Cell Attachment Research Articles

The impact of 45S5 bioglass vs. β-TCP nanoparticles ratio on rheological behavior of formulated printing inks and 3D printed polycaprolactone-based scaffolds final properties

Extrusion based 3-D printing has been extensively applied to create geometrically complex composite polymer-ceramic structures as bone tissue substitute. The rheological features of the formulated bioink that regulate the printability and resolution of the printed scaffolds, rely on physicochemical properties of ink components, mainly their composition and chemical structure. The aim of this study was to evaluate the effect of different content of 45S5 bioglass (BG) and β-tricalcium phosphate (β-TCP) nanoparticles on the rheological behavior of printing inks and final composite scaffolds based on polycaprolactone (PCL)/BG/β-TCP. Ceramic nano-powders were first characterized and the composite bioinks were prepared by mixing various ratios of BG and β-TCP powders into the 50% w/v of PCL solution (β-TCP/BG: 70/30, 50/50, 30/70, and 10/90 w/w %). All formulated inks showed a thixotropic behavior and viscosity significantly increased by applying higher fraction of BG. Interestingly highly loaded β-TCP ink (β-TCP/BG: 70/30) revealed a rubbery nature with high surface tension which reduced final scaffolds resolution. All printed scaffolds possessed highly porous structure with interconnected pores. However, porosity percentage and shape stability improved by increasing BG content which was accompanied with lower mechanical strength and superior biodegradation and bioactivity of scaffolds. The biological performance of the printed scaffolds was evaluated using osteoblast cell line MG-63. MTT assay and cell attachment observation by SEM confirmed that printed scaffolds are well biocompatible and properly support cell colonization and proliferation. In overall, besides more appropriate materialistic properties, scaffolds with β-TCP/BG: 30/70 composition provided the most favorable microenvironment for cell colonization and growth.

Development of a Human Recombinant Collagen for Vat Polymerization-Based Bioprinting.

In light-based 3D-bioprinting, gelatin methacrylate (GelMA) is one of the most widely used materials, as it supports cell attachment, and shows good biocompatibility and degradability in vivo. However, as an animal-derived material, it also causes safety concerns when used in medical applications. Gelatin is a partial hydrolysate of collagen, containing high amounts of hydroxyproline. This causes the material to form a thermally induced gel at ambient temperatures, a behavior also observed in GelMA. This temperature-dependent gelation requires precise temperature control during the bioprinting process to prevent the gelation of the material. To avoid safety concerns associated with animal-derived materials and reduce potential issues caused by thermal gelation, a recombinant human alpha-1 collagen I fragment was expressed in Komagataella phaffii without hydroxylation. The resulting protein was successfully modified with methacryloyl groups and underwent rapid photopolymerization upon ultraviolet light exposure. The developed material exhibited slightly slower polymerization and lower storage modulus compared to GelMA, while it showed higher stretchability. However, unlike the latter, the material did not undergo physical gelation at ambient temperatures, but only when cooled down to below 10°C, a characteristic that has not been described for comparable materials so far. This gelation was not caused by the formation of triple-helical structures, as shown by the absence of the characteristic peak at 220nm in CD spectra. Moreover, the developed recombinant material facilitated cell adherence with high cell viability after crosslinking via light to a 3D structure. Furthermore, desired geometries could be easily printed on a stereolithographic bioprinter.

Improvement in bioactivity, hardness and friction resistance of 3 % manganese-doped hydroxyapatite coated on alumina using radio frequency magnetron sputtering

