Collagen Matrices Research Articles

Automated Nanodroplet Dispensing for Large-Scale Spheroid Generation via Hanging Drop and Parallelized Lossless Spheroid Harvesting.

Creating model systems that replicate in vivo tissues is crucial for understanding complex biological pathways like drug response and disease progression. Three-dimensional (3D) in vitro models, especially multicellular spheroids (MCSs), offer valuable insights into physiological processes. However, generating MCSs at scale with consistent properties and efficiently recovering them pose challenges. We introduce a workflow that automates large-scale spheroid production and enables parallel harvesting into individual wells of a microtiter plate. Our method, based on the hanging-drop technique, utilizes a non-contact dispenser for dispensing nanoliter droplets of a uniformly mixed-cell suspension. The setup allows for extended processing times of up to 45 min without compromising spheroid quality. As a proof of concept, we achieved a 99.3% spheroid generation efficiency and maintained highly consistent spheroid sizes, with a coefficient of variance below 8% for MCF7 spheroids. Our centrifugation-based drop transfer for spheroid harvesting achieved a sample recovery of 100%. We successfully transferred HT29 spheroids from hanging drops to individual wells preloaded with collagen matrices, where they continued to proliferate. This high-throughput workflow opens new possibilities for prolonged spheroid cultivation, advanced downstream assays, and increased hands-off time in complex 3D cell culture protocols.

Mitigation of BMP-induced inflammation in craniofacial bone regeneration and improvement of bone parameters by dietary hesperidin

Based on anti-inflammatory and osteogenic properties of hesperidin (HE), we hypothesized its systemic administration could be a cost-effective method of improving BMP-induced bone regeneration. Sprague–Dawley rats were allocated into 4 groups (n = 10/group): a 5-mm critical-sized mandible defect + collagen scaffold or, scaffold + 1 µg of BMP2 with and without dietary HE at 100 mg/kg. HE was administered by oral gavage 4 weeks prior to surgeries until euthanasia at day 7 or 14 post-surgery. The healing tissue within the defect collected at day 7 was subjected to gene expression analysis. Mandibles harvested at day 14 were subjected to microcomputed tomography and histology. HE + BMP2-treated rats had a statistically significant decrease in expression of inflammatory genes compared to BMP2 alone. The high-dose BMP2 alone caused cystic-like regeneration with incomplete defect closure. HE + BMP2 showed virtually complete bone fusion. Collagen fibril birefringence pattern (red color) under polarized light indicated high organization in BMP2-induced newly formed bone (NFB) in HE-supplemented group (p < 0.05). Clear changes in osteocyte lacunae as well as a statistically significant increase in osteoclasts were found around NFB in HE-treated rats. A significant increase in trabecular volume and thickness, and trabecular and cortical density was found in femurs of HE-supplemented rats (p < 0.05). Our findings show, for the first time, that dietary HE has a remarkable modulatory role in the function of locally delivered high-dose BMP2 in bone regeneration possibly via control of inflammation, osteogenesis, changes in osteocyte and osteoclast function and collagen maturation in regenerated and native bone. In conclusion, HE had a significant skeletal bone sparing effect and the ability to provide a more effective BMP-induced craniofacial regeneration.

Structural and mechanical behavior of type-I collagen fibrils in presence of induced electrostatic interactions through ionic liquids

Tuning the self-assembly of collagen has broad applications in the biomedical field owing to their desired biological performance as collagenous materials with tunable functionalities can further determine cellular responses. In this work, an attempt has been made to tune the self-assembly of collagen using ionic liquids, viz., imidazolium chloride (IC) and choline dihydrogen phosphate (CDHP) at its physiological pH, followed by probing assembled systems using various characterization methods. Turbidity measurements of fibrillar networks were performed to ascertain the rate of fibril formation in addition of imidazolium chloride and choline dihydrogen phosphate to collagen at physiological pH. Morphological changes were examined using Scanning Electron Microscope (SEM), binding affinities were measured by Microscale Thermophoresis (MST), in addition to, changes in the shear viscosity, mechanical strength of collagen fibrils when interacted with imidazolium and choline based ILs were carried out using rotational rheometer and Quartz Crystal Microbalance (QCM) measurements. Experimental result depicts that CDHP imparts better crosslinking as well as mechanical strength compare to IC, which is already known for destabilizing the triple helix structure is inhibiting the fibril formation. This self-assembled, ionic-liquid treated collagen-fibrillar system would accelerate various force modulated fibrillar network study, for mimicking the ECM and tissue engineering application.

