New Strategies In Regenerative Medicine Research Articles

New strategies in regenerative medicine and surgery and their application to orthopedic surgery field: The bio-active composite therapies (BACTs)

Regenerative Medicine and Surgery is a rapidly expanding branch of translational research in tissue engineering, cellular and molecular biology. To date, the methods to improve cell intake, survival and isolation need to comply with a complex and still unclear regulatory frame, becoming everyday more restrictive and often limiting effectiveness and outcome of the therapeutic choices. Thus, the author developed a novel regenerative strategy, based on the synergic action of several bio-active components, called the Bio-Active Composite Therapies (BACTs) to improve grafted cells intake and survival in total compliance with the legal and ethical limits of the current regulatory frame. The rationale at the origin of this new technology is based on the evidence that cells need supportive substrate to survive in vitro and this observation, applying the concept of translational medicine, is true also in vivo. Many different sources have been used in the past for MSCs, molecules and growth factors (GF) isolation and extraction, but the Adipose Tissue and its Stromal Vascular Fraction (SVF) definitely remains the most valuable, abundant, safe and reliable. Bio-Active Composite Mixtures (BACMs) are tailor-made injectable “cocktails” containing several bio-active components to support cells survival and induce a strong regenerative response in vivo by stimulating the recipient site to act as an in-situ real Bioreactor. In this article, the author analyze the main causes of cell’s death and the strategies for preventing it, and outline all the technical steps for preparing the main components of BACMs and the different mixing modalities to obtain the most efficient regenerative action on different clinical and pathological conditions in several surgical specialties. Orthopedic Surgery is definitely the one that most can benefit of these new therapeutic strategies. The final part of this work is anticipating the logical and sequential evolution toward other fundamental technical steps for further supporting and enhancing the most efficient regenerative activity.

Effects of Hair Follicle Stem Cells on Partial-Thickness Burn Wound Healing and Tensile Strength

Background:The recent improvements in wound healing have led to new strategies in regenerative medicine. Burn wound healing is an important issue in skin regeneration and has multiple indications for stem cell therapy. HFSCs are a highly promising source of stem cells for healing use, as these cells are accessible, active and pluripotent adult stem cells. Methods:HFSCs of the rat whisker were isolated, cultured, and labeled with DiI. Flow cytometry method was used to detect special markers of HFSCs. Deep partial-thickness burn wound was created, and labeled HFSCs were injected around the wound bed. Wound closure was recorded via digital photographs. The inflicted rats were sacrificed at 3, 7, or 14 days post burn and used for subsequent histological and tensiometry analysis. Results:Our results indicated that HFSCs were positive for Nestin and CD34 markers, but negative for Kr15. Morphological and histological photographs revealed that wound closure rate was accelerated in stem cell-treated group compared with other groups. In addition, faster re-epithelialization and collagen deposition were observed. The immunohistochemical analysis suggested that CD31 expression and vascular density enhanced in the stem cell-treated group. Further, tissue tensile strength increased in HFSCs-treated rats in comparison to the control group. Conclusion:The present study demonstrates that HFSCs could accelerate burn wound healing as well as tensile strength in rats.

Tracking Neural Progenitor Cell Migration in the Rodent Brain Using Magnetic Resonance Imaging.

The study of neurogenesis and neural progenitor cells (NPCs) is important across the biomedical spectrum, from learning about normal brain development and studying disease to engineering new strategies in regenerative medicine. In adult mammals, NPCs proliferate in two main areas of the brain, the subventricular zone (SVZ) and the subgranular zone, and continue to migrate even after neurogenesis has ceased within the rest of the brain. In healthy animals, NPCs migrate along the rostral migratory stream (RMS) from the SVZ to the olfactory bulb, and in diseased animals, NPCs migrate toward lesions such as stroke and tumors. Here we review how MRI-based cell tracking using iron oxide particles can be used to monitor and quantify NPC migration in the intact rodent brain, in a serial and relatively non-invasive fashion. NPCs can either be labeled directly in situ by injecting particles into the lateral ventricle or RMS, where NPCs can take up particles, or cells can be harvested and labeled in vitro, then injected into the brain. For in situ labeling experiments, the particle type, injection site, and image analysis methods have been optimized and cell migration toward stroke and multiple sclerosis lesions has been investigated. Delivery of labeled exogenous NPCs has allowed imaging of cell migration toward more sites of neuropathology, which may enable new diagnostic and therapeutic opportunities for as-of-yet untreatable neurological diseases.