Hydroxyapatite (HAP) is a common hard tissue implant material known for its superior biocompatibility and osteoconductivity. However, its poor mechanical strength, brittleness and slow degradation limit the applications. This study explores the enhancement of HAP mechanical properties and bioactivity by coating 3 wt% manganese-doped HAP (Mn-HAP) on another inert biomaterial alumina (Mn-HAP/Al2O3) substrates using the RF magnetron sputtering technique. Characterization of these samples was performed using Field Emission Scanning Electron Microscopy (FESEM), Energy Dispersive X-ray Spectroscopy (EDS), Grazing Incidence X-ray Diffraction (GIXRD), Fourier Transform Infrared Spectroscopy (FTIR) and Brunauer-Emmett-Teller (BET) techniques. Mechanical property was assessed through Vicker's hardness and adhesion of the film was studied by scratch testing. Corrosion resistance was evaluated using Tafel plots in Ringer's solution by Electrochemical analyser (ECA), and dielectric properties were measured using Impedance analyser. Biocompatibility was examined by wettability tests, thrombogenicity, antioxidant test, antimicrobial investigation and MTT [3-(4, 5-dimethythiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] assay. The results show that Mn-HAP/Al2O3 coatings exhibit superior properties as compared to pure HAP, alumina, and HAP/Al2O3. Mn-HAP showed enhanced crystallinity and grain refinement, leading to improved hardness of 1198 HV for Mn-HAP/Al2O3 as compared to 39.84 HV for pure HAP and 1028 HV for HAP/Al2O3. The friction coefficient was found to be best in the Mn-HAP/Al2O3 sample. Corrosion rate significantly decreases in Mn-HAP/Al2O3 (1.63 ± 0.28) mmpy after coating on alumina. In vitro studies demonstrated enhanced cell attachment, proliferation, and differentiation after Mn-HAP coating on alumina. Antimicrobial tests revealed improved resistance against E. coli and S. aureus, with Mn-HAP/Al2O3 showing a larger zone of inhibition. The study concludes that 3 wt% Mn-HAP coatings deposited by RF magnetron sputtering hold great promise for enhancing the performance and longevity of hard tissue implants, paving the way for advanced biomedical applications.

Influence of copper ion cross-linked CMC-PVA film on cell viability and cell proliferation study

In this study, films composed of carboxymethyl cellulose and polyvinyl alcohol were fabricated using the solution casting method. Citric acid (4 %) was employed as a cross-linking agent, while glycerol (3 %) as a plasticizer. Cupric chloride (CuCl2·2H2O) was used for cross-linking at concentrations 0.5 %, 1 %, and 3 % over different times. The cross-linking with copper ions led to a noticeable reduction in elasticity, with the breaking strain ranging from 17.9 %–52.9 %. The ion hydration phenomenon increased the contact angle and swelling ratio. Fourier-transform infrared (FTIR) spectroscopy confirmed esterification reactions and copper ion cross-linking with sodium carboxymethyl cellulose (Na-CMC). The films showed antibacterial activity against Staphylococcus aureus and Escherichia coli. The ion-released mechanism followed was the non-Fickian super case-II type. The concentration and duration of cross-linking significantly influenced cell viability and proliferation. FE-SEM analysis revealed that effective concentrations of CuCl2.2H2O were 0.5 % and 1 %, and cross-linking times were 5–15 min, facilitating cell attachment and proliferation. Films are non-adhesive with water vapor permeation 800–900 g/m2/day. These results indicate the potential use of the films in treating second-degree burn wounds with low to medium exudate levels. This study provides valuable insights into the development of copper-infused materials for advanced wound healing applications.

Mycolactone causes destructive Sec61-dependent loss of the endothelial glycocalyx and vessel basement membrane: a new indirect mechanism driving tissue necrosis in Mycobacterium ulcerans infection.

The drivers of tissue necrosis in Mycobacterium ulcerans infection (Buruli ulcer disease) have historically been ascribed solely to the directly cytotoxic action of the diffusible exotoxin, mycolactone. However, its role in the clinically-evident vascular component of disease aetiology remains poorly explained. We have now dissected mycolactone's effects on primary vascular endothelial cells in vitro and in vivo. We show that mycolactone-induced changes in endothelial morphology, adhesion, migration, and permeability are dependent on its action at the Sec61 translocon. Unbiased quantitative proteomics identified a profound effect on proteoglycans, driven by rapid loss of type II transmembrane proteins of the Golgi, including enzymes required for glycosaminoglycan (GAG) synthesis, combined with a reduction in the core proteins themselves. Loss of the glycocalyx is likely to be of particular mechanistic importance, since knockdown of galactosyltransferase II (beta-1,3-galactotransferase 6; B3GALT6), the GAG linker-building enzyme, phenocopied the permeability and phenotypic changes induced by mycolactone. Additionally, mycolactone depleted many secreted basement membrane components and microvascular basement membranes were disrupted in vivo. Remarkably, exogenous addition of laminin-511 reduced endothelial cell rounding, restored cell attachment and reversed the defective migration caused by mycolactone. Hence supplementing mycolactone-depleted extracellular matrix may be a future therapeutic avenue, to improve wound healing rates.