Melt electrowriting (MEW)-PCL composite Three-Dimensional exosome hydrogel scaffold for wound healing

While mesenchymal stem cell-derived exosomes hold substantial potential in wound healing, challenges persist in terms of large-scale production and activity of 2D-culture derived exosome, as well as addressing their inactivation and loss during application. 3D exosomes can be produced more efficiently and possess higher activity. However, there lacks a delivery patch mimicking nanofibrous architecture of the extracellular matrix while facilitating the in situ delivery of exosomes, thereby minimizing dissipation of exosomes and accelerating the process of wound healing. In this study, we devised a controllable GelMA hydrogel-combined Melt Electrowriting (MEW)-PCL scaffold for in situ 3D-exosome release. We showed that biocompatible scaffolds prepared by MEW have a simulated extracellular matrix with a highly controllable arrangement of nanofibers that can support cell adhesion, proliferation and differentiation. Through cell proliferation, scratch assay, and tube formation experiments, we verified that 3D exosomes could effectively stimulate cell proliferation, migration, and tube formation, with dose-dependent effects. In vivo outcomes exhibited accelerated re-epithelialization, improved collagen maturation, and enhanced angiogenesis. Our findings suggest that 3D-cultured exosomes within the scaffold significantly enhance wound repair. This innovative delivery strategy opens up new avenues for the application of MSC-derived exosomes in wound healing.

PIEZO1-mediated mechanosensing governs NK-cell killing efficiency and infiltration in three-dimensional matrices.

Natural killer (NK) cells play a vital role in eliminating tumorigenic cells. Efficient locating and killing of target cells in complex three-dimensional (3D) environments are critical for their functions under physiological conditions. However, the role of mechanosensing in regulating NK-cell killing efficiency in physiologically relevant scenarios is poorly understood. Here, we report that the responsiveness of NK cells is regulated by tumor cell stiffness. NK-cell killing efficiency in 3D is impaired against softened tumor cells, whereas it is enhanced against stiffened tumor cells. Notably, the durations required for NK-cell killing and detachment are significantly shortened for stiffened tumor cells. Furthermore, we have identified PIEZO1 as the predominantly expressed mechanosensitive ion channel among the examined candidates in NK cells. Perturbation of PIEZO1 abolishes stiffness-dependent NK-cell responsiveness, significantly impairs the killing efficiency of NK cells in 3D, and substantially reduces NK-cell infiltration into 3D collagen matrices. Conversely, PIEZO1 activation enhances NK killing efficiency as well as infiltration. In conclusion, our findings demonstrate that PIEZO1-mediated mechanosensing is crucial for NK killing functions, highlighting the role of mechanosensing in NK-cell killing efficiency under 3D physiological conditions and the influence of environmental physical cues on NK-cell functions.

Human Coronary Artery Endothelial Cell Response to Porphyromonas gingivalis W83 in a Collagen Three-Dimensional Culture Model.

