In vivo tissue engineering of functional skeletal muscle by freshly isolated satellite cells embedded in a photopolymerizable hydrogel.

The success of skeletal muscle reconstruction depends on finding the most effective, clinically suitable strategy to engineer myogenic cells and biocompatible scaffolds. Satellite cells (SCs), freshly isolated or transplanted within their niche, are presently considered the best source for muscle regeneration. Here, we designed and developed the delivery of either SCs or muscle progenitor cells (MPCs) via an in situ photo-cross-linkable hyaluronan-based hydrogel, hyaluronic acid-photoinitiator (HA-PI) complex. Partially ablated tibialis anterior (TA) of C57BL/6J mice engrafted with freshly isolated satellite cells embedded in hydrogel showed a major improvement in muscle structure and number of new myofibers, compared to muscles receiving hydrogel + MPCs or hydrogel alone. Notably, SCs embedded in HA-PI also promoted functional recovery, as assessed by contractile force measurements. Tissue reconstruction was associated with the formation of both neural and vascular networks and the reconstitution of a functional SC niche. This innovative approach could overcome previous limitations in skeletal muscle tissue engineering.

Micro-bioreactor arrays for controlling cellular environments: design principles for human embryonic stem cell applications.

We discuss the utilization of micro-bioreactor arrays for controlling cellular environments in studies of factors that regulate the differentiation of human embryonic stem cells. To this end, we have designed a simple and practical system that couples a microfluidic platform with an array of micro-bioreactors, and has the size of a microscope slide [E. Figallo, C. Cannizzaro, S. Gerecht, J.A. Burdick, R. Langer, N. Elvassore, G. Vunjak-Novakovic, Lab Chip 7 (2007) 710-719]. The system allows quantitative studies of cells cultured in monolayers or encapsulated in three-dimensional hydrogels. We review the operating requirements for studies of human embryonic stem cells (hESCs) under steady-state and dynamic conditions, and the related control of the mass transport and hydrodynamic shear. We describe the design and fabrication of the individual bioreactor components, and the criteria for selecting the bioreactor configuration and operating parameters, based on the analysis of the characteristic times and scales of reaction, convection and diffusion. To illustrate the utility of the bioreactor, we present a "case study" of hESC cultivation with detailed experimental methods and representative biological readouts.

Electrical stimulation of human embryonic stem cells: cardiac differentiation and the generation of reactive oxygen species.

Exogenous electric fields have been implied in cardiac differentiation of mouse embryonic stem cells and the generation of reactive oxygen species (ROS). In this work, we explored the effects of electrical field stimulation on ROS generation and cardiogenesis in embryoid bodies (EBs) derived from human embryonic stem cells (hESC, line H13), using a custom-built electrical stimulation bioreactor. Electrical properties of the bioreactor system were characterized by electrochemical impedance spectroscopy (EIS) and analysis of electrical currents. The effects of the electrode material (stainless steel, titanium-nitride-coated titanium, titanium), length of stimulus (1 and 90 s) and age of EBs at the onset of electrical stimulation (4 and 8 days) were investigated with respect to ROS generation. The amplitude of the applied electrical field was 1 V/mm. The highest rate of ROS generation was observed for stainless steel electrodes, for signal duration of 90 s and for 4-day-old EBs. Notably, comparable ROS generation was achieved by incubation of EBs with 1 nM H(2)O(2). Cardiac differentiation in these EBs was evidenced by spontaneous contractions, expression of troponin T and its sarcomeric organization. These results imply that electrical stimulation plays a role in cardiac differentiation of hESCs, through mechanisms associated with the intracellular generation of ROS.

Bioreactor-manufactured cartilage grafts repair acute and chronic osteochondral defects in large animal studies.

OBJECTIVES: Bioreactor-based production systems have the potential to overcome limitations associated with conventional tissue engineering manufacturing methods, facilitating regulatory compliant and cost-effective production of engineered grafts for widespread clinical use. In this work, we established a bioreactor-based manufacturing system for the production of cartilage grafts. MATERIALS & METHODS: All bioprocesses, from cartilage biopsy digestion through the generation of engineered grafts, were performed in our bioreactor-based manufacturing system. All bioreactor technologies and cartilage tissue engineering bioprocesses were transferred to an independent GMP facility, where engineered grafts were manufactured for two large animal studies. RESULTS: The results of these studies demonstrate the safety and feasibility of the bioreactor-based manufacturing approach. Moreover, grafts produced in the manufacturing system were first shown to accelerate the repair of acute osteochondral defects, compared to cell-free scaffold implants. We then demonstrated that grafts produced in the system also facilitated faster repair in a more clinically relevant chronic defect model. Our data also suggested that bioreactor-manufactured grafts may result in a more robust repair in the longer term. CONCLUSION: By demonstrating the safety and efficacy of bioreactor-generated grafts in two large animal models, this work represents a pivotal step towards implementing the bioreactor-based manufacturing system for the production of human cartilage grafts for clinical applications. Read the Editorial for this article on doi:10.1111/cpr.12625.

