Tissue Repair Applications Research Articles

Effects of an in vitro low-oxygen-tension preconditioning of adipose stem cells on their in vivo chondrogenic potential: application in cartilage tissue repair

Effects of an in vitro low-oxygen-tension preconditioning of adipose stem cells on their in vivo chondrogenic potential: application in cartilage tissue repair

Effects of In Vitro Low Oxygen Tension Preconditioning of Adipose Stromal Cells on Their In Vivo Chondrogenic Potential: Application in Cartilage Tissue Repair

PurposeMultipotent stromal cell (MSC)-based regenerative strategy has shown promise for the repair of cartilage, an avascular tissue in which cells experience hypoxia. Hypoxia is known to promote the early chondrogenic differentiation of MSC. The aim of our study was therefore to determine whether low oxygen tension could be used to enhance the regenerative potential of MSC for cartilage repair.MethodsMSC from rabbit or human adipose stromal cells (ASC) were preconditioned in vitro in control or chondrogenic (ITS and TGF-β) medium and in 21 or 5% O2. Chondrogenic commitment was monitored by measuring COL2A1 and ACAN expression (real-time PCR). Preconditioned rabbit and human ASC were then incorporated into an Si-HPMC hydrogel and injected (i) into rabbit articular cartilage defects for 18 weeks or (ii) subcutaneously into nude mice for five weeks. The newly formed tissue was qualitatively and quantitatively evaluated by cartilage-specific immunohistological staining and scoring. The phenotype of ASC cultured in a monolayer or within Si-HPMC in control or chondrogenic medium and in 21 or 5% O2 was finally evaluated using real-time PCR.Results/Conclusions5% O2 increased the in vitro expression of chondrogenic markers in ASC cultured in induction medium. Cells implanted within Si-HPMC hydrogel and preconditioned in chondrogenic medium formed a cartilaginous tissue, regardless of the level of oxygen. In addition, the 3D in vitro culture of ASC within Si-HPMC hydrogel was found to reinforce the pro-chondrogenic effects of the induction medium and 5% O2. These data together indicate that although 5% O2 enhances the in vitro chondrogenic differentiation of ASC, it does not enhance their in vivo chondrogenesis. These results also highlight the in vivo chondrogenic potential of ASC and their potential value in cartilage repair.

Compatibility and Crystallinity of Electrospun Aligned PHBV/PEG Ultrafine Fibers Scaffolds

Aligned PHBV/PEG ultrafine fibers are fabricated for potential applications in tissue repair. In this paper we find that the optimum spinning parameters to prepare aligned PHBV/PEG fibers is as follows: the concentration of 8wt%, voltage at 10kv, receiving distance at 18cm, the boost speed at 0.8ml/h and the best linear velocity of the rotating drum at 9.5m/s. The content of PEG is less than 50%. In addition, we investigate the interaction between PHBV and PEG in its entirety; we also discuss the surface morphology of the blends, compatibility, thermal decomposition and crystallization behavior by the help of different test methods.

Transplantation-potential-related biological properties of decidua basalis mesenchymal stem cells from maternal human term placenta

Human placental decidua basalis originates from the maternal side of the placenta and has been described as a source of mesenchymal stem cells (MSCs). However, for its application in tissue regeneration and repair, the transplantation-potential-related biological properties of decidua-basalis-derived mesenchymal stem cells (DBMSCs) remain to be elucidated. We obtained DBMSCs through enzymatic digestion and density gradient centrifugation and confirmed their capacity to differentiate into cell types of the mesodermal lineage, such as osteoblasts, adipocytes and chondroblasts. Karyotype analysis showed that the isolated DBMSCs maintained chromosomal stability after long-term culture in vitro. Growth kinetics and ultrastructural observation revealed a high level of DBMSC proliferative activity. In addition, DBMSCs showed immunosuppressive properties by suppressing both mitogen- and alloantigen-induced peripheral lymphocyte proliferation. All of these properties suggest that DBMSCs, which are abundant and easily accessible, are a novel potential source of seed cells for cell transplantation treatments.