P. gingivalis has been reported to be an endothelial cell inflammatory response inducer that can lead to endothelial dysfunction processes related to atherosclerosis; however, these studies have been carried out in vitro in cell culture models on two-dimensional (2D) plastic surfaces that do not simulate the natural environment where pathology develops. This work aimed to evaluate the pro-inflammatory response of human coronary artery endothelial cells (HCAECs) to P. gingivalis in a 3D cell culture model compared with a 2D cell culture. HCAECs were cultured for 7 days on type I collagen matrices in both cultures and were stimulated at an MOI of 1 or 100 with live P. gingivalis W83 for 24 h. The expression of the genes COX-2, eNOS, and vWF and the levels of the pro-inflammatory cytokines thromboxane A2 (TXA-2) and prostaglandin I2 (PGI2) were evaluated. P. gingivalis W83 in the 2D cell culture increased IL-8 levels at MOI 100 and decreased MCP-1 levels at both MOI 100 and MOI 1. In contrast, the 3D cell culture induced an increased gene expression of COX-2 at both MOIs and reduced MCP-1 levels at MOI 100, whereas the gene expression of eNOS, vWF, and IL-8 and the levels of TXA2 and PGI2 showed no significant changes. These data suggest that in the collagen 3D culture model, P. gingivalis W83 induces a weak endothelial inflammatory response.

Augmentation of keratinized tissue using autogenous soft-tissue grafts and collagen-based soft-tissue substitutes at teeth and dental implants: Histological findings in a pilot pre-clinical study.

To histomorphometrically assess three treatment modalities for gaining keratinized tissue (KT) at teeth and at dental implants. In five dogs, the distal roots of the mandibular second, third and fourth premolars were extracted. Dental implants were placed at the distal root areas 2 months later. After another 2 months, KT augmentation was performed at both distal (implants) and at mesial root (teeth) areas in the presence (wKT groups) or absence (w/oKT groups) of a KT band at the mucosal/gingival level. Three treatment modalities were applied randomly: apically positioned flap only (APF), free gingival grafts (FGGs) and xenogeneic collagen matrices (XCMs). A combination of the above produced six groups. Two months later, tissue sections were harvested and analysed histomorphometrically. The median KT height and length were greatest at implants with FGG in both wKT (3.7 and 5.1 mm, respectively) and w/oKT groups (3.7 and 4.6 mm), and at teeth with FGG in wKT groups (3.7 and 6.1 mm) and with APF in the w/oKT groups (3.9 and 4.4 mm). The XCM and APF produced more favourable results at teeth than at implants. FGG was advantageous in gaining KT, especially at implants.

Bilirubin/morin self-assembled nanoparticle-engulfed collagen/polyvinyl alcohol hydrogel accelerates chronic diabetic wound healing by modulating inflammation and ameliorating oxidative stress

Chronic diabetic wounds pose a serious threat to human health and safety because of their refractory nature and high recurrence rates. The formation of refractory wounds is associated with wound microenvironmental factors such as increased expression of proinflammatory factors and oxidative stress. Bilirubin is a potent endogenous antioxidant, and morin is a naturally active substance that possesses anti-inflammatory and antioxidant effects. Both hold the potential for diabetic wound treatment by intervening in pathological processes. In this study, we developed bilirubin/morin-based carrier-free nanoparticles (BMn) to treat chronic diabetic wounds. In vitro studies showed that BMn could effectively scavenge overproduced reactive oxygen species and suppress elevated inflammation, thereby exerting a protective effect. BMn was then loaded into a collagen/polyvinyl alcohol gel (BMn@G) for an in vivo study to maintain a moist environment for the skin and convenient biomedical applications. BMn@G exhibits excellent mechanical properties, water retention capabilities, and in vivo safety. In type I diabetic mice, BMn@G elevated the expression of the anti-inflammatory factor IL-10 and concurrently diminished the expression of the proinflammatory factor TNF-α in the tissues surrounding the wounds. Furthermore, BMn@G efficiently mediated macrophage polarization from the M1-type to the M2-type, thereby fostering anti-inflammatory effects. Additionally, BMn@G facilitated the conversion of type III collagen fiber bundles to type I collagen fiber bundles, resulting in a more mature collagen fiber structure. This study provides a promising therapeutic alternative for diabetic wound healing.

Modulating TRPV4 Channel Activity in Pro-Inflammatory Macrophages within the 3D Tissue Analog.

