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Chronic, non-healing, dermal wounds form a worldwide medical problem with limited and inadequate treatment options and high societal burden and costs. With the advent of regenerative therapies exploiting extracellular matrix (ECM) components, its efficacy to augment wound healing is to be explored. This systematic review was performed to assess and compare the current therapeutic efficacy of ECM hydrogels on dermal wound healing. The electronic databases of Embase, Medline Ovid, and Cochrane Central were searched for in vivo and clinical studies on the therapeutic effect of ECM-composed hydrogels on dermal wound healing (April 13, 2021). Two reviewers selected studies independently. Studies were assessed based on ECM content, ECM hydrogel composition, additives, and wound healing outcomes, such as wound size, angiogenesis, and complications. Of the 2102 publications, 9 rodent-based studies were included while clinical studies were not published at the time of the search. Procedures to decellularize tissue or cultured cells and subsequently generate hydrogels were highly variable and in demand of standardization. ECM hydrogels with or without additives reduced wound size and also seem to enhance angiogenesis. Serious complications were not reported. To date, preclinical studies preclude to draw firm conclusions on the efficacy and working mechanism of ECM-derived hydrogels on dermal wound healing. The use of ECM hydrogels can be considered safe. Standardization of decellularization protocols and implementation of quality and cytotoxicity controls will enable obtaining a generic and comparable ECM product. Impact statementExtracellular matrix (ECM)-based hydrogels are biocompatible and harbor growth factors that can instruct tissue healing. Their application is a novelty in (pre)clinical wound healing treatment. This systematic review provides an overview of the current evidence for ECM hydrogels in enhancing wound healing and an extensive overview of the decellularization procedures used. Lastly, challenges and future directions to standardize decellularization procedures and implement quality controls are proposed.

Extracellular matrix (ECM) is a dynamic network of proteins, proteoglycans and glycosaminoglycans, providing structure to the tissue and biochemical and biomechanical instructions to the resident cells. In fibrosis, the composition and the organization of the ECM are altered, and these changes influence cellular behaviour. Biochemical (i. e. protein composition) and biomechanical changes in ECM take place simultaneously in vivo. Investigating these changes individually in vitro to examine their (patho)physiological effects has been difficult. In this study, we generated an in vitro model to reflect the altered mechanics of a fibrotic microenvironment through applying fibre crosslinking via ruthenium/sodium persulfate crosslinking on native lung ECM-derived hydrogels. Crosslinking of the hydrogels without changing the biochemical composition of the ECM resulted in increased stiffness and decreased viscoelastic stress relaxation. The altered stress relaxation behaviour was explained using a generalized Maxwell model. Fibre analysis of the hydrogels showed that crosslinked hydrogels had a higher percentage of matrix with a high density and a shorter average fibre length. Fibroblasts seeded on ruthenium-crosslinked lung ECM-derived hydrogels showed myofibroblastic differentiation with a loss of spindle-like morphology together with greater α-smooth muscle actin (α-SMA) expression, increased nuclear area and circularity without any decrease in the viability, compared with the fibroblasts seeded on the native lung-derived ECM hydrogels. In summary, ruthenium crosslinking of native ECM-derived hydrogels provides an exciting opportunity to alter the biomechanical properties of the ECM-derived hydrogels while maintaining the protein composition of the ECM to study the influence of mechanics during fibrotic lung diseases. STATEMENT OF SIGNIFICANCE: Fibrotic lung disease is characterized by changes in composition and excessive deposition of extracellular matrix (ECM). ECM fibre structure also changes due to crosslinking, which results in mechanical changes. Separating the changes in composition and mechanical properties has been difficult to date. In this study, we developed an in vitro model that allows alteration of the mechanical changes alone by applying fibre crosslinking in native lung ECM-derived hydrogels. Characterisations of the crosslinked hydrogels indicated the model mimicked mechanical properties of fibrotic lung tissue and reflected altered fibre organisation. This ECM-based fibrosis model provides a method to preserve the native protein composition while altering the mechanical properties providing an important tool not only for lung but also other organ fibrosis.