Optimisation of cryopreservation conditions, including storage duration and revival methods, for the viability of human primary cells

BackgroundCryopreservation is a crucial procedure for safeguarding cells or other biological constructs, showcasing considerable potential for applications in tissue engineering and regenerative medicine.AimsThis study aimed to evaluate the effectiveness of different cryopreservation conditions on human cells viability.MethodsA set of cryopreserved data from Department of Tissue Engineering and Regenerative Medicine (DTERM) cell bank were analyse for cells attachment after 24 h being revived. The revived cells were analysed based on different cryopreservation conditions which includes cell types (skin keratinocytes and fibroblasts, respiratory epithelial, bone marrow mesenchymal stem cell (MSC); cryo mediums (FBS + 10% DMSO; commercial medium); storage durations (0 to > 24 months) and locations (tank 1–2; box 1–5), and revival methods (direct; indirect methods). Human dermal fibroblasts (HDF) were then cultured, cryopreserved in different cryo mediums (HPL + 10% DMSO; FBS + 10% DMSO; Cryostor) and stored for 1 and 3 months. The HDFs were revived using either direct or indirect method and cell number, viability and protein expression analysis were compared.ResultsIn the analysis cell cryopreserved data; fibroblast cells; FBS + 10% DMSO cryo medium; storage duration of 0–6 months; direct cell revival; storage in vapor phase of cryo tank; had the highest number of vials with optimal cell attachment after 24 h revived. HDFs cryopreserved in FBS + 10% DMSO for 1 and 3 months with both revival methods, showed optimal live cell numbers and viability above 80%, higher than other cryo medium groups. Morphologically, the fibroblasts were able to retain their phenotype with positive expression of Ki67 and Col-1. HDFs cryopreserved in FBS + 10% DMSO at 3 months showed significantly higher expression of Ki67 (97.3% ± 4.62) with the indirect revival method, while Col-1 expression (100%) was significantly higher at both 1 and 3 months compared to other groups.ConclusionIn conclusion, fibroblasts were able to retain their characteristics after various cryopreservation conditions with a slight decrease in viability that may be due to the thermal-cycling effect. However, further investigation on the longer cryopreservation periods should be conducted for other types of cells and cryo mediums to achieve optimal cryopreservation outcomes.

BPP43_05035 is a Brachyspira pilosicoli cell surface adhesin that weakens the integrity of the epithelial barrier during infection

ABSTRACT The anaerobic spirochete Brachyspira causes intestinal spirochetosis, characterized by the intimate attachment of bacterial cells to the colonic mucosa, potentially leading to symptoms such as diarrhea, abdominal pain, and weight loss. Despite the clinical significance of Brachyspira infections, the mechanism of the interaction between Brachyspira and the colon epithelium is not known. We characterized the molecular mechanism of the B. pilosicoli-epithelium interaction and its impact on the epithelial barrier during infection. Through a proteomics approach, we identified BPP43_05035 as a candidate B. pilosicoli surface protein that mediates bacterial attachment to cultured human colonic epithelial cells. The crystal structure of BPP43_05035 revealed a globular lipoprotein with a six-bladed beta-propeller domain. Blocking the native BPP43_05035 on B. pilosicoli, either with a specific antibody or via competitive inhibition, abrogated its binding to epithelial cells, which required cell surface-exposed N-glycans. Proximity labeling and interaction assays revealed that BPP43_05035 bound to tight junctions, thereby increasing the permeability of the epithelial monolayer. Extending our investigation to humans, we discovered a downregulation of tight junction and brush border genes in B. pilosicoli-infected patients carrying detectable levels of epithelium-bound BPP43_05035. Collectively, our findings identify BPP43_05035 as a B. pilosicoli adhesin that weakens the colonic epithelial barrier during infection.