Investigating macrophage plasticity emerges as a promising strategy for promoting tissue regeneration and can be exploited by regulating the transient receptor potential vanilloid 4 (TRPV4) channel. The TRPV4 channel responds to various stimuli including mechanical, chemical, and selective pharmacological compounds. It is well documented that treating cells such as epithelial cells and fibroblasts with a TRPV4 agonist enhances the Ca2+ influx to the cells, which leads to secretion of pro-inflammatory cytokines, while a TRPV4 antagonist reduces both Ca2+ influx and pro-inflammatory cytokine secretion. In this work, we investigated the effect of selective TRPV4 modulator compounds on U937-differentiated macrophages encapsulated within three-dimensional (3D) matrices. Despite offering a more physiologically relevant model than 2D cultures, pharmacological treatment of macrophages within 3D collagen matrices is largely overlooked in the literature. In this study, pro-inflammatory macrophages were treated with an agonist, 500 nM of GSK1016790A (TRPV4(+)), and an antagonist, 10 mM of RN-1734 (TRPV4(-)), to elucidate the modulation of the TRPV4 channel at both cellular and extracellular levels. To evaluate macrophage phenotypic alterations within 3D collagen matrices following TRPV4 modulator treatment, we employed structural techniques (SEM, Masson's trichrome, and collagen hybridizing peptide (CHP) staining), quantitative morphological measures for phenotypic assessment, and genotypic methods such as quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC). Our data reveal that pharmacological modulation of the macrophage TRPV4 channel alters the cytoskeletal structure of macrophages and influences the 3D structure encapsulating them. Moreover, we proved that treating macrophages with a TRPV4 agonist and antagonist enhances the expression of pro- and anti-inflammatory genes, respectively, leading to the upregulation of surface markers CD80 and CD206. In the TRPV4(-) group, the CD206 gene and CD206 surface marker were significantly upregulated by 9- and 2.5-fold, respectively, compared to the control group. These findings demonstrate that TRPV4 modulation can be utilized to shift macrophage phenotype within the 3D matrix toward a desired state. This is an innovative approach to addressing inflammation in musculoskeletal tissues.

Platelet-rich fibrin for rehydration and pre-vascularization of an acellular, collagen membrane of porcine origin

ObjectivesPre-vascularization of the collagen membranes with autologous platelet concentrates is a standard procedure in oral and maxillofacial surgery. This study analyzed the possible interaction of an acellular collagen membrane of porcine origin (NM) with platelet-rich fibrin (PRF) regarding its rehydration protocol with differences in pH values and effect on angiogenesis.Materials and methodsNM was analyzed alone and combined with solid PRF by plotting or co-culturing with injectable PRF. Different media (venous blood, buffer solution with a fixed pH value of 7, saline solution, and injectable PRF) were used to analyze the influence on pH value during rehydration. Chorion allantois membrane assay (CAM) was applied to check pro-angiogenic effects after 24, 48, and 72 h, followed by immunohistochemical analysis.ResultsRehydration in injectable PRF showed acidity over time (p < 0.05). A definite pro-angiogenic effect of NM alone was found regarding neo-vessel formation supported by the respective light microscopically analysis without significant differences to PRF alone (p > 0.005). This pro-angiogenic effect could not be exaggerated when NM was combined with liquid/solid PRF (each p > 0.005).ConclusionsRehydration with liquid PRF of the collagen membrane results in acidity compared to a saline solution or patient’s blood. The significant pro-angiogenic potential of the membrane alone resulted in enhanced neo-vessel formation that could not be optimized with the addition of PRF.Clinical relevance statementUsing injectable PRF for rehydration protocol of the collagen membrane leads to acidosis that can ultimately optimize wound healing. Differences in the physio-mechanical interplay of collagen matrices and autologous platelet concentrates must result in clinical algorithms if pre-vascularization can maximize outcomes.