Background Wound healing and scar formation depends on a plethora of factors. Given the impact of abnormal scar formation, interventions aimed to improve scar formation would be most advantageous. The tissue stromal vascular fraction (tSVF) of adipose tissue is composed of a heterogenous mixture of cells embedded in extracellular matrix. It contains growth factors and cytokines involved in wound-healing processes, eg, parenchymal proliferation, inflammation, angiogenesis, and matrix remodeling. Objectives The aim of this study was to investigate the hypothesis that tSVF reduces postsurgical scar formation. Methods This prospective, double-blind, placebo-controlled, randomized trial was conducted between 2016 and 2020. Forty mammoplasty patients were enrolled and followed for 1 year. At the end of the mammoplasty procedure, all patients received tSVF in the lateral 5 cm of the horizontal scar of 1 breast and a placebo injection in the contralateral breast to serve as an intrapatient control. Primary outcome was scar quality measure by the Patient and Observer Scar Assessment Scale (POSAS). Secondary outcomes were obtained from photographic evaluation and histologic analysis of scar tissue samples. Results Thirty-four of 40 patients completed follow-up. At 6 months postoperation, injection of tSVF had significantly improved postoperative scar appearance as assessed by the POSAS questionnaire. No difference was observed at 12 months postoperation. No improvement was seen based on the evaluation of photographs and histologic analysis of postoperative scars between both groups. Conclusions Injection of tSVF resulted in improved wound healing and reduced scar formation at 6 months postoperation, without any noticeable advantageous effects seen at 12 months. Level of Evidence: 2

Background For decades, facial fat grafting has been used in clinical practice for volume restoration. The main challenge of this technique is variable volume retention. The addition of supplements to augment fat grafts and increase volume retention has been reported in recent years. Objectives The aim of this systematic review was to investigate which supplements increase volume retention in facial fat grafting as assessed by volumetric outcomes and patient satisfaction. Methods Embase, Medline, Ovid, Web of Science Core Collection, Cochrane Central Register of Controlled Trials, and Google Scholar were searched up to November 30, 2020. Only studies assessing volume after facial fat grafting with supplementation in human subjects were included. Outcomes of interest were volume or patient satisfaction. The quality of the studies was assessed with the Effective Public Health Practice Project tool. Results After duplicates were removed 3724 studies were screened by title and abstract. After reading 95 full-text articles, 27 studies were eligible and included for comparison. Supplementation comprised of platelet-rich plasma, platelet-rich fibrin, adipose tissue–derived stromal cells or bone marrow–derived stromal cells, cellular or tissue stromal vascular fraction, or nanofat. In 13 out of 22 studies the supplemented group showed improved volumetric retention and 5 out of 16 studies showed greater satisfaction. The scientific quality of the studies was rated as weak for 20 of 27 studies, moderate for 6 of 27 studies, and strong for 1 study. Conclusions It remains unclear if additives contribute to facial fat graft retention and there is a need to standardize methodology. Level of Evidence: 4

Background Lipofilling has become popular as a treatment to improve aging-related skin characteristics (eg, wrinkles, pigmentation spots, pores, or rosacea). Different additives such as platelet-rich plasma (PRP) or stromal vascular fraction (SVF) have been combined with lipofilling to increase the therapeutic effect of adipose-derived stromal cells (ASCs). Objectives The aim of this study was to examine the hypothesis that mechanically isolated SVF augments the therapeutic effect of PRP-supplemented lipofilling to improve facial skin quality. Methods This prospective, double-blind, placebo-controlled, randomized trial was conducted between 2016 and 2019. In total, 28 female subjects were enrolled; 25 completed the follow-up. All patients received PRP-supplemented lipofilling with either mechanically isolated SVF or saline. SVF was isolated by fractionation of adipose tissue (tSVF). Results were evaluated by changes in skin elasticity and transepidermal water loss, changes in skin-aging-related features, ie, superficial spots, wrinkles, skin texture, pores, vascularity, and pigmentation, as well as patient satisfaction (FACE-Q), recovery, and number of complications up to 1 year postoperative. Results The addition of tSVF to PRP-supplemented lipofilling did not improve skin elasticity, transepidermal water loss, or skin-aging-related features. No improvement in patient satisfaction with overall facial appearance or facial skin quality was seen when tSVF was added to PRP-supplemented lipofilling. Conclusions In comparison to PRP-supplemented lipofilling, PRP-supplemented lipofilling combined with tSVF does not improve facial skin quality or patient satisfaction in a healthy population. PRP-supplemented lipofilling combined with tSVF can be considered a safe procedure. Level of Evidence: 2