Evaluation of Additives on the Cell Metabolic Activity of New PHB/PLA-Based Formulations by Means of Material Extrusion 3D Printing for Scaffold Applications

In this study, specific additives were incorporated in polyhydroxyalcanoate (PHB) and polylactic acid (PLA) blend to improve its compatibility, and so enhance the cell metabolic activity of scaffolds for tissue engineering. The formulations were manufactured through material extrusion (MEX) additive manufacturing (AM) technology. As additives, petroleum-based poly(ethylene) with glicidyl metacrylate (EGM) and methyl acrylate-co-glycidyl methacrylate (EMAG); poly(styrene-co-maleic anhydride) copolymer (Xibond); and bio-based epoxidized linseed oil (ELO) were used. On one hand, standard geometries manufactured were assessed to evaluate the compatibilizing effect. The additives improved the compatibility of PHB/PLA blend, highlighting the effect of EMAG and ELO in ductile properties. The processability was also enhanced for the decrease in melt temperature as well as the improvement of thermal stability. On the other hand, manufactured scaffolds were evaluated for the purpose of bone regeneration. The mean pore size and porosity exhibited values between 675 and 718 μm and 50 and 53%, respectively. According to the results, the compression stress was higher (11–13 MPa) than the required for trabecular bones (5–10 MPa). The best results in cell metabolic activity were obtained by incorporating ELO and Xibond due to the decrease in water contact angle, showing a stable cell attachment after 7 days of culture as observed in SEM.

Biocompatibility and Enamel Remineralization Potential of Titanium-Doped Phosphate Glasses With/Without Diode Laser Irradiation

This study investigates the biocompatibility and enamel remineralization potential of titanium-doped phosphate glasses (TDPG), third-generation materials with promising applications in dentistry. The biocompatibility of TDPG paste and its components (TDPG powder and polyacrylic acid liquid) was assessed using human gingival fibroblast cells (hGF) and human dental pulp stem cells (hDPSC). Cell attachment and viability were examined at 1, 3, and 7 days, comparing results with Nupro® Prophy paste and Teethmate, commonly used desensitizers, and positive control cells in normal tissue culture medium. Furthermore, the remineralization potential of these glasses with or without the use of diode laser irradiation on white spot lesions in enamel was explored at 12- and 21-days using Raman Spectroscopy and Field Emission Scanning Electron Microscopy (FESEM). The remineralization potential of TDPG was compared with Nupro® Prophy paste and Teethmate. Furthermore, both sound and demineralized enamel were also used as other controls.Results indicated that cells exposed to TDPG extracts displayed similar morphology and viability to positive control cells. While all tested pastes did not show remineralizing action after 12 days, Nupro® Prophy and Teethmate exhibited such action after 21 days. However, laser application enhanced remineralization potential for all pastes at both time points, with TDPG showing comparable results to Nupro® Prophy paste and significantly higher potential than Teethmate when used with laser at 21 days.In summary, this study reveals the biocompatibility and enhanced remineralization potential of TDPG when combined with diode laser irradiation. These findings signify innovative strides in dental materials and treatment modalities, offering new possibilities for advanced dental applications.

Bioactive stem cell-laden 3D nanofibrous scaffolds for tissue engineering

This study presents biomimetic nanoscaffolds composed of electrospun polycaprolactone-collagen (PCL-Coll) nanofibers, loaded with bioactive Arnebia euchroma (AE) extract and stem cells, to develop cell-based tissue engineering constructs. The incorporation of AE extract, known for its antioxidant and anti-inflammatory properties, into the PCL-Coll nanofibers resulted in nanoscaffolds denoted as PCL-Coll/AE0, PCL-Coll/AE5, PCL-Coll/AE10, and PCL-Coll/AE15, corresponding to AE extract concentrations of 0.0, 5.0, 10.0, and 15.0 wt%, respectively. The PCL-Coll/AE nanoscaffolds exhibited high porosity values of 62.81 ± 4.78 %, 59.9 ± 2.10 %, 52.44 ± 2.66 %, and 46.32 ± 1.35 %, respectively, thereby providing an optimal 3D environment for cell attachment and proliferation. Analysis of the AE extract revealed the presence of abundant shikonin, a key bioactive compound, as indicated by its characteristic absorption bands at 490, 520, and 560 nm. FE-SEM data confirmed the homogeneous immobilization of AE compounds within the 3D nanofiber scaffolds, with fibers averaging 316 ± 137 nm in diameter, falling within the range of natural collagen fibers. To evaluate the structural integrity of the fully stem cell-laden scaffolds, we assessed cell growth, cell attachment within the PCL-Coll/AE nanoscaffolds, the cytoprotective effects of AE extract, and the expression levels of stemness-related genes, including Nanog, Rex1, Sox2, Oct4, Klf4, and C-Myc. Real-time PCR assays indicated that key indicators of stemness were upregulated, with average increases from 120 ± 15 % in PCL-Coll/AE0 to 250 ± 30 % in PCL-Coll/AE15 over a two-week period. These upregulations promote cell proliferation and facilitate cell cycle progression. Data indicate that increasing the concentration of bioactive AE extract within the scaffolds enhanced cell adhesion on the scaffold surface and improved cell viability after 7 days of culture, with viability rising from 109 ± 6.15 % in PCL-Coll/AE0 to 145.5 ± 10.11 % in PCL-Coll/AE15. Cytoprotective assays against oxidative stress induced by H₂O₂ revealed relative cell viability for PCL-Coll/AE0, PCL-Coll/AE5, PCL-Coll/AE10, and PCL-Coll/AE15 as 42.78 ± 5.85 %, 49.29 ± 6.79 %, 64.98 ± 3.32 %, and 78.68 ± 3.09 %, respectively. These results correlate with the antioxidant potency of AE extract compounds, particularly shikonin and its derivatives. Therefore, the cell-laden biomimetic PCL-Coll/AE15 scaffolds, which demonstrate sustained stemness features and a continuous local release of bioactive AE compounds, represent a promising candidate for tissue engineering applications.

