Vascularization of tissue-engineered skeletal muscle constructs.

Skeletal muscle tissue can be created in vitro by tissue engineering approaches, based on differentiation of muscle stem cells. Several approaches exist and generally result in three dimensional constructs composed of multinucleated myofibers to which we refer as myooids. Engineering methods date back to 3 decades ago and meanwhile a wide range of cell types and scaffold types have been evaluated. Nevertheless, in most approaches, myooids remain very small to allow for diffusion-mediated nutrient supply and waste product removal, typically less than 1 mm thick. One of the shortcomings of current in vitro skeletal muscle organoid development is the lack of a functional vascular structure, thus limiting the size of myooids. This is a challenge which is nowadays applicable to almost all organoid systems. Several approaches to obtain a vascular structure within myooids have been proposed. The purpose of this review is to give a concise overview of these approaches.

Sonoprinting liposomes on tumor spheroids by microbubbles and ultrasound.

Ultrasound-triggered drug-loaded microbubbles have great potential for drug delivery due to their ability to locally release drugs and simultaneously enhance their delivery into the target tissue. We have recently shown that upon applying ultrasound, nanoparticle-loaded microbubbles can deposit nanoparticles onto cells grown in 2D monolayers, through a process that we termed "sonoprinting". However, the rigid surfaces on which cell monolayers are typically growing might be a source of acoustic reflections and aspherical microbubble oscillations, which can influence microbubble-cell interactions. In the present study, we aim to reveal whether sonoprinting can also occur in more complex and physiologically relevant tissues, by using free-floating 3D tumor spheroids as a tissue model. We show that both monospheroids (consisting of tumor cells alone) and cospheroids (consisting of tumor cells and fibroblasts, which produce an extracellular matrix) can be sonoprinted. Using doxorubicin-liposome-loaded microbubbles, we show that sonoprinting allows to deposit large amounts of doxorubicin-containing liposomes to the outer cell layers of the spheroids, followed by doxorubicin release into the deeper layers of the spheroids, resulting in a significant reduction in cell viability. Sonoprinting may become an attractive approach to deposit drug patches at the surface of tissues, thereby promoting the delivery of drugs into target tissues.

Human decellularized dermal matrix seeded with adipose-derived stem cells enhances wound healing in a murine model: Experimental study.

OBJECTIVE: Full-thickness cutaneous wounds treated with split-thickness skin grafts often result in unaesthetic and hypertrophic scars. Dermal substitutes are currently used together with skin grafts in a single treatment to reconstruct the dermal layer of the skin, resulting in improved quality of scars. Adipose-derived stem cells (ASCs) have been described to enhance wound healing through structural and humoral mechanisms. In this study, we investigate the compatibility of xenogen-free isolated human ASCs seeded on human acellular dermal matrix (Glyaderm®) in a murine immunodeficient wound model. METHODS: Adipose tissue was obtained from abdominal liposuction, and stromal cells were isolated mechanically and cultured xenogen-free in autologous plasma-supplemented medium. Glyaderm® discs were seeded with EGFP-transduced ASCs, and implanted on 8 mm full-thickness dorsal wounds in an immunodeficient murine model, in comparison to standard Glyaderm® discs. Re-epithelialization rate, granulation thickness and vascularity were assessed by histology on days 3, 7 and 12. Statistical analysis was conducted using the Wilcoxon signed-rank test. EGFP-staining allowed for tracking of the ASCs in vivo. Hypoxic culture of the ASCs was performed to evaluate cytokine production. RESULTS: ASCs were characterized with flowcytometric analysis and differentiation assay. EGFP-tranduction resulted in 95% positive cells after sorting. Re-epithelialization in the ASC-seeded Glyaderm® side was significantly increased, resulting in complete wound healing in 12 days. Granulation thickness and vascularization were significantly increased during early wound healing. EGFP-ASCs could be retrieved by immunohistochemistry in the granulation tissue in early wound healing, and lining vascular structures in later stages. CONCLUSION: Glyaderm® is an effective carrier to deliver ASCs in full-thickness wounds. ASC-seeded Glyaderm® significantly enhances wound healing compared to standard Glyaderm®. The results of this study encourage clinical trials for treatment of full-thickness skin defects. Furthermore, xenogen-free isolation and autologous plasma-augmented culture expansion of ASCs, combined with the existing clinical experience with Glyaderm®, aid in simplifying the necessary procedures in a GMP-laboratory setting.

