Bioactive composite hydrogels as 3D mesenchymal stem cell encapsulation environment for bone tissue engineering: in vitro and in vivo studies.

Although decellularized bone matrix (DBM) has often been used in scaffold form for osteogenic applications, its use as a stem cell encapsulation matrix adaptable to surgical shaping procedures has been neglected. This study aimed to investigate the feasibility of utilizing solubilized DBM and nanohydroxyapatite (nHAp)-incorporated DBM hydrogels as encapsulation matrix for bone marrow-derived MSCs (BM-MSCs). First, DBM and DBM/nHAp hydrogels were assessed by physical, chemical, turbidimetric, thermal, and mechanical methods; then, in vitro cytocompatibility and in vitro hemocompatibility were investigated. An in vivo study was performed to evaluate the osteogenic properties of hydrogels alone or with BM-MSCs encapsulated in them. The findings revealed that hydrogels retained high levels of collagen and glycosaminoglycans after successful decellularization. They were found to be cytocompatible and hemocompatible in vitro, and were able to gel with sufficient mechanical stability at physiological temperature. BM-MSCs survived in culture for at least 2 weeks as metabolically active when encapsulated in both DBM and DBM/nHAp. Preliminary in vivo study showed that DBM-nHAp has higher osteogenicity than DBM. Moreover, BM-MSC encapsulated DMB/nHAp showed predominant bone-like tissue formation at 30 days in the rat ectopic site compared to its cell-free form.

Decellularized tendon-based heparinized nanocomposite scaffolds for prospective regenerative applications: Chemical, physical, thermal, mechanical and in vitro biological evaluations.

The development of cell-free regenerative biomaterials is among the current approaches of tissue engineering (TE). While materials targeting homogeneous continuous tissues can be produced more easily, significant difficulties are encountered in composite tissue interfaces such as the tendon-bone. The complex bioactive and chemical contents of the microenvironment in neighboring tissues make this situation even more difficult. While target tissue can be significantly mimicked with decellularized tissues, there is a need for incorporation of inorganic components into composite interfaces. The regenerative properties of biomaterials can be regulated by enriching them with growth factors, and the mechanical properties can be imparted by developing nanocomposites of right composition. In the first phase of the study, protocols were optimized to obtain bovine tendon-based scaffolds with high bioactive content from decellularized hydrogels. The results showed that DNA could be removed from tissues (<50 ng/mg ECM) using 0.1% SDS and 0.1% EDTA after freeze-thaw, and the content of sGAGs, an important component for tendon tissue repair, was preserved in the final product at >50%. In the second stage, the scaffolds were produced in composite form containing different amounts of nanohydroxyapatite (HAp). In the final stage, tendon-based nanocomposite scaffolds were activated with heparin to impart growth factor binding affinity. The physical, chemical, thermal, mechanical and in vitro biological properties of the scaffolds were studied in detail. The findings revealed that HAp increased the thermal stability and compressive strength; and heparin could be successfully integrated into scaffolds. Nanocomposite scaffolds were found to be highly hemocompatible.

Development of a multicellular 3D-bioprinted microtissue model of human periodontal ligament-alveolar bone biointerface: Towards a pre-clinical model of periodontal diseases and personalized periodontal tissue engineering.