Alkali surface modification of titanium for biomedical implants

AbstractNanostructures were successfully created on titanium (Ti) substrates through alkali treatment. By varying the concentration, temperature, and duration of the alkali treatment, different nanostructure surfaces were achieved on Ti. FE‐SEM analysis revealed a transition from a smooth to a nanoporous surface structure. The alkali treatment notably decreased the contact angle of Ti from 64° to 48°. Raman spectroscopy of Ti treated with 5 M NaOH at 80 °C for 24 h showed distinct titanate peaks at 188, 190, and 270 cm−1, confirming the formation of titanate compounds on the Ti surface. EIS measurements further demonstrated that the treated Ti exhibited reduced chemical activity, increased surface roughness, and porosity of approximately 40–50% compared to untreated Ti. In vitro biocompatibility tests using baby hamster kidney cells indicated positive cell attachment and proliferation after 72 h of culturing on the alkali‐treated Ti. These modifications enhance the surface properties and expand the potential applications of titanium in biomedical, industrial, and environmental technologies.

Synthesis, Characterization and Osteogenic Properties of Chitin-Polydioxanone Composite Gel.

In this study, we have developed a Chitin(Ch)-Poly(dioxanone)(PDO) gel system, which can be potentially used for tissue engineering. Hydrogel has been widely used in biomedical applications for its tuneable properties and biocompatibility. Chitin (Ch) is a natural biopolymer used for its ability to mimic the natural extracellular matrix due to N-acetyl glucosamine structural units. Poly (dioxanone) (PDO) is an FDA-approved synthetic biopolymer known for its mechanical properties, good biocompatibility, and poor inflammatory response. Based on this, we have developed Ch-PDO composite gel using simple regeneration chemistry and characterized it using FT-IR and SEM. The developed composite gel showed improved gel strength, good swelling ability,and controlled degradation behaviour. It also showed good injectability with shear thinning properties and hemocompatibility. Further, the biocompatibility and cell adhesion studies of the prepared gels were studied using dental follicle stem cells (DFSCs). The prepared Ch-PDO gel was biocompatible and showed DFSCs cell attachment. Osteogenic mineralization and RUNX2 expression of the prepared Ch and Ch-PDO gel was studied and Ch-PDO gel showed an enhanced mineralization and RUNX2 expression. Therefore, the developed chitin-PDO gel could be potentially used for bone tissue engineering.

Human Mesofluidic Intestinal Model for Studying Transport of Drug Carriers and Bacteria Through a Live Mucosal Barrier.

