Regenerative treatments for scleroderma in cutaneous manifestations of the face: a systematic review.

OBJECTIVES: Scleroderma is a heterogeneous chronic autoimmune disease affecting connective tissue, characterised by chronic inflammation and fibrosis, particularly affecting internal organs and skin. Orofacial involvement is common, leading to facial atrophy, mask-like appearance and difficulties in function that significantly impact patients' quality of life. This systematic review evaluates different autologous regenerative treatments of facial manifestations of scleroderma, aiming to provide comprehensive understanding of their effectiveness in reducing fibrosis, and thereby improving function and skin quality. METHODS: A search in PubMed, Embase, Web of Science Core Collection, Cochrane CENTRAL, and CINAHL was conducted. Studies assessing autologous regenerative treatments in cutaneous manifestations of the face in scleroderma patients were included. Outcomes of interest were treatment characteristics, characterisation of biomaterials, outcome measurements and patient satisfaction. Methodological quality was assessed with the Effective Public Health Practice Project tool. RESULTS: In total 18 studies were included. Methodological quality of studies was weak (n=15) and moderate (n=3). Treatments consisted of autologous fat grafting, platelet-rich plasma, stromal vascular fraction, and adipose-derived stem cells. In general, most studies showed improvements of symptoms, but no treatment was considered superior. CONCLUSIONS: Autologous regenerative treatments hold potential for alleviating cutaneous manifestations of the face in scleroderma. Further clinical trials should be well-designed to improve the quality of clinical evidence.

Fibroblast alignment and matrix remodeling induced by a stiffness gradient in a skin-derived extracellular matrix hydrogel.

Large skin injuries heal as scars. Stiffness gradually increases from normal skin to scar tissue (20x higher), due to excessive deposition and crosslinking of extracellular matrix (ECM) mostly produced by (myo)fibroblasts. Using a custom mold, skin-derived ECM hydrogels (dECM) were UV crosslinked after diffusion of ruthenium (Ru) to produce a Ru-dECM gradient hydrogel. The Ru diffusion gradient equates to a stiffness gradient and models physiology of the scarred skin. Crosslinking in Ru-dECM hydrogels results in a 23-fold increase in stiffness from a stiffness similar to that of normal skin. Collagen fiber density increases in a stiffness-dependent fashion while stress relaxation also alters, with one additional Maxwell element necessary for characterizing Ru-dECM. Alignment of fibroblasts encapsulated in hydrogels suggests that the stiffness gradient directs fibroblasts to orientate at ∼45 ° in regions below 120 kPa. In areas above 120 kPa, fibroblasts decrease the stiffness prior to adjusting their orientation. Furthermore, fibroblasts remodel their surrounding ECM in a gradient-dependent fashion, with rearrangement of cell-surrounding ECM in high-stiffness areas, and formation of interlaced collagen bundles in low-stiffness areas. Overall, this study shows that fibroblasts remodel their local environment to generate an optimal ECM mechanical and topographical environment. STATEMENT OF SIGNIFICANCE: This study developed a versatile in vitro model with a gradient stiffness using skin-derived ECM hydrogel with unchanged biochemical environment. Using Ruthenium crosslinking, a 20-fold stiffness increase was achieved as observed in fibrotic skin. The interaction between fibroblasts and matrix depends on changes in the matrix stiffness. The stiffness gradient directed the alignment of fibroblasts with ∼45° in regions with≤ 120 kPa. The cells in regions with the higher stiffness decreased stiffness first and then oriented themselves. Furthermore, fibroblasts remodeled surrounding ECM and regulated its mechanics in a gradient-dependent fashion to reach an optimal condition. Our study highlights the dynamic interplay between cells and surrounding matrix, shedding light on potential mechanisms and strategies to target scar formation and remodeling.

Physical Properties and Biochemical Composition of Extracellular Matrix-Derived Hydrogels Dictate Vascularization Potential in an Organ-Dependent Fashion.