Osteopontine-derived functional fragments coupled to RADA16 self-assembled peptide hydrogels promotes bone and vascular regeneration in vivo

Biomaterial scaffolds have been widely used in tissue engineering. A functionalized self-assembled peptide scaffold named RADA16-OPD was designed by linking the short functional motif of osteopontine (OPN)-derived functional fragments SVVYGLR (OPD) to the C-terminus of the self-assembled peptide RADA16. Atomic force microscopy (AFM) was used to analyze the self-assembling peptide’s structural composition. The live/dead staining results showed that RADA16-OPD is not toxic to rASC. After creating a rat skull defect model artificially, micro-CT results revealed that the defect area treated with RADA16-OPD hydrogel had higher bone volume/total volume (BV/TV), a higher trabecular number (TB.N.), and higher bone density (BMD) at different treatment time points. Histological evaluation found that there was more new bone and mature collagen production in the RADA16-OPD group. Meanwhile, the RADA16-OPD group had higher expression of alkaline phosphatase (ALP) and osteocalcin (OCN) than the other two groups. Additionally, immunofluorescence revealed that the RADA16-OPD group had higher levels of platelet/endothelial cell adhesion molecule 1 (CD31) expression than the other two groups. It demonstrated the potential for clinical use of the RADA16-OPD peptide scaffold by promoting bone regeneration and blood vessel development in vivo.

Influence of photoactivation on tissue response to different dyes used in photodynamic therapy and laser ablation therapy

Laser ablation therapy (LA) uses Indocyanine Green dye (ICG) which efficiently absorbs laser energy and the increased temperature results in an instantaneous flame that chars tissue and microbes. Photodynamic therapy (PDT) uses different dyes that are activated by light to kill bacteria. This study evaluated the biocompatibility of the dye Curcumin (CUR), Methylene Blue (MB), and Indocyanine Green (ICG) before and after laser activation (ACT). Polyethylene tubes containing one of the dyes were implanted in the subcutaneous tissue of 32 rats (4 tubes per rat) which were divided into 8 groups: C - control (saline solution); C + ACT (Red Laser 660 nm); CUR; CUR + ACT (480 nm blue LED); MB; MB + ACT (Red Laser 660 nm); ICG; ICG + ACT (810 nm Infrared Laser). After 7 and 30 days (n = 8/time), the rats were euthanized and the tubes with the surrounding tissue were removed and processed for histological analysis of inflammation using H&E stain, and collagen fiber maturation using picrosirius red (PSR). A two-way analysis of variance statistical test was applied (p < 0.05). At 7 days, regardless of laser activation, the CUR group showed a greater inflammatory infiltrate compared to the ICG and control groups, and the MB group had a greater inflammation only in relation to the control (p < 0.05). At 30 days, CUR and MB groups showed a greater inflammatory infiltrate than the control (p < 0.05). ICG group was equal to the control in both periods, regardless of the laser activation (p > 0.05). Laser activation induced the proliferation of collagen immature fibers at 7 days, regardless of the dye (p < 0.05). The CUR group showed a lower percentage of immature and mature fibers at 7 days, compared to ICG and control (p < 0.05) and, at 30 days, compared to control (p < 0.05). Regardless of laser activation, the ICG showed the results of collagen maturation closest to the control (p > 0.05). It was concluded that all dyes are biocompatible and that laser activation did not interfere with biocompatibility. In addition, the maturity of collagen was adequate before and after the laser activation. These results demonstrate that the clinical use of dyes is safe even when activated with a laser.

Quantifying the Influence of X-Ray Irradiation on Cell-Size-Scale Viscoelasticity of Collagen Type 1.