Xenogen-free isolation and culture of human adipose mesenchymal stem cells.

BACKGROUND: Adipose-derived Stem Cells (ASCs) present great potential for reconstructive procedures. Currently, isolation by enzyme digestion and culturing using xenogenic substances remain the gold standard, impairing clinical use. METHODS: Abdominal lipo-aspirate and blood samples were obtained from healthy patients. A novel mechanical isolation method for ASCs was compared to (the standard) collagenase digestion. ASCs are examined by flowcytometry and multilineage differentiation assays. Cell cultures were performed without xenogenic or toxic substances, using autologous plasma extracted from peripheral blood. After eGFP-transfection, an in vivo differentiation assay was performed. RESULTS: Mechanical isolation is more successful in isolating CD34+/CD31-/CD45-/CD13+/CD73+/CD146- ASCs from lipo-aspirate than isolation via collagenase digestion (p <0 .05). ASCs display multilineage differentiation potential in vitro. Autologous plasma is a valid additive for ASCs culturing. eGFP-ASCs, retrieved after 3 months in vivo, differentiated in adipocytes and endothelial cells. CONCLUSION: A practical method for human ASC isolation and culturing from abdominal lipo-aspirate, without the addition of xenogenic substances, is described. The mechanical protocol is more successful than the current gold standard protocol of enzyme digestion. These results are important in the translation of laboratory-based cell cultures to clinical reconstructive and aesthetic applications.

Texture profile analysis reveals a stiffer ovarian cortex after testosterone therapy: a pilot study.

PURPOSE: The importance of the surrounding ovarian stromal cells and extracellular matrix in the development and maturation of follicles has recently gained attention. An aberrant extracellular matrix has been described in ovaries of patients with polycystic ovary syndrome where a more rigid structural environment, possibly induced by endogenous testosterone, impairs normal folliculogenesis. In this context, we describe the textural parameters of the ovarian cortex of transgender men after prolonged testosterone administration compared to the textural parameters of the non-exposed ovarian cortex originating from female oncological patients. METHODS: Texture profile analysis (TPA) was performed on ovarian cortex (5 × 5 mm) of oncological and transgender patients in order to measure stiffness, hardness, cohesiveness, and springiness of the ovarian cortex (LRXplus universal testing system). Statistical analysis was performed using repeated measurements mixed models and the Spearman rank order correlation test (IBM SPSS Statistics 23). RESULTS: A total of 36 frozen-thawed cortical strips (5 × 5 mm) were subjected to TPA. The superficial part of cortex fragments originating from transgender persons (fragments < 1.4 mm; N = 10) appeared to be significantly stiffer compared to cortex derived from oncology patients (fragments < 1.4 mm; N = 7) (6.78 ± 1.38 N/mm versus 5.41 ± 0.9 N/mm respectively, p = 0.036). CONCLUSIONS: This is the first application of TPA in ovarian cortex to study the physical properties. Comparing the physical properties, we objectively describe an increased cortical stiffness in the most outer part of the ovarian cortex following prolonged testosterone administration in transgender men compared to the ovarian cortex of oncological patients. This preliminary and novel approach could be the start of future research to understand the physical properties of ovarian tissue.

Improving the surface properties of an UHMWPE shoulder implant with an atmospheric pressure plasma jet.