The inherent extracellular matrix (ECM) originating from a specific tissue impacts the process of vascularization, specifically vascular network formation (VNF) orchestrated by endothelial cells (ECs). The specific contribution toward these processes of ECM from highly disparate organs such as the skin and lungs remains a relatively unexplored area. In this study, we compared VNF and ECM remodeling mediated by microvascular ECs within gel, lung, and combinations thereof (hybrid) ECM hydrogels. Irrespective of the EC source, the skin-derived ECM hydrogel exhibited a higher propensity to drive and support VNF compared to both lung and hybrid ECM hydrogels. There were distinct disparities in the physical properties of the three types of hydrogels, including viscoelastic properties and complex architectural configurations, including fiber diameter, pore area, and numbers among the fibers. The hybrid ECM hydrogel properties were unique and not the sum of the component ECM parts. Furthermore, cellular ECM remodeling responses varied with skin ECM hydrogels promoting matrix metalloproteinase 1 (MMP1) secretion, while hybrid ECM hydrogels exhibited increased MMP9, fibronectin, and collagen IV deposition. Principal component analysis (PCA) indicated that the influence of a gel's mechanical properties on VNF was stronger than the biochemical composition. These data indicate that the organ-specific properties of an ECM dictate its capacity to support VNF, while intriguingly showing that ECs respond to more than just the biochemical constituents of an ECM. The study suggests potential applications in regenerative medicine by strategically selecting ECM origin or combinations to manipulate vascularization, offering promising prospects for enhancing wound healing through pro-regenerative interventions.

H3K27Me3 abundance increases fibrogenesis during endothelial-to-mesenchymal transition via the silencing of microRNA-29c.

Objective: Endothelial-to-mesenchymal transition (EndMT) is a transdifferentiation process in which endothelial cells (ECs) adopt a mesenchymal-like phenotype. Over the past few years, it became clear that EndMT can contribute to several cardiovascular pathologies. However, the molecular pathways underlying the development of EndMT remain incompletely understood. Since the epigenetic enzyme Enhancer of Zeste Homolog 2 (EZH2) and its concomitant mark H3K27Me3 have been shown to be elevated in many cardiovascular diseases that associate with EndMT, we hypothesized that H3K27Me3 is a determinant for the susceptibility of EndMT. Methods: To study the association between H3K27Me3 and EndMT, a knockdown model of EZH2 in human endothelial cells (HUVEC) was utilized to reduce H3K27Me3 abundance, followed by induction of EndMT using TGFß1. The expression of molecular markers of EndMT and fibrogenesis were analysed. Results: In cultured HUVECs, a reduction of H3K27Me3 abundance facilitates EndMT but mitigates fibrogenesis as shown by a decreased expression of collagen I and III. In HUVEC, H3K27Me3 abundance directly affects the expression of miR29c, a collagen-targeting miRNA. Additionally, knockdown of miR-29c in HUVEC with low H3K27Me3 abundance partly restored the expression of collagen I and III. Expectedly, in rats with perivascular fibrosis an increased abundance of H3K27Me3 associated with a decreased expression of miR-29c. Conclusion: our data shows that endothelial fibrogenesis underlies an epigenetic regulatory pathway and we demonstrate that a decreased abundance of H3K27Me3 in ECs blunts fibrogenesis in part in a miR-29c dependent manner. Therefore, a reduction of H3K27Me3 could serve as a novel therapeutical strategy to mitigate fibrogenesis and may prove to be beneficial in fibrogenic diseases including atherosclerosis, cardiac fibrosis, and PAH.

Hyperglycemia amplifies TLR-mediated inflammatory response of M(IL4) macrophages to dyslipidemic ligands.

Hyperglycemia is critical for initiation of diabetic vascular complications. We systemically addressed the role of hyperglycemia in the regulation of TLRs in primary human macrophages. Expression of TLRs (1-9) was examined in monocyte-derived M(NC), M(IFNγ), and M(IL4) differentiated in normoglycemic and hyperglycemic conditions. Hyperglycemia increased expression of TLR1 and TLR8 in M(NC), TLR2 and TLR6 in M(IFNγ), and TLR4 and TLR5 in M(IL4). The strongest effect of hyperglycemia in M(IL4) was the upregulation of the TLR4 gene and protein expression. Hyperglycemia amplified TLR4-mediated response of M(IL4) to lipopolysaccharide by significantly enhancing IL1ß and modestly suppressing IL10 production. In M(IL4), hyperglycemia in combination with synthetic triacylated lipopeptide (TLR1/TLR2 ligand) amplified expression of TLR4 and production of IL1ß. In summary, hyperglycemia enhanced the inflammatory potential of homeostatic, inflammatory, and healing macrophages by increasing specific profiles of TLRs. In combination with dyslipidemic ligands, hyperglycemia can stimulate a low-grade inflammatory program in healing macrophages supporting vascular diabetic complications.