Liquid Crystalline Human Recombinant Collagen: The Challenge and the Opportunity

Collagen is a key component of the extracellular matrix, and by far the most prominent constituent of all load-bearing tissues. Its abundance and self-assembly capacities render it a practical scaffold material for tissue repair and regeneration applications. However, some difficulties exist in artificially regenerating functional collagen structures to match native tissues and their respective performances. There are two major limitations of existing collagen-based scaffolds: The first one is poor mechanical performance, and the second one is the failure to closely mimic natural tissues as to provide the necessary topographic and mechanical cues required for cell propagation and differentiation. The complexity of inducing sufficient order and alignment stands at the base of the impediments to successful formation of artificial collagen scaffolds, which closely match native tissue strength and morphology. Recombinant human collagen produced in transgenic tobacco plants has the capacity of forming highly concentrated liquid crystalline dope that can be aligned by application of shear force. Leveraging shear alignment of liquid crystalline recombinant human collagen opens new possibilities toward obtaining scaffolds that may be able to provide the necessary mechanical support, while closely mimicking the molecular signals and mechanical cues displayed to natural cell milieu. Such scaffolds may prove advantageous in the development of improved medical devices in fields, such as ophthalmology, neurology, and orthopedics.

Array of Biodegradable Microraftsfor Isolation and Implantation of Living, Adherent Cells.

A new strategy for efficient sorting and implantation of viable adherent cells into animals is described. An array of biodegradable micro-structures (microrafts) was fabricated using a polydimethylsiloxane substrate for micromolding poly(lactic-co-glycolic acid) (PLGA). Screening various forms of PLGA determined that the suitability of PLGA for microraft manufacture, biocompatibility and in vitro degradation was dependent on molecular weight and lactic/glycolic ratio. Cells plated on the array selectively attached to the microrafts and could be identified by their fluorescence, morphology or other criteria. The cells were efficiently dislodged and collected from the array using a microneedle device. The platform was used to isolate specific cells from a mixed population establishing the ability to sort target cells for direct implantation. As a proof of concept, fluorescently conjugated microrafts carrying tumor cells stably expressing luciferase were isolated from an array and implanted subcutaneously into mice. In vivo bio-luminescence imaging confirmed the growth of a tumor in the recipient animals. Imaging of tissue sections from the tumors demonstrated in vivo degradation of the implanted microrafts. The process is a new strategy for isolating and delivering a small number of adherent cells for animal implantation with potential applications in tissue repair, tumor induction, in vivo differentiation of stem cells and other biomedical research.

Cells derived from murine induced pluripotent stem cells (iPSC) by treatment with members of TGF-beta family give rise to osteoblasts differentiation and form bone in vivo

BackgroundInduced pluripotent stem cells (iPSC) are generated by reprogramming somatic cells into embryonic like state (ESC) using defined factors. There is great interest in these cells because of their potential for application in regenerative medicine.ResultsiPSC reprogrammed from murine tail tip fibroblasts were exposed to retinoic acid alone (RA) or in combination with TGF-β1 and 3, basic fibroblast growth factor (bFGF) or bone morphogenetic protein 2 (BMP-2). The resulting cells expressed selected putative mesenchymal stem cells (MSCs) markers; differentiated toward osteoblasts and adipocytic cell lineages in vitro at varying degrees. TGF-beta1 and 3 derived-cells possessed higher potential to give rise to osteoblasts than bFGF or BMP-2 derived-cells while BMP-2 derived cells exhibited a higher potential to differentiate toward adipocytic lineage. TGF-β1 in combination with RA derived-cells seeded onto HA/TCP ceramics and implanted in mice deposited typical bone. Immunofluorescence staining for bone specific proteins in cell seeded scaffolds tissue sections confirmed differentiation of the cells into osteoblasts in vivo.ConclusionsThe results demonstrate that TGF-beta family of proteins could potentially be used to generate murine iPSC derived-cells with potential for osteoblasts differentiation and bone formation in vivo and thus for application in musculoskeletal tissue repair and regeneration.