The intestinal mucosal barrier forms a critical interface between lumen contents such as bacteria, drugs, and drug carriers and the underlying tissue. Current in vitro intestinal models, while recapitulating certain aspects of this barrier, generally present challenges with respect to imaging transport across mucus and uptake into enterocytes. A human mesofluidic small intestinal chip was designed to enable facile visualization of a mucosal interface created by growing primary human intestinal cells on a vertical hydrogel wall separating channels representing the intestinal lumen and circulatory flow. Type I collagen, fortified via cross-linking to prevent deformation and leaking during culture, was identified as a suitable gel wall material for supporting primary organoid-derived human duodenal epithelial cell attachment and monolayer formation. Addition of DAPT and PGE2 to culture medium paired with air-liquid interface culture increased the thickness of the mucus layer on epithelium grown within the device for 5 days from approximately 5 mm to 50 μm, making the model suitable for revealing intriguing features of interactions between luminal contents and the mucus barrier using live cell imaging. Time-lapse imaging of nanoparticle diffusion within mucus revealed a zone adjacent to the epithelium largely devoid of nanoparticles up to 4.5 hr after introduction to the lumen channel, as well as pockets of dimly lectin-stained mucus within which particles freely diffused, and apparent clumping of particles by mucus components. Multiple particle tracking conducted on the intact mucus layer in the chip revealed significant size-dependent differences in measured diffusion coefficients. E. coli introduced to the lumen channel were freely mobile within the mucus layer and appeared to intermittently contact the epithelial surface over 30 minute periods of culture. Mucus shedding into the lumen and turnover of mucus components within cells were visualized. Taken together, this system represents a powerful tool for visualization of interactions between luminal contents and an intact live mucosal barrier.

The Role of Chitosan and Gelatin-Based Scaffolds in Bone Regeneration: A Systematic Review.

Guided bone regeneration facilitates the growth of new bone in areas where there is a bone defect or insufficiency. This technique involves placing a barrier membrane over the bone graft site, and the membrane prevents the invasion of soft tissue (such as gingival tissue) into the bone graft area. This allows the slower-growing bone cells to populate the area without competition, promoting proper bone regeneration. When combined, chitosan and gelatin create composite scaffolds that leverage the strengths of both materials. Chitosan provides structural integrity and antimicrobial properties, and gelatin enhances cell attachment and proliferation, which improves mechanical properties and makes it more suitable for supporting bone regeneration in load-bearing areas.This systematic review aims to evaluate the effectiveness ofchitosan and gelatin-based scaffolds in bone regeneration. Various databases such as PubMed, Cochrane Library, LILAC, and Google Scholar were screened to adhere to the eligibility criteria. The included studies in the review were the in vivo and in vitro assessment of the chitosan and gelatin efficiency as a scaffold. Six studies were investigated for the involvement of chitosan and gelatin-based scaffolds in bone regeneration. Of these, two in vivostudies examined bone regeneration by measuring alkaline phosphatase activity (ALP) using different staining techniques, while the remaining four in vitro studies used histologic and histometric analysis where stem cells, chemicals, and other biopolymers were compared. Chitosan and gelatin scaffolds consistently showed better results in terms of bone repair throughout all six experiments. Gelatin's capacity for regeneration can be increased by mixing itwith chitosan. For additional advancement, future researchers need to focus on incorporating biopolymers.The potential of scaffolds composed of gelatin and chitosan to replace tissue lost due to periodontitis shows great clinical significance.

Evaluation of chicken embryo extract and egg yolk extract as alternatives to basic cell culture medium supplement

BackgroundFetal calf serum (FCS), an existing cell culture supplement, is effective but has several drawbacks, including being expensive, requiring a lengthy process of production, and requiring a hard currency. With this in mind, we planned to evaluate chick embryo extract and egg yolk extracts in cell culture as alternatives to fetal calf serum (FCS).MethodsSpecific pathogen-free eggs were purchased from the National Veterinary Institute, Bishoftu, Ethiopia, and incubated in a humidified incubator at 37 °C for 11 days. Egg yolk extract (EYE) and chick embryo extract (CEE) were collected after the egg was opened with caution not to destroy the yolk sack or the chick embryo itself. Chick fibroblasts and Vero cells were cultured in minimum essential medium (MEM) supplemented with egg yolk extract or chick embryo extract at ratios of 0:10, 1:9, 2.5:7.5, and 5:5% fetal calf serum.ResultsFibroblast cell attachment was better in media supplemented with 5% CEE and 5% FCS. The confluency was also greater than 50% at this concentration. Vero cells cultured with 5% CEE and 5% FCS also exhibited very good cell attachment and a confluency of up to 70%. Viability and confluency were also observed at 5%:5% ratios of 50 and 70%, respectively.ConclusionThis investigation evaluated these two extracts as cell culture supplements and revealed promising results as alternatives to fetal calf serum. The limitation of this study is that it only used two cell types and additional cell lines, and different ratios should be tested. With the above findings, further research using different cell lines, ratios and conditions is warranted.