Insufficient glenoid fixation is one of the main reasons for failure in total shoulder arthroplasty. This is predominantly caused by the inert nature of the ultra-high molecular weight polyethylene (UHMWPE) used in the glenoid component of the implant, which makes it difficult to adhesively bind to bone cement or bone. Previous studies have shown that this adhesion can be ameliorated by changing the surface chemistry using plasma technology. An atmospheric pressure plasma jet is used to treat UHMWPE substrates and to modify their surface chemistry. The modifications are investigated using several surface analysis techniques. The adhesion with bone cement is assessed using pull-out tests while osteoblast adhesion and proliferation is also tested making use of several cell viability assays. Additionally, the treated samples are put in simulated body fluid and the resulting calcium phosphate (CaP) deposition is evaluated as a measure of the in vitro bioactivity of the samples. The results show that the plasma modifications result in incorporation of oxygen in the surface, which leads to a significant improved adhesion to bone cement, an enhanced osteoblast proliferation and a more pronounced CaP deposition. The plasma-treated surfaces are therefore promising to act as a shoulder implant.

Synergistic effect of κ-carrageenan and gelatin blends towards adipose tissue engineering.

The current paper focuses on the functionalization of κ-carrageenan and gelatin as extracellular matrix polysaccharide and protein mimic respectively to produce hydrogel films for adipose tissue engineering. More specifically, κ-carrageenan as well as gelatin have been functionalized with methacrylate and methacrylamide moieties respectively to enable subsequent UV-induced crosslinking in the presence of a photo-initiator. The gel fraction, the mass swelling ratio and the mechanical properties of both the one-component hydrogels and the protein/polysaccharide blends have been evaluated. The mechanical and swelling properties of the blends could be tuned by varying the hydrogel composition as well as the crosslinking method applied. The in vitro biocompatibility assays indicated a significantly higher cell viability of adipose tissue-derived mesenchymal stem cells seeded onto the blends as compared to the one-component hydrogels. The results show that the blends of gelatin and κ-carrageenan clearly outperform the one-component hydrogels in terms of adipose tissue engineering potential.

The role of endothelial cells in the vasculopathy of systemic sclerosis: A systematic review.

INTRODUCTION: Systemic sclerosis (SSc) is an autoimmune connective tissue disorder characterized by fibroproliferative vasculopathy, immunological abnormalities and progressive fibrosis of multiple organs including the skin. In this study, all English speaking articles concerning the role of endothelial cells (ECs) in SSc vasculopathy and representing biomarkers are systematically reviewed and categorized according to endothelial cell (EC) (dys)function in SSc. METHODS: A sensitive search on behalf of the EULAR study group on microcirculation in Rheumatic Diseases was developed in Pubmed, The Cochrane Library and Web of Science to identify articles on SSc vasculopathy and the role of ECs using the following Mesh terms: (systemic sclerosis OR scleroderma) AND pathogenesis AND (endothelial cells OR marker). All selected papers were read and discussed by two independent reviewers. The selection process was based on title, abstract and full text level. Additionally, both reviewers further searched the reference lists of the articles selected for reading on full text level for supplementary papers. These additional articles went through the same selection process. RESULTS: In total 193 resulting articles were selected and the identified biomarkers were categorized according to description of EC (dys)function in SSc. The most representing and reliable biomarkers described by the selected articles were adhesion molecules for EC activation, anti-endothelial cell antibodies for EC apoptosis, vascular endothelial growth factor (VEGF), its receptor VEGFR-2 and endostatin for disturbed angiogenesis, endothelial progenitors cells for defective vasculogenesis, endothelin-1 for disturbed vascular tone control, Von Willebrand factor for coagulopathy and interleukin (IL)-33 for EC-immune system communication. Emerging, relatively new discovered biomarkers described in the selected articles, are VEGF165b, IL-17A and the adipocytokines. Finally, myofibroblasts involved in tissue fibrosis in SSc can derive from ECs or epithelial cells through a process known as endothelial-to-mesenchymal transition. CONCLUSION: This systematic review emphasizes the growing evidence that SSc is primarily a vascular disease where EC dysfunction is present and prominent in different aspects of cell survival (activation and apoptosis), angiogenesis and vasculogenesis and where disturbed interactions between ECs and various other cells contribute to SSc vasculopathy.

Addition of bioactive glass to glass ionomer cements: Effect on the physico-chemical properties and biocompatibility.