Human Elastin‐Based Recombinant Biopolymers Improve Mesenchymal Stem Cell Differentiation

Elastin-based polypeptides are a class of smart biopolymers representing an important model in the design of biomaterials. The combination of biomimetic materials with cells that have great plasticity provides a promising strategy for the realization of highly engineered cell-based constructs for regenerative medicine and tissue repair applications. Two recombinant biopolymers inspired by human elastin are assessed as coating agents to prepare biomimetic surfaces for cell culture. These substrates are assayed for hBM MSC culture. The coated surfaces are also characterized with AFM to evaluate the topographical features of the deposited biopolymers. The results suggest that the elastin-derived biomimetic surfaces play a stimulatory role on osteogenic differentiation of MSCs.

The stat3/socs3a Pathway Is a Key Regulator of Hair Cell Regeneration in Zebrafish stat3/socs3a Pathway: Regulator of Hair Cell Regeneration

All nonmammalian vertebrates studied can regenerate inner ear mechanosensory receptors (i.e., hair cells) (Corwin and Cotanche, 1988; Lombarte et al., 1993; Baird et al., 1996), but mammals possess only a very limited capacity for regeneration after birth (Roberson and Rubel, 1994). As a result, mammals experience permanent deficiencies in hearing and balance once their inner ear hair cells are lost. The mechanisms of hair cell regeneration are poorly understood. Because the inner ear sensory epithelium is highly conserved in all vertebrates (Fritzsch et al., 2007), we chose to study hair cell regeneration mechanism in adult zebrafish, hoping the results would be transferrable to inducing hair cell regeneration in mammals. We defined the comprehensive network of genes involved in hair cell regeneration in the inner ear of adult zebrafish with the powerful transcriptional profiling technique digital gene expression, which leverages the power of next-generation sequencing ('t Hoen et al., 2008). We also identified a key pathway, stat3/socs3, and demonstrated its role in promoting hair cell regeneration through stem cell activation, cell division, and differentiation. In addition, transient pharmacological inhibition of stat3 signaling accelerated hair cell regeneration without overproducing cells. Taking other published datasets into account (Sano et al., 1999; Schebesta et al., 2006; Dierssen et al., 2008; Riehle et al., 2008; Zhu et al., 2008; Qin et al., 2009), we propose that the stat3/socs3 pathway is a key response in all tissue regeneration and thus an important therapeutic target for a broad application in tissue repair and injury healing.

Biomaterial applications in cardiovascular tissue repair and regeneration

Cardiovascular disease physically damages the heart, resulting in loss of cardiac function. Medications can help alleviate symptoms, but it is more beneficial to treat the root cause by repairing injured tissues, which gives patients better outcomes. Besides heart transplants, cardiac surgeons use a variety of methods for repairing different areas of the heart such as the ventricular septal wall and valves. A multitude of biomaterials are used in the repair and replacement of impaired heart tissues. These biomaterials fall into two main categories: synthetic and natural. Synthetic materials used in cardiovascular applications include polymers and metals. Natural materials are derived from biological sources such as human donor or harvested animal tissues. A new class of composite materials has emerged to take advantage of the benefits of the strengths and minimize the weaknesses of both synthetic and natural materials. This article reviews the current and prospective applications of biomaterials in cardiovascular therapies.