Background Autologous lipofilling is an emerging procedure to treat and possibly reverse dermal scars and to reduce scar-related pain, but its efficacy and mechanisms are poorly understood. Objectives The aim of this study was to test the hypothesis that repeated lipografts reverse dermal scars by reinitiation of wound healing. Methods In a prospective, non-placebo-controlled clinical study, 27 adult patients with symptomatic scars were given 2 lipofilling treatments at 3-month intervals. As primary outcome, clinical effects were measured with the Patient and Observer Scar Assessment Scale (POSAS). Scar biopsies were taken before and after treatments to assess scar remodeling at a cellular level. Results Twenty patients completed the study. Patients’ scars improved after lipofilling. The total POSAS scores (combined patient and observer scores) decreased from 73.2 [14.7] points (mean [standard deviation]) pretreatment to 46.1 [14.0] and 32.3 [13.2] points after the first and second lipofilling treatment, respectively. Patient POSAS scores decreased from 37.3 [8.8] points to 27.2 [11.3] and 21.1 [11.4] points, whereas observer POSAS scores decreased from 35.9 [9.5] points to 18.9 [6.0] and 11.3 [4.5] points after the first and second treatment, respectively. After each lipofilling treatment, T lymphocytes, mast cells, and M2 macrophages had invaded scar tissue and were associated with increased vascularization. In addition, the scar-associated epidermis showed an increase in epidermal cell proliferation to levels similar to that normal in skin. Moreover, lipofilling treatment caused normalization of the extracellular matrix organization towards that of normal skin. Conclusions Autologous lipofilling improves the clinical outcome of dermal scars through the induction of a pro-regenerative immune response, increased vascularization, and epidermal proliferation and remodeling of scar tissue extracellular matrix. Level of Evidence: 4

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.

The rate of the remodeling of the arterialized saphenous vein conduit limits the outcomes of coronary artery bypass graft surgery (CABG), which may be influenced by endothelial dysfunction. We tested the hypothesis that high stretch (HS) induces human saphenous vein endothelial cell (hSVEC) dysfunction and examined candidate underlying mechanisms. Our results showed that in vitro HS reduces NO bioavailability, increases inflammatory adhesion molecule expression (E-selectin and VCAM1) and THP-1 cell adhesion. HS decreases F-actin in hSVECs, but not in human arterial endothelial cells, and is accompanied by G-actin and cofilin’s nuclear shuttling and increased reactive oxidative species (ROS). Pre-treatment with the broad-acting antioxidant N-acetylcysteine (NAC) supported this observation and diminished stretch-induced actin remodeling and inflammatory adhesive molecule expression. Altogether, we provide evidence that increased oxidative stress and actin cytoskeleton remodeling play a role in HS-induced saphenous vein endothelial cell dysfunction, which may contribute to predisposing saphenous vein graft to failure.