OBJECTIVES: Glass ionomer cements (GICs) are a subject of research because of their inferior mechanical properties, despite their advantages such as fluoride release and direct bonding to bone and teeth. Recent research aims to improve the bioactivity of the GICs and thereby improve mechanical properties on the long term. In this study, two types of bioactive glasses (BAG) (45S5F and CF9) are combined with GICs to evaluate the physico-chemical properties and biocompatibility of the BAG-GIC combinations. The effect of the addition of Al3+ to the BAG composition and the use of smaller BAG particles on the BAG-GIC properties was also investigated. MATERIALS AND METHODS: Conventional aluminosilicate glass (ASG) and (modified) BAG were synthesized by the melt method. BAG-GIC were investigated on setting time, compressive strength and bioactivity. Surface changes were evaluated by Fourier transform infrared (FT-IR), scanning electron microscopy (SEM), EDS and PO43- -and Ca2+ uptake in SBF. Biocompatibility of selected BAG-GICs was determined by a direct toxicity assay. RESULTS: The addition of BAG improves the bioactivity of the GIC, which can be observed by the formation of an apatite (Ap) layer, especially in CF9-containing GICs. More BAG leads to more bioactivity but decreases strength. The addition of Al3+ to the BAG composition improves strength, but decreases bioactivity. BAGs with smaller particle sizes have no effect on bioactivity and decrease strength. The formation of an Ap layer seems beneficial to the biocompatibility of the BAG-GICs. SIGNIFICANCE: Bioactive GICs may have several advantages over conventional GICs, such as remineralization of demineralized tissue, adhesion and proliferation of bone- and dental cells, allowing integration in surrounding tissue. CF9 BAG-GIC combinations containing maximum 10mol% Al3+ are most promising, when added in ≤20wt% to a GIC.

Bioactivity and biocompatibility of two fluoride containing bioactive glasses for dental applications.

OBJECTIVE: Bioactive glasses (BAG) form, in contrast to formerly used implant materials, a stable bond with tissues, especially bone, when implanted. Nowadays BAGs are often mixed with a cement/composite that hardens in situ to broaden its applications in dentistry or orthopedics. The bioactivity and biocompatibility of possible BAG candidates for BAG-cement/composite development were evaluated. METHODS: Two fluoride containing BAGs were tested: a Na+-containing (45S5F), based on the first commercial BAG, and a Na+-free BAG (CF9), with a higher Ca2+ and PO43- content. BAGs were tested on their bioactivity upon immersion in SBF for 7days by evaluating the surface changes by FT-IR, SEM, EDS and PO43- and Ca2+ uptake and/or release from SBF. Moreover, the biocompatibility of the BAGs was investigated with a direct contact cell viability study with HFF cells and a cell adhesion study with MG-63 cells. RESULTS: The Na+-free BAG, CF9, showed the highest potential to bioactivate cements because of its high Ca2+-release and apatite (Ap) formation, as evidenced by SEM pictures and corresponding EDX patterns. FT-IR confirmed the formation of an Ap layer. Moreover CF9 had a higher biocompatibility than 45S5F. SIGNIFICANCE: For the bioactivation of GICs/composites in order to enhance bonding and remineralization of surrounding tissues, fluoride containing BAG may have advantages over other BAGs as a more stable fluorapatite can be formed. CF9 may be an excellent candidate therefore.

Absent Anterior Cruciate Ligament.

This case report presents the MRI findings of aplasia of the anterior cruciate ligament with associated hypoplasia of the posterior cruciate ligament (Manner type 2). Radiographically the presence of a shallow femoral notch and hypoplastic tibial spines (the so-called "dromedar" sign) can aid in the diagnosis. Operative treatment is often not indicated since the congenital absence of the ACL implies longstanding altered biomechanics to which the knee has well adapted in the majority of cases.

Intra-articular synovial lipoma of the knee joint.

We present a rare case of an intra-articular synovial lipoma, which was diagnosed in a patient after a knee trauma. MRI is the imaging modality of choice to suggest the diagnosis preoperatively, by demonstrating a well-delineated fat-containing lesion. The differential diagnosis of an intra-articular lipomatous lesion consists of lipoma arborescens and synovial lipoma.