Laser Surface Microstructuring of a Bio-Resorbable Polymer to Anchor Stem Cells, Control Adipocyte Morphology, and Promote Osteogenesis.

New strategies in regenerative medicine include the implantation of stem cells cultured in bio-resorbable polymeric scaffolds to restore the tissue function and be absorbed by the body after wound healing. This requires the development of appropriate micro-technologies for manufacturing of functional scaffolds with controlled surface properties to induce a specific cell behavior. The present report focuses on the effect of substrate topography on the behavior of human mesenchymal stem cells (MSCs) before and after co-differentiation into adipocytes and osteoblasts. Picosecond laser micromachining technology (PLM) was applied on poly (L-lactide) (PLLA), to generate different microstructures (microgrooves and microcavities) for investigating cell shape, orientation, and MSCs co-differentiation. Under certain surface topographical conditions, MSCs modify their shape to anchor at specific groove locations. Upon MSCs differentiation, adipocytes respond to changes in substrate height and depth by adapting the intracellular distribution of their lipid vacuoles to the imposed physical constraints. In addition, topography alone seems to produce a modest, but significant, increase of stem cell differentiation to osteoblasts. These findings show that PLM can be applied as a high-efficient technology to directly and precisely manufacture 3D microstructures that guide cell shape, control adipocyte morphology, and induce osteogenesis without the need of specific biochemical functionalization.

Impaired immune surveillance accelerates accumulation of senescent cells and aging

Cellular senescence is a stress response that imposes stable cell-cycle arrest in damaged cells, preventing their propagation in tissues. However, senescent cells accumulate in tissues in advanced age, where they might promote tissue degeneration and malignant transformation. The extent of immune-system involvement in regulating age-related accumulation of senescent cells, and its consequences, are unknown. Here we show that Prf1−/− mice with impaired cell cytotoxicity exhibit both higher senescent-cell tissue burden and chronic inflammation. They suffer from multiple age-related disorders and lower survival. Strikingly, pharmacological elimination of senescent-cells by ABT-737 partially alleviates accelerated aging phenotype in these mice. In LMNA+/G609G progeroid mice, impaired cell cytotoxicity further promotes senescent-cell accumulation and shortens lifespan. ABT-737 administration during the second half of life of these progeroid mice abrogates senescence signature and increases median survival. Our findings shed new light on mechanisms governing senescent-cell presence in aging, and could motivate new strategies for regenerative medicine.

High efficiency penetration of antibody-immobilized nanoneedle thorough plasma membrane for in situ detection of cytoskeletal proteins in living cells.

BackgroundThe field of structural dynamics of cytoskeletons in living cells is gathering wide interest, since better understanding of cytoskeleton intracellular organization will provide us with not only insights into basic cell biology but may also enable development of new strategies in regenerative medicine and cancer therapy, fields in which cytoskeleton-dependent dynamics play a pivotal role. The nanoneedle technology is a powerful tool allowing for intracellular investigations, as it can be directly inserted into live cells by penetrating through the plasma membrane causing minimal damage to cells, under the precise manipulation using atomic force microscope. Modifications of the nanoneedles using antibodies have allowed for accurate mechanical detection of various cytoskeletal components, including actin, microtubules and intermediate filaments. However, successful penetration of the nanoneedle through the plasma membrane has been shown to vary greatly between different cell types and conditions. In an effort to overcome this problem and improve the success rate of nanoneedle insertion into the live cells, we have focused here on the fluidity of the membrane lipid bilayer, which may hinder nanoneedle penetration into the cytosolic environment.ResultsWe aimed to reduce apparent fluidity of the membrane by either increasing the approach velocity or reducing experimental temperatures. Although changes in approach velocity did not have much effect, lowering the temperature was found to greatly improve the detection of unbinding forces, suggesting that alteration in the plasma membrane fluidity led to increase in nanoneedle penetration.ConclusionsOperation at a lower temperature of 4 °C greatly improved the success rate of nanoneedle insertion to live cells at an optimized approach velocity, while it did not affect the binding of antibodies immobilized on the nanoneedle to vimentins for mechanical detection. As these experimental parameters can be applied to various cell types, these results may improve the versatility of the nanoneedle technology to other cell lines and platforms.Electronic supplementary materialThe online version of this article (doi:10.1186/s12951-016-0226-5) contains supplementary material, which is available to authorized users.