Chronic lung diseases such as idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD) are associated with changes in extracellular matrix (ECM) composition and abundance affecting the mechanical properties of the lung. This study aimed to generate ECM hydrogels from control, severe COPD [Global Initiative for Chronic Obstructive Lung Disease (GOLD) IV], and fibrotic human lung tissue and evaluate whether their stiffness and viscoelastic properties were reflective of native tissue. For hydrogel generation, control, COPD GOLD IV, and fibrotic human lung tissues were decellularized, lyophilized, ground into powder, porcine pepsin solubilized, buffered with PBS, and gelled at 37°C. Rheological properties from tissues and hydrogels were assessed with a low-load compression tester measuring the stiffness and viscoelastic properties in terms of a generalized Maxwell model representing phases of viscoelastic relaxation. The ECM hydrogels had a greater stress relaxation than tissues. ECM hydrogels required three Maxwell elements with slightly faster relaxation times (τ) than that of native tissue, which required four elements. The relative importance (Ri) of the first Maxwell element contributed the most in ECM hydrogels, whereas for tissue the contribution was spread over all four elements. IPF tissue had a longer-lasting fourth element with a higher Ri than the other tissues, and IPF ECM hydrogels did require a fourth Maxwell element, in contrast to all other ECM hydrogels. This study shows that hydrogels composed of native human lung ECM can be generated. Stiffness of ECM hydrogels resembled that of whole tissue, while viscoelasticity differed.

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.

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

The extracellular matrix provides mechanical cues to cells within it, not just in terms of stiffness (elasticity) but also time-dependent responses to deformation (viscoelasticity). In this work, we determined the viscoelastic transformation of gelatine methacryloyl (GelMA) hydrogels caused by adipose tissue-derived stromal cells (ASCs) through mathematical modelling. GelMA-ASCs combination is of interest to model stem cell-driven repair and to understand cell-biomaterial interactions in 3D environments. Immortalised human ASCs were embedded in 5%, 10%, and 15% (w/v) GelMA hydrogels and evaluated for 14 d. GelMA had a concentration-dependent increase in stiffness, but cells decreased this stiffness over time, across concentrations. Viscoelastic changes in terms of stress relaxation increased progressively in 5% GelMA, while mathematical Maxwell analysis showed that the relative importance (Ri) of the fastest Maxwell elements increased proportionally. The 10% GelMA only showed differences at 7 d. In contrast, ASCs in 15% GelMA caused slower stress relaxation, increasing the Ri of the slowest Maxwell element. We conclude that GelMA concentration influenced the stiffness and number of Maxwell elements. ASCs changed the percentage stress relaxation and Ri of Maxwell elements transforming hydrogel viscoelasticity into a more fluid environment over time. Overall, 5% GelMA induced the most favourable ASC response.

The proteins and polysaccharides of the extracellular matrix (ECM) provide architectural support as well as biochemical and biophysical instruction to cells. Decellularized, ECM hydrogels replicate in vivo functions. The ECM’s elasticity and water retention renders it viscoelastic. In this study, we compared the viscoelastic properties of ECM hydrogels derived from the skin, lung and (cardiac) left ventricle and mathematically modelled these data with a generalized Maxwell model. ECM hydrogels from the skin, lung and cardiac left ventricle (LV) were subjected to a stress relaxation test under uniaxial low-load compression at a 20%/s strain rate and the viscoelasticity determined. Stress relaxation data were modelled according to Maxwell. Physical data were compared with protein and sulfated GAGs composition and ultrastructure SEM. We show that the skin-ECM relaxed faster and had a lower elastic modulus than the lung-ECM and the LV-ECM. The skin-ECM had two Maxwell elements, the lung-ECM and the LV-ECM had three. The skin-ECM had a higher number of sulfated GAGs, and a highly porous surface, while both the LV-ECM and the lung-ECM had homogenous surfaces with localized porous regions. Our results show that the elasticity of ECM hydrogels, but also their viscoelastic relaxation and gelling behavior, was organ dependent. Part of these physical features correlated with their biochemical composition and ultrastructure.