Intra-Articular Injection of Adipose-Derived Stromal Vascular Fraction in Osteoarthritic Temporomandibular Joints: Study Design of a Randomized Controlled Clinical Trial.

INTRODUCTION: Temporomandibular joint (TMJ) osteoarthritis is a degenerative disease of the TMJ. It is characterized by progressive degradation of the extracellular matrix components of articular cartilage, with secondary inflammatory components leading to pain in the temporomandibular region and reduced mouth opening. Current treatments do not halt disease progression, hence the need for new therapies to reduce inflammation and, consequently, improve symptoms. The aim of our randomized controlled clinical trial protocol is to investigate the efficacy of adjuvant intra-articular injections of autologous tissue-like stromal vascular fraction (tSVF), compared to arthrocentesis alone, in reducing pain and improving mouth opening in TMJ osteoarthritis patients. MATERIALS AND METHODS: The primary endpoint analysis will consist of the visual analogue scale (VAS) for pain. The secondary endpoint analyses will include maximal interincisal mouth opening measurements; assessment of oral health and mandibular function based on the oral health impact profile (OHIP) questionnaire and mandibular functional impairment questionnaire (MFIQ); complications during the follow up; synovial cytokine analysis at baseline and after 26 weeks; and nucleated cells and tSVF (immuno)histochemistry analyses of the intervention group. DISCUSSION: Our randomized clinical trial protocol will be applied to evaluate the efficacy of a new promising tSVF injection therapy for TMJ osteoarthritis. The safety of intra-articular injections of tSVF has been proven for knee osteoarthritis. However, since a tSVF injection is considered a heterologous application of cell therapy, the regulatory requirements are strict, which makes medical ethical approval challenging.

Extracellular vesicles derived from liver sinusoidal endothelial cells inhibit the activation of hepatic stellate cells and Kupffer cells in vitro.

Liver sinusoidal endothelial cells (LSECs) play a crucial role in maintaining liver microcirculation and exchange of nutrients in the liver and are thought to be involved in the pathogenesis of metabolic dysfunction-associated steatotic liver disease (MASLD). The activation of hepatic stellate cells (HSCs) and Kupffer cells (KCs) has been considered to be responsible for the onset of liver fibrosis and the aggravation of liver injury. However, the paracrine regulatory effects of LSECs in the development of MASLD, in particular the role of LSEC-derived extracellular vesicles (EVs) remains unclear. Therefore, the aim of the present study was to investigate the influence of LSEC-derived EVs on HSCs and KCs. Primary rat LSECs, HSCs and KCs were isolated from male Wistar rats. LSEC-derived EVs were isolated from conditioned medium by ultracentrifugation and analyzed by nanoparticle tracking analysis, and expression of specific markers. LSEC-derived EVs reduced the expression of activation markers in activated HSCs but did not affect quiescent HSCs. Also, LSEC-derived EVs suppressed proliferation of activated HSCs activation, as assessed by Xcelligence and BrdU assay. LSEC-derived EVs also increased the expression of inflammatory genes in HSCs that normally are lowly expression during their activation. In contrast, EVs decreased the expression of inflammatory genes in activated KCs. In summary, our results suggest that LSEC-derived EVs may attenuate the fibrogenic phenotype of activated HSCs and the inflammatory phenotype of KCs. Our results show promise for LSEC-derived EVs as therapeutic moieties to treat MASLD. In addition, these EVs might prove of diagnostic value.

3-D culture of human lung fibroblasts decreases proliferative and increases extracellular matrix remodeling genes.

