p < 0.05), and 54.3 ± 12.2 for DP (n.s.). Conclusion: MRI and histological analysis indicated that TEC treatment led to superior cartilage coverage and quality compared to the defect group. TECs from SM demonstrated better results than the defect group in the histological assessment. Full article

Background: Bone marrow aspiration concentrate (BMAC) has garnered increasing interest due to its potential for healing musculoskeletal injuries. While the iliac crest remains a common harvest site, the aspiration technique’s efficacy in offering the highest yield and prevalence of mesenchymal stem cells (MSCs) is controversial. This study aimed to compare two different techniques of bone marrow aspiration over the anterior iliac crest from a single level versus multiple levels. Methods: Anterior iliac crests were selected in seven adult patients (aged between 31 and 59 years old). Aspiration was achieved using an 11-gauge needle (length: 100 mm; diameter: 2.3 mm) specifically manufactured for bone marrow collection (BD, Becton, Franklin Lakes, NJ, USA) connected to a 10 mL syringe. On one side, 4cc of bone marrow was aspirated at a single level to a depth of 7 cm without changing the needle direction. On the other side, over the same portion of the iliac crest, 1 cc of bone marrow was obtained from multiple levels of different depths during needle retrieval, maintaining a distance of 1 cm and changing the tip direction. The samples were blindly sent to the laboratory without indicating whether they came from an single level or multiple levels. Fluorescence-activated cell sorting (FACS) and osteoblast differentiation were analyzed and compared. Results: In the FACS analysis, the single level resulted in a higher population of MSCs that were positive for CD105, CD73, and CD90 and negative for CD34, compared to the multiple-level method. In the process of osteoblast differentiation, it was observed that MSCs exhibited more advanced features of enhanced osteoblastic abilities in the single-level method rather than the multiple-level method. Conclusions: A single-level aspiration technique at the anterior iliac crest may produce a high-quality bone marrow aspirate. This technique may help obtain specific populations of MSCs with the desired characteristics for use in regenerative therapies for musculoskeletal injuries. Full article

The simple oxides like titania, zirconia, and ZnO are famous with their antibacterial (or even antimicrobial) properties as well as their biocompatibility. They are broadly used for air and water filtering, in food packaging, in medicine (for implants, prostheses, and scaffolds), etc. However, these application fields can be broadened by switching to the composite multicomponent compounds (for example, titanates) containing in their unit cell, together with oxygen, several different metallic ions. This review begins with a description of the synthesis methods, starting from wet chemical conversion through the manufacturing of oxide (nano)powders toward mechanosynthesis methods. The morphology of these multicomponent oxides can also be very different (like thin films, complicated multilayers, or porous scaffolds). Further, we discuss in vitro tests. The antimicrobial properties are investigated with Gram-positive or Gram-negative bacteria (like Escherichia coli or Staphylococcus aureus) or fungi. The cytotoxicity can be studied, for example, using mouse mesenchymal stem cells, MSCs (C3H10T1/2), or human osteoblast-like cells (MG63). Other human osteoblast-like cells (SaOS-2) can be used to characterize the cell adhesion, proliferation, and differentiation in vitro. The in vitro tests with individual microbial or cell cultures are rather far away from the real conditions in the human or animal body. Therefore, they have to be followed by in vivo tests, which permit the estimation of the real applicability of novel materials. Further, we discuss the physical, chemical, and biological mechanisms determining the antimicrobial properties and biocompatibility. The possible directions of future developments and novel application areas are described in the concluding section of the review. Full article

Objective: The safety, feasibility, and potential functional improvement following the intravenous infusion of mesenchymal stem cells (MSCs) were investigated in patients with chronic severe spinal cord injury (SCI). Methods: The intravenous infusion of autologous MSCs cultured in auto-serum under Good Manufacturing Practices (GMP) was administered to seven patients with chronic SCI (ranging from 1.3 years to 27 years after the onset of SCI). In addition to evaluating feasibility and safety, neurological function was evaluated using the American Spinal Injury Association Impairment Scale (AIS), International Standards for Neurological Classification of Spinal Cord Injury (ISCSCI-92), and Spinal Cord Independence Measure III (SCIM-III). Results: No serious adverse events occurred. Neither CNS tumors, abnormal cell growth, nor neurological deterioration occurred in any patients. While this initial case series was not blinded, significant functional improvements and increased quality of life (QOL) were observed at 90 and 180 days post-MSC infusion compared to pre-infusion status. One patient who had an AIS grade C improved to grade D within six months after MSC infusion. Conclusions: This case series suggests that the intravenous infusion of autologous MSCs is a safe and feasible therapeutic approach for chronic SCI patients. Furthermore, our data showed significant functional improvements and better QOL after MSC infusion in patients with chronic SCI. A blind large-scale study will be necessary to fully evaluate this possibility. Full article

Mesenchymal stromal cells (MSCs) are promising candidates for cartilage repair therapy due to their self-renewal, chondrogenic, and immunomodulatory capacities. It is widely recognized that a shift from fetal bovine serum (FBS)-containing medium toward a fully chemically defined serum-free (SF) medium would be necessary for clinical applications of MSCs to eliminate issues such as xeno-contamination and batch-to-batch variation. However, there is a notable gap in the literature regarding the evaluation of the chondrogenic ability of SF-expanded MSCs (SF-MSCs). In this study, we compared the in vivo regeneration effect of FBS-MSCs and SF-MSCs in a rat osteochondral defect model and found poor cartilage repair outcomes for SF-MSCs. Consequently, a comparative analysis of FBS-MSCs and SF-MSCs expanded using two SF media, MesenCult™-ACF (ACF), and Custom StemPro™ MSC SFM XenoFree (XF) was conducted in vitro. Our results show that SF-expanded MSCs constitute variations in morphology, surface markers, senescence status, differentiation capacity, and senescence/apoptosis status. Highly proliferative MSCs supported by SF medium do not always correlate to their chondrogenic and cartilage repair ability. Prior determination of the SF medium’s ability to support the chondrogenic ability of expanded MSCs is therefore crucial when choosing an SF medium to manufacture MSCs for clinical application in cartilage repair. Full article