Endothelial–mesenchymal transition (EndMT) is a form of endothelial dysfunction wherein endothelial cells acquire a mesenchymal phenotype and lose endothelial functions, which contributes to the pathogenesis of intimal hyperplasia and atherosclerosis. The mitogen activated protein kinase 7 (MAPK7) inhibits EndMT and decreases the expression of the histone methyltransferase Enhancer-of-Zeste homologue 2 (EZH2), thereby maintaining endothelial quiescence. EZH2 is the catalytic subunit of the Polycomb Repressive Complex 2 that methylates lysine 27 on histone 3 (H3K27me3). It is elusive how the crosstalk between MAPK7 and EZH2 is regulated in the endothelium and if the balance between MAPK7 and EZH2 is disturbed in vascular disease. In human coronary artery disease, we assessed the expression levels of MAPK7 and EZH2 and found that with increasing intima/media thickness ratio, MAPK7 expression decreased, whereas EZH2 expression increased. In vitro, MAPK7 activation decreased EZH2 expression, whereas endothelial cells deficient of EZH2 had increased MAPK7 activity. MAPK7 activation results in increased expression of microRNA (miR)-101, a repressor of EZH2. This loss of EZH2 in turn results in the increased expression of the miR-200 family, culminating in decreased expression of the dual-specificity phosphatases 1 and 6 who may repress MAPK7 activity. Transfection of endothelial cells with miR-200 family members decreased the endothelial sensitivity to TGFβ1-induced EndMT. In endothelial cells there is reciprocity between MAPK7 signaling and EZH2 expression and disturbances in this reciprocal signaling associate with the induction of EndMT and severity of human coronary artery disease.

Tissue engineering of skeletal muscle aims to replicate the parallel alignment of myotubes on the native tissue. Directional topography gradients allow the study of the influence of topography on cellular orientation, proliferation, and differentiation, resulting in yield cues and clues to develop a proper in vitro environment for muscle tissue engineering. In this study, we used a polydimethylsiloxane-based substrate containing an aligned topography gradient with sinusoidal features ranging from wavelength (lambda) = 1,520 nm and amplitude (A) =176 nm to lambda = 9,934 nm and A = 2,168 nm. With this topography gradient, we evaluated the effect of topography on human myoblasts distribution, dominant orientation, cell area, nuclei coverage, cell area per number of nuclei, and nuclei area of myotubes. We showed that human myoblasts aligned and differentiated irrespective of the topography section. In addition, aligned human myotubes showed functionality and maturity by contracting spontaneously and nuclei peripheral organization resembling natural myotubes.

Background Adipose tissue has been widely used in regenerative surgery for its therapeutic potential. This potential is often ascribed to the stromal vascular fraction (SVF), which can be mechanically isolated. Mechanical isolation results in an SVF that retains intact cell-cell communication including extracellular matrix and is therefore named tissue-SVF (tSVF). Objectives The aim of this study was to evaluate a new disposable 1-hole fractionator for fractionation of adipose tissue (FAT), and compare this new device with the existing reusable 3-hole fractionator. Methods The composition of tSVF obtained via the 1-hole fractionator was histologically and histochemically compared to unprocessed adipose tissue. The number of viable nuclear cells in tSVF obtained by the 1-hole and 3-hole fractionators as well as unprocessed adipose tissue were compared after enzymatic isolation and tested for colony-forming capacity. Flow cytometry was used to compare different cell compositions based on surface marker expression between tSVF isolated by the two types of fractionators. Results Fractionation of adipose tissue with the 1-hole fractionator condenses vasculature and extracellular matrix by disrupting adipocytes. The number of viable nuclear cells in tSVF obtained with the two fractionators was comparable and significantly higher than unprocessed lipoaspirate. Furthermore, tSVF isolated by both fractionators showed similar cell compositions and comparable colony-forming capacities. Conclusions FAT with a disposable 1-hole fractionator effectively isolates tSVF with a cell count and cell composition comparable to the fraction obtained with the 3-hole reusable fractionator. The disposable 1-hole fractionator, however, is safer and more user friendly.