Fibroblasts are the main producers of extracellular matrix (ECM) responsible for ECM maintenance and repair, a process often disrupted in chronic lung diseases. The accompanying mechanical changes adversely affect resident cells and overall lung function. Numerous models have been used to elucidate fibroblast behavior that are now evolving toward complex three-dimensional (3-D) models incorporating ECM, aiming to replicate the cells' native environment. Little is known about the cellular changes that occur when moving from two-dimensional (2-D) to 3-D cell culture. This study compared the gene expression profiles of primary human lung fibroblasts from seven subjects with normal lung function, that were cultured for 24 h on 2-D collagen I-coated tissue culture plastic and in 3-D collagen I hydrogels, which are commonly used to mimic ECM in various models, from contraction assays to intricate organ-on-a-chip models. Comparing 3-D with 2-D cell culture, 6,771 differentially expressed genes (2,896 up, 3,875 down) were found; enriched gene sets within the downregulated genes, identified through Gene Set Enrichment Analysis and Ingenuity Pathway Analysis, were involved in the initiation of DNA replication which implied downregulation of fibroblast proliferation in 3-D. Observation of cells for 72 h in 2-D and 3-D environments confirmed the reduced progression through the cell cycle in 3-D. A focused analysis, examining the Hippo pathway and ECM-associated genes, showed differential patterns of gene expression in the 3-D versus 2-D culture. Altogether, the transcriptional response of fibroblasts cultured in 3-D indicated inhibition of proliferation, and alterations in Hippo and ECM pathways indicating a complete switch from proliferation to ECM remodeling.NEW & NOTEWORTHY With the introduction of complex three-dimensional (3-D) lung models, comes a need for understanding cellular behavior in these models. We compared gene expression profiles of human lung fibroblasts grown on two-dimensional (2-D) collagen I-coated surfaces with those in 3-D collagen I hydrogels. RNA sequencing and subsequent pathway analyses showed decreased proliferation, increased extracellular matrix (ECM) remodeling, and altered Hippo signaling and ECM deposition-related gene signatures. These findings highlight unique responses of fibroblasts in 3-D models.

Fibroblasts alter the physical properties of dermal ECM-derived hydrogels to create a pro-angiogenic microenvironment.

This study aimed to investigate the impact of fibroblasts (MRC-5) on the extracellular matrix (ECM) microenvironment of endothelial cells (ECs) during the vascularization of skin-derived ECM hydrogel in vitro. Two types of ECs were studied: human dermal microvascular endothelial cells (HMEC) and human pulmonary microvascular endothelial cells (HPMEC). Results showed that the presence of MRC-5 fibroblasts increased the stiffness of the hydrogel and led to larger fiber diameters and increased porosity. Extensive collagen fiber remodeling occurred in the ECM hydrogel with MRC-5 fibroblasts. Additionally, higher levels of fibulin-1 and fibronectin were deposited in the hydrogel when co-cultured with MRC-5 fibroblasts. These findings suggest that MRC-5 fibroblasts play a role in modifying the ECM microenvironment, promoting vascularization through dynamic ECM remodeling.

Towards standardization of human adipose-derived stromal cells secretomes.

The secretome of adipose-derived stromal cells (ASC) is a heterogeneous mixture of components with a beneficial influence on cellular microenvironments. As such, it represents a cell-free alternative in regenerative medicine therapies. Pathophysiological conditions increase the therapeutic capacity of ASC and, with this, the benefits of the secretome. Such conditions can be partially mimicked in vitro by adjusting culturing conditions. Secretomics, the unbiased analysis of a cell secretome by mass spectrometry, is a powerful tool to describe the composition of ASC secretomes. In this proteomics databases review, we compared ASC secretomic studies to retrieve persistently reported proteins resulting from the most explored types of culturing conditions used in research, i.e., exposure to normoxia, hypoxia, or cytokines. Our comparisons identified only eight common proteins within ASC normoxic secretomes, no commonalities within hypoxic ASC secretomes, and only nine within secretomes of ASC exposed to proinflammatory cytokines. Within these, and regardless of the culturing condition that stimulated secretion, a consistent presence of extracellular matrix-related pathways associated with such proteins was identified. Confounders such as donors' age, sex, body mass index, the anatomical area where ASC were harvested, secretome collection method, data description, and how the data is shared with the scientific community are discussed as factors that might explain our outcomes. We conclude that standardization is imperative as the currently available ASC secretomic studies do not facilitate solid conclusions on the therapeutic value of different ASC secretomes.