Injectable PLGA Microscaffolds with Laser-Induced Enhanced Microporosity for Nucleus Pulposus Cell Delivery.

Intervertebral disc (IVD) degeneration is a leading cause of lower back pain (LBP). Current treatments primarily address symptoms without halting the degenerative process. Cell transplantation offers a promising approach for early-stage IVD degeneration, but challenges such as cell viability, retention, and harsh host environments limit its efficacy. This study aimed to compare the injectability and biocompatibility of human nucleus pulposus cells (hNPC) attached to two types of microscaffolds designed for minimally invasive delivery to IVD. Microscaffolds are developed from poly(lactic-co-glycolic acid) (PLGA) using electrospinning and femtosecond laser structuration. These microscaffolds are tested for their physical properties, injectability, and biocompatibility. This study evaluates cell adhesion, proliferation, and survival in vitro and ex vivo within a hydrogel-based nucleus pulposus model. The microscaffolds demonstrate enhanced surface architecture, facilitating cell adhesion and proliferation. Laser structuration improved porosity, supporting cell attachment and extracellular matrix deposition. Injectability tests show that microscaffolds can be delivered through small-gauge needles with minimal force, maintaining high cell viability. The findings suggest that laser-structured PLGA microscaffolds are viable for minimally invasive cell delivery. These microscaffolds enhance cell viability and retention, offering potential improvements in the therapeutic efficiency of cell-based treatments for discogenic LBP.

Comparison of Bioengineered Scaffolds for the Induction of Osteochondrogenic Differentiation of Human Adipose-Derived Stem Cells.

Osteochondral lesions may be due to trauma or congenital conditions. In both cases, therapy is limited because of the difficulty of tissue repair. Tissue engineering is a promising approach that relies on designed scaffolds with variable mechanical attributes to favor cell attachment and differentiation. Human adipose-derived stem cells (hASCs) are a very promising cell source in regenerative medicine with osteochondrogenic potential. Based on the assumption that stiffness influences cell commitment, we investigated three different scaffolds: a semisynthetic animal-derived GelMA hydrogel, a combined scaffold made of rigid PEGDA coated with a thin GelMA layer and a decellularized plant-based scaffold. We investigated the role of different biomechanical stimulations in the scaffold-induced osteochondral differentiation of hASCs. We demonstrated that all scaffolds support cell viability and spontaneous osteochondral differentiation without any exogenous factors. In particular, we observed mainly osteogenic commitment in higher stiffness microenvironments, as in the plant-based one, whereas in a dense and softer matrix, such as in GelMA hydrogel or GelMA-coated-PEGDA scaffold, chondrogenesis prevailed. We can induce a specific cell commitment by combining hASCs and scaffolds with particular mechanical attributes. However, in vivo studies are needed to fully elucidate the regenerative process and to eventually suggest it as a potential approach for regenerative medicine.

Magnetical scafford with ROS-scavenging for bone regeneration under static magnetic field

Excessive reactive oxygen species (ROS) and bacterial infection significantly disrupt the microenvironment of tissue regeneration. Magnetic nanoparticles in combination with magnetic fields are emerging strategies to regulate tissue regeneration. In this work, ZnFe2O4 (ZFO) nanoparticles were prepared and subsequently decorated with polydopamine (PDA) and RGD peptide. The modified ZFO nanoparticles were loaded into polylactic-glycolic acid/polycaprolactone (PLGA/PCL) scaffolds as PP-ZFO/PDA-RGD magnetic scaffolds. Physicochemical, anti-ROS, antibacterial and osteogenic properties were evaluated in vitro. Under 30 mT static magnetic field, the PP-ZFO/PDA-RGD scaffold significantly enhanced the expression of ALP, RUNX2, BMP2 and mineralized nodules. It indicated that the magnetic system could promote the attachment, proliferation, differentiation and mineralization of MC3T3-E1 cells. The scaffold showed excellent ROS scavenging ability via the additive effect of ZFO, PDA and RGD. The remarkable antibacterial properties of Zn²⁺ and Fe³⁺ endowed the scaffolds with excellent antibacterial activity against E. coli and S. aureus. These results demonstrate that the PP-ZFO/PDA-RGD based magnetic system is a promising approach for bone regeneration under complex microenvironment.