In teeth with an injured pulp, dentin matrix orchestrates hard tissue repair through the release of dentin extracellular matrix components (dEMCs). dEMCs regulate the differentiation of resident mesenchymal stromal cells (MSCs), thereby affecting mineral deposition. In this study, we show that low-concentration solubilized dEMCs in osteogenic cultures of human umbilical cord mesenchymal stromal cells (UC-MSCs) and dental pulp stromal cells (DPSCs) enhanced mineral deposition, while adipose stromal cells (ASCs) were barely affected. Interestingly, UC-MSCs displayed significantly greater hydroxyapatite formation compared with DPSCs. UC-MSCs and DPSCs showed a dose-dependent viability and proliferation, whereas proliferation of ASCs remained unaffected. Qualitative analysis of the dEMC-supplemented osteogenic cultures through scanning electron microscopy demonstrated differences in the architecture of the deposited mineralized structures. Large-sized mineral accretions on a poorly organized collagen network were the prominent feature of UC-MSC cultures, while mineral nodules interspersed throughout a collagen mesh were observed in the respective DPSC cultures. The ability of dEMCs to induce mineralization varies between different human MSC types in terms of total mineral formation and architecture. Mineral formation by UC-MSCs exposed to low-concentration dEMCs proved to be the most efficient and therefore could be considered a promising combination for mineralized tissue engineering. Impact Statement This research has been conducted with the aim to contribute to the development of treatment modalities for the reconstruction of lost/damaged mineralized tissues. Currently, determining the most appropriate stromal cell population and signaling cues stands at the core of developing effective treatments. We provide new insights into the effect of innate inductive cues found in human dentin matrix components, on the osteogenic differentiation of various human stromal cell types. The effects of dentin extracellular matrix components on umbilical cord mesenchymal stromal cells have not been investigated before. The findings of this study could underpin translational research based on the development of techniques for mineralized tissue engineering and will be of great interest for the readership of Tissue Engineering Part A.

INTRODUCTION: Fat grafting is an established clinical intervention to promote tissue repair. The role of the fat's extracellular matrix (ECM) in regeneration is largely neglected. We investigated in vitro the use of human adipose tissue-derived ECM hydrogels as release platform for factors secreted by adipose derived stromal cells (ASC) MATERIAL & METHODS: Lipoaspirates from non-diabetic and diabetic donors were decellularized. Finely powdered acellular ECM was evaluated for cell remainders and DNA content. Acellular ECM was digested and hydrogels were formed at 37 °C and their viscoelastic relaxation properties investigated. Release of ASC-released factors from hydrogels was immune-assessed and bio-activity was determined by fibroblast proliferation and migration and endothelial angiogenesis.RESULTS: Acellular ECM contained no detectable cell remainders and negligible DNA contents. Viscoelastic relaxation measurements yielded no data for diabetic-derived hydrogels due to gel instability. Hydrogels released several ASC-released factors concurrently in a sustained fashion. Functionally, released factors stimulated fibroblast proliferation and migration as well as angiogenesis. No difference between non-diabetic and diabetic hydrogels in release of factors was measured.CONCLUSION: Adipose ECM hydrogels incubated with released factors by ASC are a promising new therapeutic modality to promote several important wound healing related processes by releasing factors in a controlled way.

The use of Magnesium as biomedical implant has increased in recent years due to its ability to reabsorb in the body without causing adverse effects. For this kind of application, sterilization method should be considered to ensure the appropriate performance of the implant. The present study focused on the influence of different sterilization techniques such as steam autoclaving, dry-heating (170 degrees C), UV irradiation and steam formaldehyde on surface characteristics, composition and wettability of commercially pure Mg anodized in a silicate solution. Characterization of the samples was done by SEM, EDS, and XRD. Surface free energy was calculated by contact angle measurement. Changes in biological activity were assessed in vitro as a hemolysis assay. Our results showed that UV treatment generated only minor surface changes, but that irregularly shaped surface pores might prevent full penetrance and sterilization of the assessed materials. Dry-heat induced cracks on the surfaces and formaldehyde affected the surface morphology. Despite autoclave was the treatment that showed the highest changes in the surface energy, it did not induce structural surface changes and therefore it was considered as the choice option for the sterilization of Mg samples.