The electrical stimulation of carbon nanotubes to provide a cardiomimetic cue to MSCs

Once damaged, cardiac muscle has little intrinsic repair capability due to the poor regeneration potential of remaining cardiomyocytes. One method of overcoming this issue is to deliver functional cells to the injured myocardium to promote repair. To address this limitation we sought to test the hypothesis that electroactive carbon nanotubes (CNT) could be employed to direct mesenchymal stem cell (MSC) differentiation towards a cardiomyocyte lineage. Using a two-pronged approach, MSCs exposed to medium containing CNT and MSCs seeded on CNT based polylactic acid scaffolds were electrically stimulated in an electrophysiological bioreactor. After electrical stimulation the cells reoriented perpendicular to the direction of the current and adopted an elongated morphology. Using qPCR, an upregulation in a range of cardiac markers was detected, the greatest of which was observed for cardiac myosin heavy chain (CMHC), where a 40-fold increase was observed for the electrically stimulated cells after 14 days, and a 12-fold increase was observed for the electrically stimulated cells seeded on the PLA scaffolds after 10 days. Differentiation towards a cardioprogenitor cell was more evident from the western blot analysis, where upregulation of Nkx2.5, GATA-4, cardiac troponin t (CTT) and connexin43 (C43) was seen to occur. This was echoed in immunofluorescent staining, where increased levels of CTT, CMHC and C43 protein expression were observed after electrical stimulation for both cells and cell-seeded scaffolds. More interestingly, there was evidence of increased cross talk between the cells as shown by the pattern of C43 staining after electrical stimulation. These results establish a paradigm for nanoscale biomimetic cues that can be readily translated to other electroactive tissue repair applications.

Coalescence of Extracellular Matrix (ECM) from Porcine Urinary Bladder (UBM) with a Laser-Activated Chitosan-Based Surgical Adhesive

Urinary bladder matrix (UBM) has been extensively investigated as a naturally occurring biomaterial in therapeutic applications for tissue repair or regeneration, while other strategies involve biopolymers such as chitosan for tissue reconstruction. The coalescence of UBM with chitosan has considerable potential in enhancing tissue reconstruction. Characterisation of a novel, laser-activated, chitosan-based, thin-film surgical adhesive with UBM in various morphologies showed that the films had increased surface rugosities and crystallinities (Ra approx. 0.83 um, approx. 12% crystallinity) when compared to the chitosan adhesive alone (R a = 0.74 um, 7% crystallinity). Tensile strength of the films was significantly increased by the addition of UBM in particulate form (12.1–32.4 MPa). Furthermore, tissue adhesion strengths using these hybrid biomaterials were maintained at approx. 15 kPa compared to 3 kPa for fibrin glue. Histological analysis demonstrated that laser irradiation of the UBM-chitosan adhesive biomaterial caused no thermal damage to tissue. Examination of the cellular response at the material interface showed that 3T3 fibroblasts maintained their regular morphology with enhanced growth compared to films of both chitosan and its adhesive form. These results suggest that coalescence of UBM with a chitosan-based adhesive supports the development of biomaterial devices for sutureless wound closure that could enhance tissue repair and reconstruction.

Spinal Cord Repair and Regeneration: The Application of Bioadhesives and Extracellular Matrix Bioscaffolds

Extracellular matrices (ECM) derived from urinary bladder membrane (UBM) have demonstrated substantial potential for applications in tissue repair and reconstruction. However, these materials are limited by the requirement of suturing following surgery. In this paper, the coalescence of UBM with an advanced surgical adhesive demonstrated a suitable alternative to sutures for wound closure, prevention of fluid leakage and improvement of cell growth. This novel bioadhesive contains favourable characteristics that are suitable for tissue repair, support of cell growth, and influence cellular microenvironments that are biocompatible with peripheral nerve regeneration in the spinal cord.