The Neurofilament-Derived Peptide NFL-TBS.40-63 Targets Neural Stem Cells and Affects Their Properties.

Targeting neural stem cells (NSCs) in the adult brain represents a promising approach for developing new regenerative strategies, because these cells can proliferate, self-renew, and differentiate into new neurons, astrocytes, and oligodendrocytes. Previous work showed that the NFL-TBS.40-63 peptide, corresponding to the sequence of a tubulin-binding site on neurofilaments, can target glioblastoma cells, where it disrupts their microtubules and inhibits their proliferation. We show that this peptide targets NSCs in vitro and in vivo when injected into the cerebrospinal fluid. Although neurosphere formation was not altered by the peptide, the NSC self-renewal capacity and proliferation were reduced and were associated with increased adhesion and differentiation. These results indicate that the NFL-TBS.40-63 peptide represents a new molecular tool to target NSCs to develop new strategies for regenerative medicine and the treatment of brain tumors. In the present study, the NFL-TBS.40-63 peptide targeted neural stem cells in vitro when isolated from the subventricular zone and in vivo when injected into the cerebrospinal fluid present in the lateral ventricle. The in vitro formation of neurospheres was not altered by the peptide; however, at a high concentration of the peptide, the neural stem cell (NSC) self-renewal capacity and proliferation were reduced and associated with increased adhesion and differentiation. These results indicate that the NFL-TBS.40-63 peptide represents a new molecular tool to target NSCs to develop new strategies for regenerative medicine and the treatment of brain tumors.

Polymeric Nanohybrids as a New Class of Therapeutic Biotransporters.

A possible solution to enhance existing drug and gene therapies is to develop hybrid nanocarriers capable of delivering therapeutic agents in a controlled and targeted manner. This goal can be achieved by designing nanohybrid systems, which combine organic or inorganic nanomaterials with biomacromolecules into a single composite. The unique combination of properties along with their facile fabrication enables the design of smart carriers for both drug and gene delivery. These hybrids can be further modified with cell targeting motifs to enhance their biological interactivity. In this Talents and Trends article, an overview of emerging nanohybrid-based technologies will be provided to highlight their potential use as innovative platforms for improved cancer therapies and new strategies in regenerative medicine. The clinical relevance of these systems will be reviewed to define the current challenges which still need to be addressed to allow these therapies to move from bench to bedside.

Tunable and dynamic soft materials for three-dimensional cell culture.

The human body is complex and hierarchically structured, composed of cells residing within the extracellular matrix (ECM) of tissues that are assembled into organs, all working together to complete a given function. One goal of current biomaterials research is to capture some of this complexity outside of the body for understanding the underlying biology of development, repair, and disease and to devise new strategies for regenerative medicine or disease treatment. Polymeric materials have arisen as powerful tools to mimic the native ECM, giving experimenters a way to capture key aspects of the native cellular environment outside of the body. In particular, dynamic materials allow changes in the properties of these ECM mimics during an experiment, affording an additional degree of control for the experimenter. In this tutorial review, the basic cellular processes of cell migration, proliferation, and differentiation will be overviewed to motivate design considerations for polymeric ECM mimics, and examples will be given of how classes of dynamic materials are being used to study each cellular process.

Multi-tissue microarray analysis identifies a molecular signature of regeneration.