In a diabetic milieu high levels of reactive oxygen species (ROS) are induced. This contributes to the vascular complications of diabetes. Recent studies have shown that ROS formation is exacerbated in diabetic monocytes and macrophages due to a glycolytic metabolic shift. Macrophages are important players in the progression of diabetes and promote inflammation through the release of pro-inflammatory cytokines and proteases. Because ROS is an important mediator for the activation of pro-inflammatory signaling pathways, obesity and hyperglycemia-induced ROS production may favor induction of M1-like pro-inflammatory macrophages during diabetes onset and progression. ROS induces MAPK, STAT1, STAT6 and NFκB signaling, and interferes with macrophage differentiation via epigenetic (re)programming. Therefore, a comprehensive understanding of the impact of ROS on macrophage phenotype and function is needed in order to improve treatment of diabetes and its vascular complications. In the current comprehensive review, we dissect the role of ROS in macrophage polarization, and analyze how ROS production links metabolism and inflammation in diabetes and its complications. Finally, we discuss the contribution of ROS to the crosstalk between macrophages and endothelial cells in diabetic complications.

Endothelial-mesenchymal transition occurs during intimal hyperplasia and neointima formation via mechanisms that are incompletely understood. Endothelial MAPK7 signaling is a key mechanosensitive factor that protects against endothelial-mesenchymal transition, but its signaling activity is lost in vessel areas that are undergoing pathological remodeling. At sites of vascular remodeling in mice and pigs, endothelial MAPK7 signaling was lost. The TGFβ-induced microRNA-374b targets MAPK7 and its downstream effectors in endothelial cells, and its expression induces endothelial-mesenchymal transition. Gain-of-function experiments, where endothelial MAPK7 signaling was restored, precluded endothelial-mesenchymal transition. In human coronary artery disease, disease severity is associated with decreased MAPK7 expression levels and increased miR-374b expression levels. Endothelial-mesenchymal transition occurs in intimal hyperplasia and early lesion formation and is governed in part by microRNA-374b-induced silencing of MAPK7 signaling. Restoration of MAPK7 signaling abrogated these pathological effects in endothelial cells expressing miR-374b. Thus, our data suggest that the TGFβ-miR-374b-MAPK7 axis plays a key role in the induction of endothelial-mesenchymal transition during intimal hyperplasia and early lesion formation and might pose an interesting target for antiatherosclerosis therapy. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Reconstruction of bile ducts damaged remains a vexing medical problem. Surgeons have few options when it comes to a long segment reconstruction of the bile duct. Biological scaffolds of decellularized biliary origin may offer an approach to support the replace of bile ducts. Our objective was to obtain an extracellular matrix scaffold derived from porcine extrahepatic bile ducts (dECM-BD) and to analyze its biological and biochemical properties. The efficiency of the tailored perfusion decellularization process was assessed through histology stainings. Results from 4’-6-diamidino-2-phenylindole (DAPI), Hematoxylin and Eosin (H&amp, E) stainings, and deoxyribonucleic acid (DNA) quantification showed proper extracellular matrix (ECM) decellularization with an effectiveness of 98%. Immunohistochemistry results indicate an effective decrease in immunogenic marker as human leukocyte antigens (HLA-A) and Cytokeratin 7 (CK7) proteins. The ECM of the bile duct was preserved according to Masson and Herovici stainings. Data derived from scanning electron microscopy (SEM) and thermogravimetric analysis (TGA) showed the preservation of the dECM-BD hierarchical structures. Cytotoxicity of dECM-BD was null, with cells able to infiltrate the scaffold. In this work, we standardized a decellularization method that allows one to obtain a natural bile duct scaffold with hierarchical ultrastructure preservation and adequate cytocompatibility.

Different bioinks have been used to produce cell-laden alginate-based hydrogel constructs for cell replacement therapy but some of these approaches suffer from issues with print quality, long-term mechanical instability, and bioincompatibility. In this study, new alginate-based bioinks were developed to produce cell-laden grid-shaped hydrogel constructs with stable integrity and immunomodulating capacity. Integrity and printability were improved by including the co-block-polymer Pluronic F127 in alginate solutions. To reduce inflammatory responses, pectin with a low degree of methylation was included and tested for inhibition of Toll-Like Receptor 2/1 (TLR2/1) dimerization and activation and tissue responses under the skin of mice. The viscoelastic properties of alginate-Pluronic constructs were unaffected by pectin incorporation. The tested pectin protected printed insulin-producing MIN6 cells from inflammatory stress as evidenced by higher numbers of surviving cells within the pectin-containing construct following exposure to a cocktail of the pro-inflammatory cytokines namely, IL-1β, IFN-γ, and TNF-α. The results suggested that the cell-laden construct bioprinted with pectin-alginate-Pluronic bioink reduced tissue responses via inhibiting TLR2/1 and support insulin-producing β-cell survival under inflammatory stress. Our study provides a potential novel strategy to improve long-term survival of pancreatic islet grafts for Type 1 Diabetes (T1D) treatment.