While periodontal (PD) disease is among principal causes of tooth loss worldwide, regulation of concomitant soft and mineralized PD tissues, and PD pathogenesis have not been completely clarified yet. Besides, relevant pre-clinical models and in vitro platforms have limitations in simulating human physiology. Here, we have harnessed three-dimensional bioprinting (3DBP) technology for developing a multi-cellular microtissue model resembling PD ligament-alveolar bone (PDL-AB) biointerface for the first time. 3DBP parameters were optimized; the physical, chemical, rheological, mechanical, and thermal properties of the constructs were assessed. Constructs containing gelatin methacryloyl (Gel-MA) and hydroxyapatite-magnetic iron oxide nanoparticles showed higher level of compressive strength when compared with that of Gel-MA constructs. Bioprinted self-supporting microtissue was cultured under flow in a microfluidic platform for >10 days without significant loss of shape fidelity. Confocal microscopy analysis indicated that encapsulated cells were homogenously distributed inside the matrix and preserved their viability for >7 days under microfluidic conditions. Immunofluorescence analysis showed the cohesion of stromal cell surface marker-1+ human PDL fibroblasts containing PDL layer with the osteocalcin+ human osteoblasts containing mineralized layer in time, demonstrating some permeability of the printed constructs to cell migration. Preliminary tetracycline interaction study indicated the uptake of model drug by the cells inside the 3D-microtissue. Also, the non-toxic levels of tetracycline were determined for the encapsulated cells. Thus, the effects of tetracyclines on PDL-AB have clinical significance for treating PD diseases. This 3D-bioprinted multi-cellular periodontal/osteoblastic microtissue model has potential as an in vitro platform for studying processes of the human PDL.

Decellularized liver ECM-based 3D scaffolds: Compositional, physical, chemical, rheological, thermal, mechanical, and in vitro biological evaluations.

The extracellular matrix (ECM) is involved in many critical cellular interactions through its biological macromolecules. In this study, a macroporous 3D scaffold originating from decellularized bovine liver ECM (dL-ECM), with defined compositional, physical, chemical, rheological, thermal, mechanical, and in vitro biological properties was developed. First, protocols were determined that effectively remove cells and DNA while ECM retains biological macromolecules collagen, elastin, sGAGs in tissue. Rheological analysis revealed the elastic properties of pepsin-digested dL-ECM. Then, dL-ECM hydrogel was neutralized, molded, formed into macroporous (~100-200 µm) scaffolds in aqueous medium at 37 °C, and lyophilized. The scaffolds had water retention ability, and were mechanically stable for at least 14 days in the culture medium. The findings also showed that increasing the dL-ECM concentration from 10 mg/mL to 20 mg/mL resulted in a significant increase in the mechanical strength of the scaffolds. The hemolysis test revealed high in vitro hemocompatibility of the dL-ECM scaffolds. Studies investigating the viability and proliferation status of human adipose stem cells seeded over a 2-week culture period have demonstrated the suitability of dL-ECM scaffolds as a cell substrate. Prospective studies may reveal the extent to which 3D dL-ECM sponges have the potential to create a biomimetic environment for cells.

Magneto-sensitive decellularized bone matrix with or without low frequency-pulsed electromagnetic field exposure for the healing of a critical-size bone defect.

Bioactive ECM-based materials mimic the complex composition and structure of natural tissues. Decellularized cancellous bone matrix (DBM) has potential for guiding new bone formation and accelerating the regeneration process. On the other hand, low frequency-pulsed electromagnetic field (LF-PEMF) has been shown to enhance the regeneration capacity of bone defects. The present study sought to explore the feasibility of using DBM and DBM/MNP, and LF-PEMF for treating critical-size bone defects. Firstly, decellularization protocol was optimized to obtain a bioactive DBM, then MNPs were incorporated. Later, the physical, chemical and biological properties of DBM and DBM/MNP were assessed in vitro. MNPs homogeneously distributed into the DBM were not found to be toxic to human osteoblast cultures. Finally, an in vivo study was carried out with DBM and DBM/MNP composites in a bilateral critical-size rat cranial defect model (n = 48) with or without LF-PEMF exposure for 45 and 90 days. The histomorphometric and radiographic evaluations revealed that, while the collagen (positive control) and Sham (negative control) groups showed high incidence of fibrous connective tissue together with low level of osteogenic activity, both the DBM and DBM/MNP-grafted groups significantly promoted new bone tissue formation and angiogenesis, by the appropriate use of LF-PEMF for 90 days.

Biomimetic 3D-Bone Tissue Model.