The inability to functionally repair tissues that are lost as a consequence of disease or injury remains a significant challenge for regenerative medicine. The molecular and cellular processes involved in complete restoration of tissue architecture and function are expected to be complex and remain largely unknown. Unlike humans, certain salamanders can completely regenerate injured tissues and lost appendages without scar formation. A parsimonious hypothesis would predict that all of these regenerative activities are regulated, at least in part, by a common set of genes. To test this hypothesis and identify genes that might control conserved regenerative processes, we performed a comprehensive microarray analysis of the early regenerative response in five regeneration-competent tissues from the newt Notophthalmus viridescens. Consistent with this hypothesis, we established a molecular signature for regeneration that consists of common genes or gene family members that exhibit dynamic differential regulation during regeneration in multiple tissue types. These genes include members of the matrix metalloproteinase family and its regulators, extracellular matrix components, genes involved in controlling cytoskeleton dynamics, and a variety of immune response factors. Gene Ontology term enrichment analysis validated and supported their functional activities in conserved regenerative processes. Surprisingly, dendrogram clustering and RadViz classification also revealed that each regenerative tissue had its own unique temporal expression profile, pointing to an inherent tissue-specific regenerative gene program. These new findings demand a reconsideration of how we conceptualize regenerative processes and how we devise new strategies for regenerative medicine.

Correlations Between the Biochemistry and Mechanical States of a Sea-Urchin Ligament: A Mutable Collagenous Structure

Mutable collagenous tissues (MCTs) of echinoderms can be regarded as intelligent and dynamic biomaterials, due to their ability to reversibly change their mechanical properties in a short physiological time span. This mutability phenomenon is nervously mediated and involves secreted factors of the specialized 'juxtaligamental' cells, which, when released into the extracellular matrix (ECM), change the cohesive forces between collagen fibrils. MCTs exist in nature in several forms, including some associated with echinoderm autotomy mechanisms. Since the molecular mechanism of mutability is still incompletely understood, the aim of this work was to provide a detailed biochemical analysis of a typical mutable collagenous structure and to identify possible correlations between its biochemistry and mechanical states. A better understanding of the mutability phenomena is likely to provide a unique opportunity to develop new concepts that can be applied in the design of dynamic biomaterial for tissue regeneration, leading to new strategies in regenerative medicine. The MCT model used was the compass depressor ligament (CDL) of a sea urchin (Paracentrotus lividus), which was analyzed in different mechanical states, mimicking the mutability phenomenon. Spectroscopic techniques, namely Fourier transform infrared (FT-IR) and confocal Raman microscopy, were used to identify the specific molecular components that contribute to the CDL biochemical microenvironment and to investigate the possibility that remodelling/synthesis of new ECM components occurs during the mutability phenomenon by analogy with events during pregnancy in the uterine cervix of mammals (which also consists mainly of mechanically adaptable connective tissues). The results demonstrate that CDL ECM includes collagen with biochemical similarities to mammalian type I collagen, as well as sulphated glycosaminoglycans (GAGs). CDL mutability seems to involve a molecular rearrangement of the ECM, without synthesis of new ECM components. Although there were no significant biochemical differences between CDLs in the various mechanical states were observed. However, subtle adjustments in tissue hydration seemed to occur, particularly during stiffening.

Benefits of dedifferentiated stem cells for neural regeneration

Dedifferentiation, as one of the mechanisms rerouting cell fate, regresses cells from a differentiated status to a more primitive one. Due to its potential of amplifying the stem/progenitor cell pool and reproducing sizable and desirable cellular elements, it has been attended in the field of regenerative medicine, which will hopefully provide novel therapeutic strategies for currently incurable diseases, such as varieties of central nervous system (CNS) diseases and injuries. In this article, we will first discuss naturally occurring and experimentally induced dedifferentiation, and then set forth principles in stem-cell based therapy in the neural field; beyond that, we will introduce two recent studies that show dedifferentiated stem cells contribute to neural regeneration. Moreover, we also present our recent research results of dedifferentiated muscle stem cells for neurogenic differentiation study in vitro. Further work will be conducted to elucidate the mechanism underlying the dedifferentiation process to facilitate the development of new strategies in regenerative medicine.