Rationale: The airways in chronic obstructive pulmonary disease (COPD) are characterized by inflammation, goblet cell hyperplasia, decreased ciliated cell numbers and airway remodeling with an altered composition/deposition of the extracellular matrix (ECM). We aimed to develop a lung ECM hydrogel-based spatiotemporal model to investigate bidirectional interactions with fibroblasts and epithelial cells in relation to ECM changes. Methods: Porcine lungs were decellularized, lyophilized, ground into a powder, porcine pepsin-solubilized for 48h, buffered with PBS, pH neutralized and gelated at 37°C for 30min. Non-diseased primary lung fibroblasts (250,000 per ml) were mixed with hydrogels in transwells. After 24h the hydrogels were coated with Matrigel®, Collagen type IV or left uncoated. After 24h, 90,000 non-diseased primary bronchial epithelial cells (PBEC) were seeded on top. The hydrogels were imaged using light microscopy, stained for live and dead cells and paraffin embedded for histology. Results: The fibroblasts inside the ECM hydrogels were viable for up to 5 days, displaying a spindle-shaped morphology. The Matrigel and Collagen IV formed a thin layer on top of the ECM hydrogel which was visualized by a collagen IV immunohistochemical staining. The PBECs grew to a confluent layer in 3-5 days on top of the coated and non-coated ECM hydrogels with no apparent difference in the PBEC layer between them. Conclusions: The lung ECM hydrogel in combination with a basement membrane like coating allowed for the co-culture of primary lung fibroblasts and PBECs in their correct dimensionality resembling the airway in vivo. The next step is to expose the apical surface of the PBEC layer to air to stimulate the development of a mucociliated epithelial layer.

The administration of trophic factors (TFs) released by mesenchymal stromal cells (MSCs) as therapy for cardiovascular diseases requires a delivery vehicle capable of binding and releasing the TF in a sustained manner. We hypothesized that hydrogels derived from cardiac decellularized extracellular matrix (cardiac dECM) bind MSC secretome-derived TF and release these in a sustained fashion. Pig-derived ventricular tissue was decellularized, milled to powder, digested, and assembled as a hydrogel upon warming at 37 °C. The conditioned medium (CMed) of adipose tissue-derived stromal cells (ASC) was collected, concentrated, and incorporated into the hydrogel at 1×, 10×, and 100× the original concentration. The release of 11 ASC-secreted factors (angiopoietin-1, angiopoietin-2, fibroblast growth factor-1, hepatocyte growth factor, platelet-derived growth factor-AA, vascular endothelial growth factor, interleukin-1β, interleukin-6, interleukin-8, CCL2, and matrix metalloproteinase-1) from hydrogels was immune assessed. Bioactivity was determined by endothelial cell proliferation, function, and assessment of endothelial mesenchymal transition. We showed that dECM hydrogels could be loaded with human ASC-secreted TFs, which are released in a sustained manner for several days subsequently. Different trophic factors had different release kinetics, which correlates with the initial concentration of CMed in the hydrogel. We observed that the more concentrated was the hydrogel, the more inflammation-related cytokines, and the less pro-regenerative TFs were released. Finally, we showed that the factors secreted by the hydrogel are biologically active as these influence cell behavior. The use of dECM hydrogels as a platform to bind and release paracrine factors secreted by (mesenchymal) cells is a potential alternative in the context of cardiovascular regeneration.

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 mu g/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.