Adipose Tissue-Derived Components: From Cells to Tissue Glue to Treat Dermal Damage.

In recent decades, adipose tissue transplantation has become an essential treatment modality for tissue (volume) restoration and regeneration. The regenerative application of adipose tissue has only recently proven its usefulness; for example, the method is useful in reducing dermal scarring and accelerating skin-wound healing. The therapeutic effect is ascribed to the tissue stromal vascular fraction (tSVF) in adipose tissue. This consists of stromal cells, the trophic factors they secrete and the extracellular matrix (ECM), which have immune-modulating, pro-angiogenic and anti-fibrotic properties. This concise review focused on dermal regeneration using the following adipose-tissue components: adipose-tissue-derived stromal cells (ASCs), their secreted trophic factors (ASCs secretome), and the ECM. The opportunities of using a therapeutically functional scaffold, composed of a decellularized ECM hydrogel loaded with trophic factors of ASCs, to enhance wound healing are explored as well. An ECM-based hydrogel loaded with trophic factors combines all regenerative components of adipose tissue, while averting the possible disadvantages of the therapeutic use of adipose tissue, e.g., the necessity of liposuction procedures with a (small) risk of complications, the impossibility of interpatient use, and the limited storage options.

Gene expression profiles in mesenchymal stromal cells from bone marrow, adipose tissue and lung tissue of COPD patients and controls.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by irreversible lung tissue damage. Novel regenerative strategies are urgently awaited. Cultured mesenchymal stem/stromal cells (MSCs) have shown promising results in experimental models of COPD, but differences between sources may impact on their potential use in therapeutic strategies in patients. AIM: To assess the transcriptome of lung-derived MSCs (LMSCs), bone marrow-derived MSCs (BM-MSC) and adipose-derived MSCs (AD-MSCs) from COPD patients and non-COPD controls. METHODS: We studied differences in gene expression profiles between the MSC-subtypes, as well as between COPD and control using RNA sequencing (RNA-seq). RESULTS: We show that besides heterogeneity between donors, MSCs from different sources have strongly divergent gene signatures. The growth factors FGF10 and HGF were predominantly expressed in LMSCs. MSCs from all sources displayed altered expression profiles in COPD, with most pronounced significantly up- and downregulated genes in MSCs from adipose tissue. Pathway analysis revealed that the most differentially expressed genes in COPD-derived AD-MSCs are involved in extracellular matrix (ECM) binding and expression. In LMSCs, the gene that differed most strongly between COPD and control was CSGALNACT1, an ECM modulating gene. CONCLUSION: Autologous MSCs from COPD patients display abnormalities with respect to their transcriptome, which were surprisingly most profound in MSCs from extrapulmonary sources. LMSCs may be optimally equipped for lung tissue repair because of the expression of specific growth factor genes.

A modified hydrogel production protocol to decrease cellular content.

PURPOSE: To analyze the cytotoxicity and cell in porcine-derived decellularized skin matrix. METHODS: We analyzed the effect of multiple decellularization processes by histological analysis, DNA quantification, and flow cytometry. Subsequently, we analyzed the most appropriate hydrogel concentration to minimize cytotoxicity on fibroblast culture and to maximize cell proliferation. RESULTS: After the fourth decellularization, the DNA quantification showed the lowest DNA concentration (< 50 ng/mg). Histological analysis showed no cell components in the hydrogel. Moreover, hematoxylin and eosin showed a heterogeneous structure of collagen fibers. The best hydrogel concentration ranged from 3 to 25%, and there was no significant difference between the 24 hours and seven days. CONCLUSIONS: The process of hydrogel production was effective for removing cells and DNA elements. The best hydrogel concentration ranged from 3 to 25%.

From Macro to Micro: Comparison of Imaging Techniques to Detect Vascular Network Formation in Left Ventricle Decellularized Extracellular Matrix Hydrogels.