Various approaches have been evaluated for developing three-dimensional (3D) scaffolds for modeling or engineering of the bone tissue. However, most of such attempts have come up short in mimicking the natural bone tissue extracellular matrix (ECM) microenvironment, especially its natural bioactive content. Here we describe the methodology for the preparation of a natural ECM-based multichannel construct as a biomimetic 3D bone tissue model. We elucidate the construction of the composite scaffold incorporating decellularized small intestinal submucosa ECM, synthetic hydroxyapatite and poly(ε-caprolactone), and the mechanical stimulation of the cell-seeded construct under bioreactor culture.

Decellularized Cell Culture ECMs Act as Cell Differentiation Inducers.

Decellularized tissues and organs have aroused considerable interest for developing functional bio-scaffolds as natural templates in tissue engineering applications. More recently, the use of natural extracellular matrix (ECM) extracted from the in vitro cell cultures for cellular applications have come into question. It is well known that the microenvironment largely defines cellular properties. Thus, we have anticipated that the ECMs of the cells with different potency levels should likely possess different effects on cell cultures. To test this, we have comparatively evaluated the differentiative effects of ECMs derived from the cultures of human somatic dermal fibroblasts, human multipotent bone marrow mesenchymal stem cells, and human induced pluripotent stem cells on somatic dermal fibroblasts. Although challenges remain, the data suggest that the use of cell culture-based extracellular matrices perhaps may be considered as an alternative approach for the differentiation of even somatic cells into other cell types.

A CD34+ Cell Enrichment Protocol of Hematopoietic Stem Cells in a Well-Established Quality Management System.

Allogeneic stem cell transplantation applications have improved tremendously over the past quarter of a century. The use of new immunosuppressive protocols and elimination of T cells by CD34+ cell enrichment or T cell depletion on apheresis products increases the chance of using partially matched or haploidentical grafts. This is without increasing the risk of graft-versus-host disease, which is observed as a major complication of hematopoietic stem cell transplantation. The aim of this protocol is to evaluate the results obtained from 6 different process cycles performed on 6 different days. We used the CliniMACS Plus system located in our Cell and Tissue Manufacturing Center Quality Control Unit which is already calibrated as a class D room and includes a class A microbiological safety cabinet inside. The average purity of the end products was 95.66%, excluding only one end product which was 70%; this was higher than the values in current studies in the field. Superior to the reported studies, the CD3 quantity in each end product was below the dedicated thresholds. BactecTM FX40 blood culture system test results were detected as negative for each end product. Endotoxin testing suggested the absence of endotoxin within the products. The consistent outcomes obtained from these 6 different process cycles confirmed that the CliniMACS® Plus process cycles performed in accordance with our well-defined quality management system procedure is sufficient for the routine application of high-quality and safe CD34+ enrichment processes within our clean room area.

Decellularized bSIS-ECM as a Regenerative Biomaterial for Skin Wound Repair.

Tissue engineering-based regenerative applications can involve the use of stem cells for the treatment of non-healing wounds. Multipotent mesenchymal stem cells have become a focus of skin injury treatments along with many other injury types owing to their unprecedented advantages. However, there are certain limitations concerning the solo use of stem cells in skin wound repair. Natural bioactive extracellular matrix-based scaffolds have great potential for overcoming these limitations by supporting the regenerative activity and localization of stem cells. This chapter describes the use of bone marrow mesenchymal stem cells together with decellularized bovine small intestinal submucosa (SIS), for the treatment of a critical-sized full-thickness skin defect in a small animal model.

Decellularized bovine small intestinal submucosa-PCL/hydroxyapatite-based multilayer composite scaffold for hard tissue repair.