REVITALIZATION OF CRITICAL-SIZED CALVARIAL DEFECTS IN RATS USING HEPARIN-CONJUGATED FIBRIN HYDROGEL WITH BMP-2 AND ADIPOSE-DERIVED PERICYTES

Massive bone defects present challenges in orthopedic surgery, requiring innovative solutions. Hydrogels offer promise due to their resemblance to the extracellular matrix. Adipose tissue-derived pericytes (ADPs) and osteoinductive proteins like BMP-2 show potential for bone tissue engineering. Combining them via heparin-conjugated fibrin hydrogel aims to enhance healing in critical-sized calvarial defects. The study investigated the use of a heparin-conjugated fibrin (HCF) hydrogel containing BMP-2 and adipose-derived pericytes for repairing critical-sized calvarial defects in rats. ADPs were cultured from rat adipose tissue, and the HCF hydrogel was prepared accordingly. In vitro experiments assessed BMP-2 release kinetics and bioactivity, while mineralization assays were conducted on cultured ADPs. In vivo experiments involved surgically creating calvarial defects in rats and implanting HCF gel alone or with BMP-2, pericytes, or both. Micro-CT imaging and histological analysis were performed post-implantation to evaluate bone formation. In our study, we prepared the HCF hydrogel by combining heparin-conjugated fibrinogen, human fibrinogen, human thrombin, aprotinin, and calcium chloride. Gelation occurred within 3 minutes at room temperature. SEM analysis revealed an open interconnected pore morphology and macroporous structure, facilitating cell attachment and proliferation. ELISA showed sustained release of BMP-2 from the HCF hydrogel over 28 days. Bioactivity assays demonstrated the ability of released BMP-2 to enhance ALP activity in rat neonatal calvarial osteoblasts. Additionally, BMP-2 induced osteogenic differentiation of rat ADPs, as evidenced by increased ALP activity, osteocalcin expression, and calcium deposition. Implantation of HCF hydrogels containing BMP-2 and/or ADPs into rat calvarial defects significantly promoted bone tissue regeneration compared to controls, with the greatest effect observed in the group receiving both BMP-2 and ADPs. Micro-CT and histological analysis confirmed substantial bone formation and defect closure after three months. In conclusion, our in vitro findings demonstrated that the HCF hydrogel provided sustained release of BMP-2, retaining its bioactivity and promoting osteogenesis in rat calvarial osteoblasts and ADPs. In vivo results showed that combining allogeneic ADPs with BMP-2 enhanced bone regeneration in critical-sized calvarial defects, indicating the potential of this approach for large bone defect restoration.

Composite Polycaprolactone/Gelatin Nanofiber Membrane Scaffolds for Mesothelial Cell Culture and Delivery in Mesothelium Repair.

To repair damaged mesothelium tissue, which lines internal organs and cavities, a tissue engineering approach with mesothelial cells seeded to a functional nanostructured scaffold is a promising approach. Therefore, this study explored the uses of electrospun nanofiber membrane scaffolds (NMSs) as scaffolds for mesothelial cell culture and transplantation. We fabricated a composite NMS through electrospinning by blending polycaprolactone (PCL) with gelatin. The addition of gelatin enhanced the membrane's hydrophilicity while maintaining its mechanical strength and promoted cell attachment. The in vitro study demonstrated enhanced adhesion of mesothelial cells to the scaffold with improved morphology and increased phenotypic expression of key marker proteins calretinin and E-cadherin in PCL/gelatin compared to pure PCL NMSs. In vivo studies in rats revealed that only cell-seeded PCL/gelatin NMS constructs fostered mesothelial healing. Implantation of these constructs leads to the regeneration of new mesothelium tissue. The neo-mesothelium is similar to native mesothelium from hematoxylin and eosin (H&E) and immunohistochemical staining. Taken together, the PCL/gelatin NMSs can be a promising scaffold for mesothelial cell attachment, proliferation, and differentiation, and the cell/scaffold construct can be used in therapeutic applications to reconstruct a mesothelium layer.