Enzymatic mineralization of gellan gum hydrogel for bone tissue-engineering applications and its enhancement by polydopamine.

Interest is growing in the use of hydrogels as bone tissue-engineering (TE) scaffolds due to advantages such as injectability and ease of incorporation of active substances such as enzymes. Hydrogels consisting of gellan gum (GG), an inexpensive calcium-crosslinkable polysaccharide, have been applied in cartilage TE. To improve GG suitability as a material for bone TE, alkaline phosphatase (ALP), an enzyme involved in mineralization of bone by cleaving phosphate from organic phosphate, was incorporated into GG hydrogels to induce mineralization with calcium phosphate (CaP). Incorporated ALP induced formation of apatite-like material on the submicron scale within GG gels, as shown by FTIR, SEM, EDS, XRD, ICP-OES, TGA and von Kossa staining. Increasing ALP concentration increased amounts of CaP as well as stiffness. Mineralized GG was able to withstand sterilization by autoclaving, although stiffness decreased. In addition, mineralizability and stiffness of GG was enhanced by the incorporation of polydopamine (PDA). Furthermore, mineralization of GG led to enhanced attachment and vitality of cells in vitro while cytocompatibility of the mineralized gels was comparable to one of the most commonly used bone substitute materials. The results proved that ALP-mediated enzymatic mineralization of GG could be enhanced by functionalization with PDA.

Periosteal chondroma of the proximaltibia mimicking Osgood-Schlatter's disease.

We report a case of a periosteal chondroma of the proximal tibia in an 11-year-old girl, which was initially misdiagnosed as Osgood-Schlatter's disease. The absence of pain and meticulous analysis of the imaging findings on initial and follow-up plain radiographs, ultrasound and MRI allowed to suggest the diagnosis of a periosteal chondroma, which was confirmed after biopsy. Besides the difficulty in the imaging diagnosis of the lesion, determination of the optimal treatment strategy may be challenging as well. Given the localization of this lesion close to the growth plate, decision has to be made whether the lesion will be treated surgically or a waitful watching policy will be implemented in order to prevent interference with the normal growth of the bone.

Evaluation of the behavior of murine and human embryonic stem cells in in vitro migration and invasion assays.

Cell migration and invasion are essential processes in a variety of physiological events in the body, but also in several patho-physiological events. In this paper, the behavior of murine and human embryonic stem cells is examined in in vitro migration and invasion models. mESC and hESC were applied as spheroids, also known as patches, and as single cells, to mimic possible cell therapy application strategies. Two known in vitro migration assays, the ECM (extracellular matrix) assay and the Boyden chamber migration assay were selected. These assays revealed that mESC are statistically significantly more infiltrative than hESC. Application as spheroid results in a slightly higher infiltrative capacity compared single cells. The PHF (precultured chick heart fragment) assay was selected as an invasion assay. In the PHF assay a more 3D examination of the infiltrative nature of the ESC can be observed. The mESC showed infiltrative behavior, as spheroids and as single cells. The hESC were infiltrative as single cells but not as spheroids. The results of these assays are mostly complementary and prove the applicability of these assays, which were originally applied in tumor biology, in migratory behavior studies regarding stem cells and their progeny in basic and other conditions.

Double protein-coated poly-ε-caprolactone scaffolds: successful 2D to 3D transfer.

In the past decade, tissue engineering has evolved from a promising technology to an established scientific field. Large attention has focussed on developing scaffolds from both biodegradable and nondegradable polymers to be cultivated with cells, to replace human body defects. The major drawback of most polymers is however their limited cell-interactive properties. An additional complication when developing a surface modification protocol for those materials is the transferability of protocols from 2D substrates to 3D scaffolds. In the present work, we therefore report on possible biological effects originating from the transfer of a double protein coating protocol, involving gelatin type B and fibronectin, from 2D poly-ε-caprolactone (PCL) films to 3D PCL scaffolds produced by rapid prototyping. A variety of techniques including scanning electron microscopy, X-ray photoelectron spectroscopy and confocal fluorescence microscopy confirmed a successful and homogeneous protein-coating on both 2D and 3D substrates. Interestingly, the biological performance of the double protein-coated PCL substrates, reflected by the initial cell adhesion, proliferation, and colonization was superior compared to the other surface modification steps, independent of the material dimension.