Background: Angiogenesis is a crucial process in physiological maintenance and tissue regeneration. To understand the contribution of angiogenesis, it is essential to replicate this process in an environment that reproduces the biochemical and physical properties which are largely governed by the extracellular matrix (ECM). We investigated vascularization in cardiac left ventricular ECM hydrogels to mimic post-myocardial repair. We set out to assess and compare different destructive and non-destructive methods, optical as well as non-optical, to visualize angiogenesis and associated matrix remodeling in myocardial ECM hydrogels. Methods: A total of 100,000, 300,000, and 600,000 Human Pulmonary Microvascular Endothelial Cells (HPMEC) were seeded in left ventricular cardiac ECM hydrogel in 48-well plates. After 1, 7, and 14 days of culture, the HPMEC were imaged by inverted fluorescence microscopy and 3D confocal laser scanning microscopy (Zeiss Cell Discoverer 7). In addition, cell-seeded ECM hydrogels were scanned by optical coherence tomography (OCT). Fixed and paraffin-embedded gels were thin-sectioned and assessed for ECM components via H&E, picrosirius red histochemical staining, and immunostaining for collagen type I. ImageJ-based densitometry was used to quantify vascular-like networks and GraphPad was used for statistical analyses. Results: Qualitative analyses were realized through fluoromicrographs obtained by the confocal laser scanning microscope which allowed us to visualize the extensive vascular-like networks that readily appeared at all seeding densities. Quantification of networks was only possible using fluoromicrographs from inverted microscopy. These showed that, after three days, the number of master junctions was seeding density-dependent. The resolution of optical coherence tomography was too low to distinguish between signals caused by the ECM and cells or networks, yet it did show that gels, irrespective of cells, were heterogeneous. Interestingly, (immuno)histochemistry could clearly distinguish between the cast cardiac-derived matrix and newly deposited ECM in the hydrogels. The H&E staining corroborated the presence of vascular-like network structures, albeit that sectioning inevitably led to the loss of 3D structure. Conclusions: Except for OCT, all methods had complementary merit and generated qualitative and quantitative data that allowed us to understand vascular network formation in organ-derived ECM hydrogels.

A Beginner's Guide to the Characterization of Hydrogel Microarchitecture for Cellular Applications.

The extracellular matrix (ECM) is a three-dimensional, acellular scaffold of living tissues. Incorporating the ECM into cell culture models is a goal of cell biology studies and requires biocompatible materials that can mimic the ECM. Among such materials are hydrogels: polymeric networks that derive most of their mass from water. With the tuning of their properties, these polymer networks can resemble living tissues. The microarchitectural properties of hydrogels, such as porosity, pore size, fiber length, and surface topology can determine cell plasticity. The adequate characterization of these parameters requires reliable and reproducible methods. However, most methods were historically standardized using other biological specimens, such as 2D cell cultures, biopsies, or even animal models. Therefore, their translation comes with technical limitations when applied to hydrogel-based cell culture systems. In our current work, we have reviewed the most common techniques employed in the characterization of hydrogel microarchitectures. Our review provides a concise description of the underlying principles of each method and summarizes the collective data obtained from cell-free and cell-loaded hydrogels. The advantages and limitations of each technique are discussed, and comparisons are made. The information presented in our current work will be of interest to researchers who employ hydrogels as platforms for cell culture, 3D bioprinting, and other fields within hydrogel-based research.

Matrix Metalloproteases from Adipose Tissue-Derived Stromal Cells Are Spatiotemporally Regulated by Hydrogel Mechanics in a 3D Microenvironment.