X-rays are widely used in mammography and radiotherapy of breast cancer. The research has focused on the effects of X-rays on cells in breast tissues, instead of the tissues' nonliving material, extracellular matrix. It is unclear what the influence of X-ray irradiation is on the matrix's mechanical cues, known to regulate malignant cancer-cell behaviors. Here, we developed a technique based on magnetic microrheology that can quantify the influence of X-ray irradiation on matrix viscoelasticity--or (solid-like) elastic and (liquid-like) viscous characteristics--at cell-size scales. To model breast-tissue extracellular matrix, we used the primary component of the tissue matrix, collagen type 1, as it is for control, and as irradiated by X-rays (tube voltage 50 kV). We used a magnetic microrheometer to measure collagen matrices using 10-μm-diameter magnetic probes. In each matrix, the probes were nanomanipulated using controlled magnetic forces by the microrheometer while the probes' displacements were detected to measure the viscoelasticity. The collagen-matrix data involve with a typical spatial variation in viscoelasticity. We find that higher irradiation doses (320 Gy) locally reduce stiffness (soften) collagen matrices and increase their loss tangent, indicating an elevated liquid-like nature. For lower, clinically relevant irradiation doses (54 Gy), we find insignificant matrix-viscoelasticity changes. We provide this irradiation-related technique for detection, and modification, of matrix viscoelastic cues at cell-size scales. The technique enables enhanced characterization of irradiated tissue constituents in a variety of breast-cancer radiotherapy types.

Assessment of mesh shrinkage using fibroblast-populated collagen matrices: a proof of concept for in vitro hernia mesh testing

PurposeThis study uses free-floating contractile fibroblast-populated collagen matrices (FPCMs) to test the shrinkage of different hernia mesh products. We hope to present this model as a proof of concept for the development of in vitro hernia mesh testing—a novel technology with interesting potential.MethodsFPCMs were formed by seeding Human Dermal Fibroblasts into collagen gels. FPCMs were seeded with three different cell densities and cast at a volume of 500 μl into 24-well plates. Five different mesh products were embedded within the collagen constructs. Gels were left to float freely within culture media and contract over 5 days. Photographs were taken daily and the area of the collagen gel and mesh were measured. Media samples were taken at days 2 and 4 for the purposes of measuring MMP-9 release. After 5 days, dehydrated FPCMs were also examined under light and fluorescence microscopy to assess cell morphology.ResultsTwo mesh products—the mosquito net and large pore lightweight mesh were found to shrink notably more than others. This pattern persisted across all three cell densities. There were no appreciable differences observed in MMP-9 release between products.ConclusionsThis study has successfully demonstrated that commercial mesh products can be successfully integrated into free-floating contractile FPCMs. Not only this, but FPCMs are capable of applying a contractile force upon those mesh products—eliciting different levels of contraction between mesh products. Such findings demonstrate this technique as a useful proof of concept for future development of in vitro hernia mesh testing.

Effectiveness of a collagen matrix seal and xenograft in alveolar ridge preservation: an experimental study in dogs

Majority of previous studies on alveolar ridge preservation (ARP) used collagen membranes as barrier membranes, and further evidence for ARP in dehiscent extraction sockets with a deproteinized bovine bone mineral (DBBM) and matrix is needed. The aim of this study is to assess the impact of non-cross linked collagen membranes (membrane) and crosslinked collagen matrices (matrix) on ARP using DBBM in extraction sockets with buccal dehiscence. In six mongrel dogs, the mesial roots of three mandibular premolars (P2, P3, and P4) were extracted 1 month after dehiscence defect induction. Two experimental groups were randomly assigned: (1) DBBM with a membrane (DBBM/membrane group) and (2) DBBM with a matrix (DBBM/matrix group). Three-dimensional (3D) volumetric, microcomputed tomography (μCT), and histologic analyses were performed to assess the ridge preservation. Both groups were effective to maintain the ridge width (p > 0.05), and the DBBM/matrix group showed more favorable soft tissue regeneration and bone quality in the histological analysis (p = 0.05). Based on these results, DBBM/matrix could be better choice for ARP in cases of buccal dehiscence defects.