Micro-bioreactor array for controlling cellular microenvironments.

High throughput experiments can be used to spatially and temporally investigate the many factors that regulate cell differentiation. We have developed a micro-bioreactor array (MBA) that is fabricated using soft lithography and contains twelve independent micro-bioreactors perfused with culture medium. The MBA enables cultivation of cells that are either attached to substrates or encapsulated in hydrogels, at variable levels of hydrodynamic shear, and with automated image analysis of the expression of cell differentiation markers. The flow and mass transport in the MBA were characterized by computational fluid dynamic (CFD) modeling. The representative MBA configurations were validated using the C2C12 cell line, primary rat cardiac myocytes and human embryonic stem cells (hESCs) (lines H09 and H13). To illustrate the utility of the MBA for controlled studies of hESCs, we established correlations between the expression of smooth muscle actin and cell density for three different flow configurations.

Practical aspects of cardiac tissue engineering with electrical stimulation.

Heart disease is a leading cause of death in western society. Despite the success of heart transplantation, a chronic shortage of donor organs, along with the associated immunological complications of this approach, demands that alternative treatments be found. One such option is to repair, rather than replace, the heart with engineered cardiac tissue. Multiple studies have shown that to attain functional tissue, assembly signaling cues must be recapitulated in vitro. In their native environment, cardiomyocytes are directed to beat in synchrony by propagation of pacing current through the tissue. Recently, we have shown that electrical stimulation directs neonatal cardiomyocytes to assemble into native-like tissue in vitro. This chapter provides detailed methods we have employed in taking this "biomimetic" approach. After an initial discussion on how electric field stimulation can influence cell behavior, we examine the practical aspects of cardiac tissue engineering with electrical stimulation, such as electrode selection and cell seeding protocols, and conclude with what we feel are the remaining challenges to be overcome.

Combination of Collagen-Based Scaffold and Bioactive Factors Induces Adipose-Derived Mesenchymal Stem Cells Chondrogenic Differentiation In vitro.

Recently, multipotent mesenchymal stem cells (MSCs) have attracted much attention in the field of regenerative medicine due to their ability to give rise to different cell types, including chondrocytes. Damaged articular cartilage repair is one of the most challenging issues for regenerative medicine, due to the intrinsic limited capability of cartilage to heal because of its avascular nature. While surgical approaches like chondral autografts and allografts provide symptoms and function improvement only for a short period, MSC based stimulation therapies, like microfracture surgery or autologous matrix-induced chondrogenesis demonstrate to be more effective. The use of adult chondrocytes, which are the main cellular constituent of cartilage, in medical practice, is indeed limited due to their instability in monolayer culture and difficulty to collect donor tissue (articular and nasal cartilage). The most recent cartilage engineering approaches combine cells, biomaterial scaffold and bioactive factors to promote functional tissue replacements. Many recent evidences demonstrate that scaffolds providing specific microenvironmental conditions can promote MSCs differentiation toward a functional phenotype. In the present work, the chondrogenic potential of a new Collagen I based 3D scaffold has been assessed in vitro, in combination with human adipose-derived MSCs which possess a higher chondrogenic potential compared to MSCs isolated from other tissues. Our data indicate that the scaffold was able to promote the early stages of chondrogenic commitment and that supplementation of specific soluble factors was able to induce the complete differentiation of MSCs in chondrocytes as demonstrated by the appearance of cartilage distinctive markers (Sox 9, Aggrecan, Matrilin-1, and Collagen II), as well as by the cartilage-specific Alcian Blue staining and by the acquisition of typical cellular morphology. Such evidences suggest that the investigated scaffold formulation could be suitable for the production of medical devices that can be beneficial in the field of articular cartilage engineering, thus improving the efficacy and durability of the current therapeutic options.

Effect of low-intensity pulsed ultrasound on osteogenic human mesenchymal stem cells commitment in a new bone scaffold.