Adipose tissue-derived stromal cells (ASCs) are of interest in tissue engineering and regenerative medicine (TERM) due to their easy acquisition, multipotency, and secretion of a host of factors that promote regeneration. Retention of ASCs in or around lesions is poor following direct administration. Therefore, for TERM applications, ASCs can be 'immobilized' via their incorporation into hydrogels such as gelatine methacryloyl (GelMA). Tweaking GelMA concentration is a common approach to approximate the mechanical properties found in organs or tissues that need repair. Distinct hydrogel mechanics influence the ability of a cell to spread, migrate, proliferate, and secrete trophic factors. Mesenchymal cells such as ASCs are potent remodellers of the extracellular matrix (ECM). Not only do ASCs deposit components, they also secrete matrix metalloproteases (MMPs) which degrade ECM. In this work, we investigated if GelMA polymer concentration influenced the expression of active MMPs by ASCs. In addition, MMPs' presence was interrogated with regard to ASCs morphology and changes in hydrogel ultrastructure. For this, immortalised ASCs were embedded in 5%, 10%, and 15% (w/v) GelMA hydrogels, photopolymerised and cultured for 14 d. Zymography in situ indicated that MMPs had a variable, hydrogel concentration-dependent influence on ASCs-secreted MMPs. In 5% GelMA, ASCs showed a high and sustained expression of MMPs, while, in 10% and 15% GelMA, such expression was almost null. ASCs morphology based on F-actin staining showed that increasing GelMA concentrations inhibit their spreading. Scanning electron microscopy (SEM) showed that hydrogel ultrastructure in terms of pore density, pore size, and percentage porosity were not consistently influenced by cells. Interestingly, changes in ultrastructural parameters were detected also in cell-free materials, albeit without a clear trend. We conclude that hydrogel concentration and its underlying mechanics influenced MMP expression by ASCs. The exact MMPs that respond to these mechanical cues should be defined in follow-up experiments.

Viscoelastic properties of plasma-agarose hydrogels dictate favorable fibroblast responses for skin tissue engineering applications.

Dermal wound healing relies on the properties of the extracellular matrix (ECM). Thus, hydrogels that replicate skin ECM have reached clinical application. After a dermal injury, a transient, biodegradable fibrin clot is instrumental in wound healing. Human plasma, and its main constituent, fibrin would make a suitable biomaterial for improving wound healing and processed as hydrogels albeit with limited mechanical strength. To overcome this, plasma-agarose (PA) composite hydrogels have been developed and used to prepare diverse bioengineered tissues. To date, little is known about the influence of variable agarose concentrations on the viscoelastic properties of PA hydrogels and their correlation to cell biology. This study reports the characterization of the viscoelastic properties of different concentrations of agarose in PA hydrogels: 0 %, 0.5 %, 1 %, 1.5 %, and 2 % (w/v), and their influence on the cell number and mitochondrial activity of human dermal fibroblasts. Results show that agarose addition increased the stiffness, relaxation time constants 1 (τ1) and 2 (τ2), and fiber diameter, whereas the porosity decreased. Changes in cell metabolism occurred at the early stages of culturing and correlated to the displacement of fast (τ1) and intermediate (τ2) Maxwell elements. Fibroblasts seeded in low PA concentrations spread faster during 14 d than cells cultured in higher agarose concentrations. Collectively, these results confirm that PA viscoelasticity and hydrogel architecture strongly influenced cell behavior. Therefore, viscoelasticity is a key parameter in the design of PA-based implants.

Lytic cocktail: An effective method to alleviate severe burn induced hyper-metabolism through regulating white adipose tissue browning.

Background: Browning of white adipose tissue is associated with elevated resting metabolic rates and is considered to be one of the indispensable causes of hypermetabolism in burn patients. Hypermetabolism means increased resting energy expenditure, raised body temperature and acute-phase proteins. Persistently elevated levels of circulating stress hormones have been reported to induce browning of subcutaneous white adipose tissue. The lytic cocktail is a combination of medicines pethidine, chlorpromazine, and promethazine that has been used clinically in sedation for the management of patients. As reported this sedative treatment can reduce the expression of catecholamines in major burn rats. Thus, in this paper we focused on the effects of lytic cocktail in the regulation of white adipose tissue browning and hypermetabolism and we further investigated the underlying mechanism. Methods: A 30% total body surface area (TBSA) Ⅲ degree scald rat model was used for this study. The rats were randomly divided into a sham scald group, a scalding with immediate resuscitation group, and a group of scalding with immediate resuscitation and lytic cocktail treatment. The levels of norepinephrine and epinephrine in plasma were dynamically detected. Changes of the rat body weight and food intake were recorded and compared as indexes of metabolism responses after post-scalding. For the study of white adipose tissue browning, inguinal adipose tissue was used. Metabolic changes, while indicatives of white fat browning were measured by PET/CT. The expression of white adipose browning related proteins and the changes of mitochondria number were used to assess browning of inguinal adipose. Results: The level of plasma catecholamines norepinephrine and epinephrine in the lytic cocktail-treated group was significantly lower than the other two groups. Morphology and PET/CT showed that the inguinal white adipose browning was inhibited in the lytic cocktail treated group, whereas scalding with immediate resuscitation group showed browning of white adipose. The number of mitochondria, the expressions of white adipose browning related proteins in the lytic cocktail group were also significantly lower than that of the group of scalding with immediate resuscitation. Conclusion: By reducing expression of heat-related proteins, the application of lytic cocktail medicines inhibits the white adipose tissue browning, which suppresses hypermetabolism in scalded rats. The mechanism might be related to decreased expression levels of stress hormones induced by lytic cocktail. This research suggests that lytic cocktails may be an effective treatment for hypermetabolism after severe burn injury.