3D-printed PCL@BG scaffold integrated with SDF-1α-loaded hydrogel for enhancing local treatment of bone defects

BackgroundThe long-term nonunion of bone defects is always a difficult problem in orthopaedics treatment. Artificial bone implants made of polymeric materials are expected to solve this problem due to their suitable degradation rate and good biocompatibility. However, the lack of mechanical strength, low osteogenic induction ability and poor hydrophilicity of these synthetic polymeric materials limit their large-scale clinical application.ResultsIn this study, we used bioactive glass (BG) (20%, W/W) and polycaprolactone (PCL, 80%, W/W) as raw materials to prepare a bone repair scaffold (PCL@BG20) using fused deposition modelling (FDM) three-dimensional (3D) printing technology. Subsequently, stromal cell-derived factor-1α (SDF-1α) chemokines were loaded into the PCL@BG20 scaffold pores with gelatine methacryloyl (GelMA) hydrogel. The experimental results showed that the prepared scaffold had a porous biomimetic structure mimicking that of cancellous bone, and the compressive strength (44.89 ± 3.45 MPa) of the scaffold was similar to that of cancellous bone. Transwell experiments showed that scaffolds loaded with SDF-1α could promote the recruitment of bone marrow stromal cells (BMSCs). In vivo data showed that treatment with scaffolds containing SDF-1α and BG (PCL@BG-GelMA/SDF-1α) had the best effect on bone defect repair compared to the other groups, with a large amount of new bone and mature collagen forming at the bone defect site. No significant organ toxicity or inflammatory reactions were observed in any of the experimental groups.ConclusionsThe results show that this kind of scaffold containing BG and SDF-1α serves the dual functions of recruiting stem cell migration in vivo and promoting bone repair in situ. We envision that this scaffold may become a new strategy for the clinical treatment of bone defects.

Promoting cell proliferation and collagen production with ascorbic acid 2-phosphate-releasing poly(l-lactide-co-ε-caprolactone) membranes for treating pelvic organ prolapse.

Pelvic organ prolapse (POP) afflicts millions of women globally. In POP, the weakened support of the pelvic floor results in the descent of pelvic organs into the vagina, causing a feeling of bulging, problems in urination, defaecation and/or sexual function. However, the existing surgical repair methods for relapsed POP remain insufficient, highlighting the urgent need for more effective alternatives. Collagen is an essential component in pelvic floor tissues, providing structural support, and its production is controlled by ascorbic acid. Therefore, we investigated novel ascorbic acid 2-phosphate (A2P)-releasing poly(l-lactide-co-ε-caprolactone) (PLCLA2P) membranes in vitro to promote cell proliferation and extracellular matrix protein production to strengthen the natural support of the pelvic fascia for POP applications. We analysed the mechanical properties and the impact of PLCLA2P on cellular responses through cell culture analysis using human vaginal fibroblasts (hVFs) and human adipose-derived stem/stromal cells (hASCs) compared to PLCL. In addition, the A2P release from PLCLA2P membranes was assessed in vitro. The PLCLA2P demonstrated slightly lower tensile strength (2.2 ± 0.4 MPa) compared to PLCL (3.7 ± 0.6 MPa) for the first 4 weeks in vitro. The A2P was most rapidly released during the first 48 h of in vitro incubation. Our findings demonstrated significantly increased proliferation and collagen production of both hVFs and hASCs on A2P-releasing PLCLA2P compared to PLCL. In addition, extracellular collagen Type I fibres were detected in hVFs, suggesting enhanced collagen maturation on PLCLA2P. Moreover, increased extracellular matrix protein expression was detected on PLCLA2P in both hVFs and hASCs compared to plain PLCL. In conclusion, these findings highlight the potential of PLCLA2P as a promising candidate for promoting tissue regeneration in applications aimed for POP tissue engineering applications.

Aloe vera peel-derived nanovesicles display anti-inflammatory properties and prevent myofibroblast differentiation.