This study involved the development of a multilayer osteogenic tissue scaffold by assembling decellularized bovine small intestinal submucosa (bSIS) layers, together with synthetic hydroxyapatite microparticles (HAp) and poly(ε-caprolactone) (PCL) as the binder. As a first step, the surface and mechanical properties of the developed scaffold was determined, after which the biocompatibility was evaluated through seeding with isolated rat bone marrow mesenchymal stem cells (BM-MSCs). Then, a 21-day culture study was performed to investigate the in vitro osteoinductive potential of the scaffold on BM-MSCs under standard and osteogenic culture conditions. The SEM findings indicated that a uniform multilayer and perforated structure was acquired; that the HAp microparticles were homogenously distributed within the structure; and that the PCL-bound laminar scaffold had structural integrity. Mechanical tests revealed that the scaffold maintained its mechanical stability for at least 21 days in culture, with no changes in the first-day maximum strength and maximum stress values of 625.123 ±â€¯70.531 N and 6.57762 ±â€¯0.742 MPa, respectively. MTT and SEM analyses together revealed that BM-MSCs preserved their viability and proliferated during a 14-day culture period on the multilayer scaffold. Immunofluorescence analyses indicated that cells on the scaffold differentiated into the osteogenic lineage, by the culture-time-dependent increase in osteogenic markers' expression, i.e. Alkaline phosphatase, Osteopontin, and Osteocalcin. It was also clear that, the osteoinductive effect by the composite scaffold on BM-MSCs could be achieved even without the use of any external osteogenic inducers.

Investigations on clonazepam-loaded polymeric micelle-like nanoparticles for safe drug administration during pregnancy.

Medication during pregnancy is often a necessity for women to treat their acute or chronic diseases. The goal of this study is to evaluate the potential of micelle-like nanoparticles (MNP) for providing safe drug usage in pregnancy and protect both foetus and mother from medication side effects. Clonazepam-loaded MNP were prepared from copolymers [polystyrene-poly(acrylic acid) (PS-PAA), poly(ethylene glycol)-b-poly(lactic acid) (PEG-PLA) and distearyl-sn-glycero-3-phosphoethanolamine-N-[methoxy-poly(ethylene glycol) (PEG-DSPE)] with varying monomer ratios and their drug-loading efficiency, drug release ratio, particle size, surface charge and morphology were characterised. The cellular transport and cytotoxicity experiments were conducted on clonazepam and MNP formulations using placenta-choriocarcinoma-BeWo and brain-endothelial-bEnd3 cells. Clonazepam-loaded PEG5000-PLA4500 MNP reduced the drug transport through BeWo cells demonstrating that MNP may lower foetal drug exposure, thus reduce the drug side effects. However, lipofectamine modified MNP improved the transport of clonazepam and found to be promising for brain and in-utero-specific drug treatment.

Decellularization of Bovine Small Intestinal Submucosa.

Decellularization technology promises to overcome some of the significant limitations in the regenerative medicine field by providing functional biocompatible grafts. The technique involves removal of the cells from the biological tissues or organs for further use in tissue engineering and clinical interventions. There are significant differences between decellularization protocols due to the intrinsic properties of different tissue types and purpose of use. This multistep, chemical-solution-based protocol is optimized for the preparation of decellularized bovine small intestinal submucosa (SIS).

Differential gene expression profiling of human adipose stem cells differentiating into smooth muscle-like cells by TGFß1/BMP4.

Regenerative repair of the vascular system is challenging from the perspectives of translational medicine and tissue engineering. There are fundamental hurdles in front of creating bioartificial arteries, which involve recaputilation of the three-layered structure under laboratory settings. Obtaining and maintaining smooth muscle characteristics is an important limitation, as the transdifferentiated cells fail to display mature phenotype. This study aims to shed light on the smooth muscle differentiation of human adipose stem cells (hASCs). To this end, we first acquired hASCs from lipoaspirate samples. Upon characterization, the cells were induced to differentiate into smooth muscle (SM)-like cells using a variety of inducer combinations. Among all, TGFß1/BMP4 combination had the highest differentiation efficiency, based on immunohistochemical analyses. hSM-like cell samples were compared to hASCs and to the positive control, human coronary artery-smooth muscle cells (hCA-SMCs) through gene transcription profiling. Microarray findings revealed the activation of gene groups that function in smooth muscle differentiation, signaling pathways, extracellular modeling and cell proliferation. Our results underline the effectiveness of the growth factors and suggest some potential variables for detecting the SM-like cell characteristics. Evidence in transcriptome level was used to evaluate the TGFß1/BMP4 combination as a previously unexplored effector for the smooth muscle differentiation of adipose stem cells.