Stromal vascular fraction-enriched fat grafting as treatment of adherent scars: study design of a non-randomized early phase trial.

BACKGROUND: In the last decades, autologous fat grafting has been used to treat adherent dermal scars. The observed regenerative and scar-reducing properties have been mainly ascribed to the tissue-derived stromal vascular fraction (tSVF) in adipose tissue. Adipose tissue's components augment local angiogenesis and mitosis in resident tissue cells. Moreover, it promotes collagen remodeling. We hypothesize that tSVF potentiates fat grafting-based treatment of adherent scars. Therefore, this study aims to investigate the effect of tSVF-enriched fat grafting on scar pliability over a 12-month period. METHODS AND DESIGN: A clinical multicenter non-randomized early phase trial will be conducted in two dedicated Dutch Burn Centers (Red Cross Hospital, Beverwijk, and Martini Hospital, Groningen). After informed consent, 46 patients (≥18 years) with adherent scars caused by burns, necrotic fasciitis, or degloving injury who have an indication for fat grafting will receive a sub-cicatricic tSVF-enriched fat graft. The primary outcome is the change in scar pliability measured by the Cutometer between pre- and 12 months post-grafting. Secondary outcomes are scar pliability (after 3 months), scar erythema, and melanin measured by the DSM II Colormeter; scar quality assessed by the patient and observer scales of the Patient and Observer Scar Assessment Scale (POSAS) 2.0; and histological analysis of scar biopsies (voluntary) and tSVF quality and composition. This study has been approved by the Dutch Central Committee for Clinical Research (CCMO), NL72094.000.20. CONCLUSION: This study will test the clinical efficacy of tSVF-enriched fat grafting to treat dermal scars while the underlying working mechanism will be probed into too. TRIAL REGISTRATION: Dutch Trial Register NL 8461. Registered on 16 March 2020.

Cytotoxicity Assessment of Surface-Modified Magnesium Hydroxide Nanoparticles.

Magnesium-based nanoparticles have shown promise in regenerative therapies in orthopedics and the cardiovascular system. Here, we set out to assess the influence of differently functionalized Mg nanoparticles on the cellular players of wound healing, the first step in the process of tissue regeneration. First, we thoroughly addressed the physicochemical characteristics of magnesium hydroxide nanoparticles, which exhibited low colloidal stability and strong aggregation in cell culture media. To address this matter, magnesium hydroxide nanoparticles underwent surface functionalization by 3-aminopropyltriethoxysilane (APTES), resulting in excellent dispersible properties in ethanol and improved colloidal stability in physiological media. The latter was determined as a concentration- and time-dependent phenomenon. There were no significant effects on THP-1 macrophage viability up to 1.500 µg/mL APTES-coated magnesium hydroxide nanoparticles. Accordingly, increased media pH and Mg2+ concentration, the nanoparticles dissociation products, had no adverse effects on their viability and morphology. HDF, ASCs, and PK84 exhibited the highest, and HUVECs, HPMECs, and THP-1 cells the lowest resistance toward nanoparticle toxic effects. In conclusion, the indicated high magnesium hydroxide nanoparticles biocompatibility suggests them a potential drug delivery vehicle for treating diseases like fibrosis or cancer. If delivered in a targeted manner, cytotoxic nanoparticles could be considered a potential localized and specific prevention strategy for treating highly prevalent diseases like fibrosis or cancer. Looking toward the possible clinical applications, accurate interpretation of in vitro cellular responses is the keystone for the relevant prediction of subsequent in vivo biological effects.