Aloe vera (AV) is a medicinal plant, most known for its beneficial effects on a variety of skin conditions. Its known active compounds include carbohydrates and flavonoids such as quercetin and kaempferol, among others. In the past decade, plant nanovesicles (NVs) have gained considerable interest as interkingdom communicators, presenting an opportunity for clinical standardization of natural products. In this study, we aimed to assess the potential of AVpNVs for the treatment of burn wounds. AVpNVs were isolated and characterized regarding vesicle yield (nanoparticle tracking analysis) and structure (transmission electron microscopy and atomic force microscopy), as well as their protein content with proteomics. We assessed key characteristics for treating burn wounds in vitro, such as the anti-inflammatory potential in LPS-stimulated macrophages and keratinocytes, and the effect of AVpNVs on myofibroblast differentiation and contraction. AVpNVs presented a homogenous NV population, vesicular shape, and NV-associated protein markers. AVpNVs significantly decreased the secretion of pro-inflammatory cytokines TNFα, IL-1β, and IL-6. Furthermore, AVpNVs inhibited myofibroblast differentiation and significantly decreased their contractile potential in collagen matrices. Observed effects were linked to proteins identified in the isolates through proteomics analysis. AVpNVs displayed characteristics as an inflammatory modulator, while simultaneously diminishing myofibroblast differentiation and contraction. Novel strategies for burn wound treatment seek to decrease scarring on a cellular and molecular level in the early stages of wound healing, which makes AVpNVs a promising candidate for future plant-vesicle-based treatments.

Impact of Bone Morphogenetic Protein 7 and Prostaglandin receptors on osteoblast healing and organization of collagen.

This study seeks to investigate the impact of co-administering either a Prostaglandin EP2 receptor agonist or an EP1 receptor antagonist alone with a low dose BMP7 on in vitro healing process, collagen content and maturation of human osteoblasts. Human osteoblast cells were used in this study. These cells were cultured and subjected to different concentrations of Prostaglandin EP2 receptor agonist, EP1 receptor antagonist, BMP7, Control (Ct) (Vehicle alone), and various combinations treatments. Cell viability at 24, 48 and 72 hours (h) was evaluated using the XTT assay. A wound healing assay was conducted to observe the migration ability of human osteoblast cells. Additionally, Sirius red staining and Fourier-Transform Infrared Spectroscopy Imaging (FT-IR) was employed to analyze various parameters, including total protein concentration, collagen production, mature collagen concentration, and mineral content. The combination of low dose BMP7 and Prostaglandin EP2 receptor agonist resulted to the lowest cell viability when compared to both the Ct and individual treatments. In contrast, the Prostaglandin EP1 receptor antagonist alone showed the highest cellular viability at 72 h. In the wound healing assay, the combined treatment of low dose BMP7 with the Prostaglandin EP2 receptor agonist and EP1 receptor antagonist showed a decrease in human osteoblast healing after 24 h. Analysis of FT-IR data indicated a reduction in total protein content, collagen maturity, collagen concentration and mineral content in combination treatment compared to the single or Ct treatments. The combination of a Prostaglandin EP2 receptor agonist or an EP1 receptor antagonist when combined with low dose BMP7 significantly hinders both human osteoblast healing and collagen maturity/concentration in comparison to low dose BMP7 treatment alone.

Assembled collagen films modified using polyacrylic acid with improved mechanical properties via mineralization.

The imperative task of enforcing collagen materials holds paramount significance in the field of hard tissue repair. We hereby present mineralized collagen fiber films via mineralization with improved mechanical properties. Self-extracted collagen was assembled into an array with an aligned fibrous pattern and then modified with polyacrylic acid (PAA) followed by mineralization in cationic polyacrylamide (CPAM)-SBF. Biomineralization occurred at the inner and outer surface of the assembled collagen fiber films. A tensile strength of up to 40.38 ± 3.08 MPa of mineralized collagen was obtained, for the first time, which may be attributed to the synergistic effect of polyanion and polycation on the mineralization process of assembled intrafibrillar collagen fibers. It was argued that PAA may facilitate the intra-fiber interaction of collagen, which extends the elongation at break of collagen fibers. This study introduces a pioneering approach for the preparation of mineralized collagen materials with superior mechanical properties, which would be beneficial for hard tissue repair.