Decellularization of bovine small intestinal submucosa and its use for the healing of a critical-sized full-thickness skin defect, alone and in combination with stem cells, in a small rodent model.

In this study, we initially described an efficient decellularization protocol for bovine-derived small intestinal submucosa (bSIS), involving freeze-thaw cycles, acid/base treatment and alcohol and buffer systems. We compared the efficacy of our protocol to some previously established ones, based on DNA content and SEM and histochemical analyses. DNA content was reduced by ~89.4%, significantly higher than compared protocols. The sulphated GAG content of the remaining interconnected fibrous structure was 5.738 ± 0.207 µg/mg (55% retained). An in vitro study was performed to evaluate whether rat bone marrow mesenchymal stem cells (MSCs) could attach and survive on bSIS membranes. Our findings revealed that MSCs can preserve their viability and proliferate on bSIS for > 2 weeks in culture. We conducted in vivo applications for the treatment of an experimental rat model of critical sized (7 cm2 ) full-thickness skin defect. The wound models treated with either MSCs-seeded (1.5 × 106 cells/cm2 ) or non-seeded bSIS membranes were completely closed by week 7 without significant differences in closure time; on the other hand, the open wound control was closed at ~47% at this time point. Immunohistopathology results revealed that the group which received MSCs-seeded bSIS had less scarring at the end of the healing process and was in further stages of appendage formation in comparison with the non-seeded bSIS group. Copyright © 2015 John Wiley & Sons, Ltd.

Time-Resolved Fluorescence Resonance Energy Transfer [TR-FRET] Assays for Biochemical Processes.

Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) is a fluorescence based technique which enables the analysis of molecular interactions in biochemical processes. Principle of TR-FRET is based on time-resolved fluorescence (TRF) measurement and fluorescence resonance energy transfer (FRET) between donor and acceptor molecules. To generate FRET signal, donor and acceptor molecules must show spectral overlap and should be in close proximity to each other and display suitable dipole orientation. The specific signal is acquired from molecules of interest via interactions of donor and acceptor molecules. TR-FRET technique is widely used for studying kinase assays, cellular signaling pathways, protein-protein interactions, DNA-protein interactions, and receptor-ligand binding. There are various propriety applications of TR-FRET. Two different sample protocols are summarized in this review.

Clinical applications of decellularized extracellular matrices for tissue engineering and regenerative medicine.

Decellularization is the process of removing the cellular components from tissues or organs. It is a promising technology for obtaining a biomaterial with a highly preserved extracellular matrix (ECM), which may also act as a biological scaffold for tissue engineering and regenerative therapies. Decellularized products are gaining clinical importance and market space due to their ease of standardized production, constant availability for grafting and mechanical or biochemical superiority against competing clinical options, yielding clinical results ahead of the ones with autografts in some applications. Current drawbacks and limitations of traditional treatments and clinical applications can be overcome by using decellularized or acellular matrices. Several companies are leading the market with versatile acellular products designed for diverse use in the reconstruction of tissues and organs. This review describes ECM-based decellularized and acellular products that are currently in use for different branches of clinic.

Extracellular Matrix and Regenerative Therapies from the Cardiac Perspective.

Cardiovascular diseases are the leading cause of death and a major cause of financial burden. Regenerative therapies for heart diseases bring the promise of alternative treatment modalities for myocardial infarction, ischemic heart disease, and congestive heart failure. Although, clinical trials attest to the safety of stem cell injection therapies, researchers need to overcome the underlying mechanisms that are limiting the success of future regenerative options. This article aims to review the basic scientific concepts in the field of mechanobiology and the effects of extracellular functions on stem cell fate.