Expansion of human embryonic stem cells: a comparative study.

OBJECTIVES: Human embryonic stem cells (hESC) are promising for tissue engineering (TE) purposes due to their unique properties. However, current standard mechanical passaging techniques limit rates of possible TE experiments, as it is difficult to obtain high enough numbers of the cells for experimentation. In this study, several dissociative solutions and application methods are tested for their applicability to, and influence on, hESC culture and expansion. MATERIALS AND METHODS: Expansion of two hESC lines, H1 and VUB01, subjected to different passaging techniques, was evaluated. Four dissociative solutions - TrypLE™ Express, Trypsin-EDTA, Cell Dissociation Solution and Accutase™- were combined with two application protocols. As reference conditions, manual and bead-based passaging techniques were used. RESULTS: Results showed that use of Cell Dissociation Solution in combination with a slow adaptation protocol, generated the best expansion profile for both cell lines. The hESC single cell lines remained pluripotent, had good expansion profiles and were capable of differentiation into representatives of all three germ layers. Reproducibility of the results was confirmed by adaptation for three other hESC lines. CONCLUSION: Use of Cell Dissociation Solution, combined with slow adaptation protocol, allows a fast switch from the mechanical passaging technique to a single-cell split technique, generating stable and robust hESC cell lines, which allow for large scale expansion of hESC for TE purposes.

Effect of 17beta-estradiol on the in vitro differentiation of murine embryonic stem cells into the osteogenic lineage.

Embryonic stem (ES) cells have the potential to differentiate into various cell types of the three germ layers. They are therefore a useful cell source for transplantation and tissue engineering. In the present paper, we studied the influences of ascorbic acid (AA), dexamethasone (Dex), and 17beta-estradiol (E(2)) on the osteogenic differentiation of ES cells. Differentiation into the osteoblastic phenotype was demonstrated by the appearance of osteoblastic markers such as alkaline phosphatase (ALP), the transcription factor core binding factor alpha 1 (Cbfa1), and osteocalcin, which were detected by immunohistochemistry. Bone nodule formation, including the deposition of collagen fibrils and matrix mineralization, was studied by transmission electron microscopy. In all our cultures, a progressive upregulation of ALP activity was observed, followed by a decline after 21 d of culture. Cbfa1 was first detected after 14 d in culture and increased during the culture time. The addition of E(2) resulted in a decrease in the formation of bone-like nodules in the embryoid bodies (EBs) compared with the EBs cultured in the presence of AA and AA supplemented with Dex. An increased osteocalcin concentration was observed in the EBs cultured with Dex and E(2) compared with the EBs cultured in a control medium. EBs cultured in the presence of E(2) resulted in a culture with a high amount of osteoblast-like cells not entrapped in bone-like nodules, creating the possibility to obtain a purified osteoblast population for bone tissue engineering.

Gelatin-based microcarriers as embryonic stem cell delivery system in bone tissue engineering: an in-vitro study.

Mouse embryonic stem cells were cultured on commercially available biodegradable macroporous microcarriers. A culture period of 1-2 weeks was needed to colonize the microcarriers. Embryonic stem cells retained their pluripotency for up to 14 days when cultured in medium supplemented with leukemia inhibitory factor. Replacing this medium by differentiation medium for 2 weeks initiated osteogenic differentiation. Encapsulation of the cell-loaded microcarriers in photopolymerizable polymers (methacrylate-endcapped poly-D,L-lactide-co-caprolactone), triacetin/hydroxyethylmethacrylate (HEMA) as solvent and with/without gelatin as porogen, resulted in a homogeneous distribution of the microcarriers in the polymer. As observed by transmission electron microscopy, viability of the cells was optimal when gelatin was omitted and when using triacetin instead of HEMA.