Microwave-sintered 3D printed tricalcium phosphate scaffolds for bone tissue engineering

This study reports the manufacturing process of 3D interconnected macroporous tricalcium phosphate (TCP) scaffolds with controlled internal architecture by direct 3D printing (3DP), and high mechanical strength obtained by microwave sintering. TCP scaffolds with 27%, 35% and 41% designed macroporosity with pore sizes of 500 μm, 750 μm and 1000 μm, respectively, were manufactured by direct 3DP. These scaffolds were then sintered at 1150 °C and 1250 °C in conventional electric muffle and microwave furnaces, respectively. Total open porosity between 42% and 63% was obtained in the sintered scaffolds due to the presence of intrinsic micropores along with designed pores. A significant increase in compressive strength between 46% and 69% was achieved by microwave compared to conventional sintering as a result of efficient densification. Maximum compressive strengths of 10.95 ± 1.28 MPa and 6.62 ± 0.67 MPa were achieved for scaffolds with 500 μm designed pores (~ 400 μm after sintering) sintered in microwave and conventional furnaces, respectively. An increase in cell density with a decrease in macropore size was observed during in vitro cell-material interactions using human osteoblast cells. Histomorphological analysis revealed that the presence of both micro- and macropores facilitated osteoid-like new bone formation when tested in femoral defects of Sprague-Dawley rats. Our results show that bioresorbable 3D-printed TCP scaffolds have great potential in tissue engineering applications for bone tissue repair and regeneration.

Mesenchymal stromal cells derived from umbilical cord blood migrate in response to complement C1q

Mesenchymal stromal cells (MSC) have great potential for tissue regeneration and cellular therapy. They migrate preferentially to sites of inflammation and tissue injury, but the molecular signals that guide them to their target tissue remain to be elucidated. We have shown that complement component 1 subcomponent q (C1q) enhances the homing-related response of hematopoietic stem/progenitor cells. In this study, we investigated whether C1q elicits directional signals that could influence the migration of MSC to injured tissues/organs. We found that C1q chemoattracted human umbilical cord blood-derived MSC in a dose-dependent manner and that the receptor for the global domains of Clq (gC1qR) is present on the surface of MSC. Specific gC1qR antibody blocked the chemotactic response of MSC to C1q, indicating that the C1q/gC1qR interaction may be responsible for the C1q-mediated migration of MSC. Further, we found that C1q enhanced/primed the migration of MSC across reconstituted basement membrane Matrigel towards a low gradient of the chemokine stromal cell-derived factor-1 (SDF-1), which is also present at sites of injury, partly as a result of an increase in surface expression of the SDF-1 receptor CXCR4. Moreover, C1q increased the secretion by MSC of matrix metalloproteinase (MMP)-2 and induced the phosphorylation of ERK1/2. These results indicate that C1q mediates the migration of MSC in two ways: first, by acting as a chemoattractant, and second, by priming chemotactic responses to SDF-1. Our findings suggest new molecular mechanisms of MSC migration that may contribute to their clinical application in tissue repair.

PEG-based bioresponsive hydrogels with redox-mediated formation and degradation

A hydrogel which will undergo macroscopic transition responding to redox stimuli is prepared. Mercapto precursors are prepared from 4-armed polyethylene glycol and after deprotection of thiolate anions, they can transform into disulfide crosslinked hydrogels within 3 min by responding to oxidant H(2)O(2). Desirable elasticity is exhibited with a wide range of storage modulus from 50 Pa to 14 kPa through rheological investigation. In addition, the hydrogels are found to be hydrolytically stable but degrade within 75 days when exposed to reductant such as glutathione (GSH). So gelation time and degradation behavior can be regulated by concentrations of precursor, oxidant, reductant, temperature, and pH value. Notably, interest arises from the long-period degradation under low GSH concentration of 0.01 mM that is similar to extracellular level, but not the fast disintegration under high concentration intracellular, providing the possibility of "smart" degradation responding to those cell-secreted biomacromolecules during the process of tissue regeneration. Furthermore, both hydrogels and their degradation products show cell viability above 90% culturing with C2C12 cells, representing nontoxic properties. Such a stimuli-responsive degradation strategy will give promising application in tissue repair and regeneration; especially enable the achievement of matching the degradation kinetics with physiological environment.