PURPOSE: Bone tissue engineering is helpful in finding alternatives to overcome surgery limitations. Bone growth and repair are under the control of biochemical and mechanical signals; therefore, in recent years several approaches to improve bone regeneration have been evaluated. Osteo-inductive biomaterials, stem cells, specific growth factors and biophysical stimuli are among those. The aim of the present study was to evaluate if low-intensity pulsed ultrasound stimulation (LIPUS) treatment would improve the colonization of an MgHA/Coll hybrid composite scaffold by human mesenchymal stem cells (hMSCs) and their osteogenic differentiation. LIPUS stimulation was applied to hMSCs cultured on MgHA/Coll hybrid composite scaffold in osteogenic medium, mimicking the microenvironment of a bone fracture. METHODS: hMSCs were seeded on MgHA/Coll hybrid composite scaffold in an osteo-inductive medium and exposed to LIPUS treatment for 20 min/day for different experimental times (7 days, 14 days). The investigation was focused on (i) the improvement of hMSCs to colonize the MgHA/Coll hybrid composite scaffold by LIPUS, in terms of cell viability and ultrastructural analysis; (ii) the activation of MAPK/ERK, osteogenic (ALPL,COL1A1,BGLAP,SPP1) and angiogenetic (VEGF, IL8) pathways, through gene expression and protein release analysis, after LIPUS stimuli. RESULTS: LIPUS exposure improved MgHA/Coll hybrid composite scaffold colonization and induced in vitro osteogenic differentiation of hMSCs seeded on the scaffold. CONCLUSIONS: This work shows that the combined use of new biomimetic osteo-inductive composite and LIPUS treatment could be a useful therapeutic approach in order to accelerate bone regeneration pathways.

In Vivo Evaluation of Biocompatibility and Chondrogenic Potential of a Cell-Free Collagen-Based Scaffold.

Injured articular cartilage has a limited innate regenerative capacity, due to the avascular nature and low cellularity of the tissue itself. Although several approaches have been proposed to repair the joint cartilage, none of them has proven to be effective. The absence of suitable therapeutic options has encouraged tissue-engineering approaches combining specific cell types and biomaterials. In the present work, we have evaluated the potential of a cell-free Collagen I-based scaffold to promote the augmentation of cartilage-like phenotype after subcutaneous implantation in the mouse. Forty female mice were grafted subcutaneously with scaffolds, while four additional mice without scaffold were used as negative controls. The effects of scaffold were evaluated at 1, 2, 4, 8, or 16 weeks after implantation. Immunohistochemical analysis shows the expression of typical cartilage markers, including type-II Collagen, Aggrecan, Matrilin-1 and Sox 9. These data are also confirmed by qRT-PCR that further show that both COL2A1 and COL1A1 increase over time, but the first one increases more rapidly, thus suggesting a typical cartilage-like address. Histological analysis shows the presence of some pericellular lacunae, after 8 and 16 weeks. Results suggest that this scaffold (i) is biocompatible in vivo, (ii) is able to recruit host cells (iii) induce chondrogenic differentiation of host cells. Such evidences suggest that this cell-free scaffold is promising and represents a potential approach for cartilage regeneration.

Human adipose-derived mesenchymal stem cells seeded into a collagen-hydroxyapatite scaffold promote bone augmentation after implantation in the mouse.

Traumatic injury or surgical excision of diseased bone tissue usually require the reconstruction of large bone defects unable to heal spontaneously, especially in older individuals. This is a big challenge requiring the development of biomaterials mimicking the bone structure and capable of inducing the right commitment of cells seeded within the scaffold. In particular, given their properties and large availability, the human adipose-derived stem cells are considered as the better candidate for autologous cell transplantation. In order to evaluate the regenerative potential of these cells along with an osteoinductive biomaterial, we have used collagen/hydroxyapatite scaffolds to test ectopic bone formation after subcutaneous implantation in mice. The process was analysed both in vivo, by Fluorescent Molecular Tomography (FMT), and ex vivo, to evaluate the formation of bone and vascular structures. The results have shown that the biomaterial could itself be able of promoting differentiation of host cells and bone formation, probably by means of its intrinsic chemical and structural properties, namely the microenvironment. However, when charged with human mesenchymal stem cells, the ectopic bone formation within the scaffold was increased. We believe that these results represent an important advancement in the field of bone physiology, as well as in regenerative medicine.

Collagen-Hydroxyapatite Scaffolds Induce Human Adipose Derived Stem Cells Osteogenic Differentiation In Vitro.

Mesenchymal stem cells (MSCs) play a crucial role in regulating normal skeletal homeostasis and, in case of injury, in bone healing and reestablishment of skeletal integrity. Recent scientific literature is focused on the development of bone regeneration models where MSCs are combined with biomimetic three-dimensional scaffolds able to direct MSC osteogenesis. In this work the osteogenic potential of human MSCs isolated from adipose tissue (hADSCs) has been evaluated in vitro in combination with collagen/Mg doped hydroxyapatite scaffolds. Results demonstrate the high osteogenic potential of hADSCs when cultured in specific differentiation induction medium, as revealed by the Alizarin Red S staining and gene expression profile analysis. In combination with collagen/hydroxyapatite scaffold, hADSCs differentiate into mature osteoblasts even in the absence of specific inducing factors; nevertheless, the supplement of the factors markedly accelerates the osteogenic process, as confirmed by the expression of specific markers of pre-osteoblast and mature osteoblast stages, such as osterix, osteopontin (also known as bone sialoprotein I), osteocalcin and specific markers of extracellular matrix maturation and mineralization stages, such as ALPL and osteonectin. Hence, the present work demonstrates that the scaffold per se is able to induce hADSCs differentiation, while the addition of osteo-inductive factors produces a significant acceleration of the osteogenic process. This observation makes the use of our model potentially interesting in the field of regenerative medicine for the treatment of bone defects.

Bone augmentation after ectopic implantation of a cell-free collagen-hydroxyapatite scaffold in the mouse.

The bone grafting is the classical way to treat large bone defects. Among the available techniques, autologous bone grafting is still the most used but, however, it can cause complications such as infection and donor site morbidity. Alternative and innovative methods rely on the development of biomaterials mimicking the structure and properties of natural bone. In this study, we characterized a cell-free scaffold, which was subcutaneously implanted in mice and then analyzed both in vivo and ex vivo after 1, 2, 4, 8 and 16 weeks, respectively. Two types of biomaterials, made of either collagen alone or collagen plus magnesium-enriched hydroxyapatite have been used. The results indicate that bone augmentation and angiogenesis could spontaneously occur into the biomaterial, probably by the recruitment of host cells, and that the composition of the scaffolds is crucial. In particular, the biomaterial more closely mimicking the native bone drives the process of bone augmentation more efficiently. Gene expression analysis and immunohistochemistry demonstrate the expression of typical markers of osteogenesis by the host cells populating the scaffold. Our data suggest that this biomaterial could represent a promising tool for the reconstruction of large bone defects, without using exogenous living cells or growth factors.

Micropatterned biopolymer 3D scaffold for static and dynamic culture of human fibroblasts.

During in vivo tissue regeneration, cell behavior is highly influenced by the surrounding environment. Thus, the choice of scaffold material and its microstructure is one of the fundamental steps for a successful in vitro culture. An efficacious method for scaffold fabrication should prove its versatility and the possibility of controlling micro- and nanostructure. In this paper, hyaluronic acid 3D scaffolds were developed through lamination of micropatterned membranes, fabricated after optimization of a soft-lithography method. The scaffold presented here is characterized by a homogeneous hexagonal lattice with porosity of 69%, specific surface area of 287 cm-1, and permeability of 18.9 microm2. The control over the geometry was achieved with an accuracy of 20 mum. This technique allowed not only fabrication of planar 3D scaffolds but also production of thin wall tubular constructs. Mechanical tests, performed on dry tubular scaffolds, show high rupture tensile strength. This construct could be promising not only as engineered vascular grafts but also for regeneration of skin, urethra, and intestinal walls. The biocompatibility of a 3D planar scaffold was tested by seeding human fibroblasts. The cells were cultured in both static and dynamic conditions, in a perfusion bioreactor at different flow rates. Microscope analysis and MTT test showed cell proliferation and viability and a uniform cell distribution likely due to an appropriate lattice structure.

Characterization of electrical stimulation electrodes for cardiac tissue engineering.

Electrical stimulation has been shown to improve functional assembly of cardiomyocytes in vitro for cardiac tissue engineering. The goal of this study was to assess the conditions of electrical stimulation with respect to the electrode geometry, material properties and charge-transfer characteristics at the electrode-electrolyte interface. We compared various biocompatible materials, including nanoporous carbon, stainless steel, titanium and titanium nitride, for use in cardiac tissue engineering bioreactors. The faradaic and non-faradaic charge transfer mechanisms were assessed by electrochemical impedance spectroscopy (EIS), studying current injection characteristics, and examining surface properties of electrodes with scanning electron microscopy. Carbon electrodes were found to have the best current injection characteristics. However, these electrodes require careful handling because of their limited mechanical strength. The efficacy of various electrodes for use in 2-D and 3-D cardiac tissue engineering systems with neonatal rat cardiomyocytes is being determined by assessing cell viability, amplitude of contractions, excitation thresholds, maximum capture rate, and tissue morphology.