Synthesis and Characterization of Sol-Gel Derived Hydroxyapatite-Bioglass Composite Nanopowders for Biomedical Applications

The main purpose of this study is to prepare and characterize hydroxyapatite (HA)–10%wt bioglass (BG) composite nanopowders and its bioactivity. Composites of hydroxyapatite with synthesized bioglass are prepared at various temperatures. Suitable calcination temperature is chosen by evaluating of the phase composition. X-ray diffraction (XRD), Transmission electron microscopy (TEM) and Scanning electron microscopy (SEM) techniques are utilized to characterize the prepared nanopowders. The bioactivity of the prepared composite samples is evaluated in an in vitro study by immersion of samples in simulated body fluid (SBF) for predicted time. Fourier transformed infrared (FTIR) spectroscopy and inductively coupled plasma (ICP) are used for evaluation of apatite formation and the bioactivity properties. Results show that HA-BG composite nanopowders are successfully prepared without any decomposition of hydroxyapatite. The suitable temperature for calcination is 600°C and the particle size of hydroxyapatite is about 40-70 nm. The apatite phase forms after 14 days immersing of the samples in SBF. It could be concluded that this process can be used to synthesize HA-BG composite nanopowders with improved bioactivity which is much needed for hard tissue repair and biomedical applications.

Neural crest stem cells: discovery, properties and potential for therapy

Neural crest (NC) cells are a migratory cell population synonymous with vertebrate evolution. They generate a wide variety of cell and tissue types during embryonic and adult development including cartilage and bone, connective tissue, pigment and endocrine cells as well as neurons and glia amongst many others. Such incredible lineage potential combined with a limited capacity for self-renewal, which persists even into adult life, demonstrates that NC cells bear the key hallmarks of stem and progenitor cells. In this review, we describe the identification, characterization and isolation of NC stem and progenitor cells from different tissues in both embryo and adult organisms. We discuss their specific properties and their potential application in cell-based tissue and disease-specific repair.

Doubly crosslinked microgel–polyelectrolyte complexes: three simple methods to tune and improve gel mechanical properties

Doubly crosslinked microgels (DX MGs) are hydrogels composed of covalently interlinked microgels. They are injectable and have potential application in soft tissue repair. Simple methods for tuning their mechanical properties are required. Here, we investigate the effect of added polyelectrolyte (polycations or a polyanion) as well as NaCl on the mechanical properties of the gels for the first time. Addition of polycations improved ductility and decreased the storage modulus (G′) of the DX MG–polyelectrolyte complex (DX MG–PECs) gels. The best DX MG–PEC gel had a yield strain (γc) of 109%, which is the highest reported to date for any DX MG. Furthermore, our DX MG–PEC gels were robust and maintained high G′ values as well as good ductility when swollen at pH = 7.5. The DX MG–PEC gels with improved ductility contained less than 10% polycation. We also investigated the effect of addition of NaCl solution during DX MG and DX MG–PEC preparation. This caused remarkable increases in both G′ and ductility. The best gel had a G′ of 300 kPa which is the highest elasticity reported for a DX MG to date. Addition of linear polyacrylic acid during DX MG preparation also increased the G′ values. This study has provided three new simple methods for tuning the mechanical properties of DX MGs Because the MGs used here belong to a broad class of polymer colloids the results obtained in this study should be generally applicable.

Induction of Cell Migration <I>In Vitro</I> by an Electrospun PDGF-BB/PLGA/PEG-PLA Nanofibrous Scaffold

Electrospinning is a versatile technique used to fabricate potential tissue engineering scaffolds with a structure similar to the native extracellular matrix (ECM). In this study, Platelet-Derived Growth Factor (PDGF)-BB with BSA as a carrier protein was incorporated into an electrospun PLGA/PEG-PLA composite scaffold for induction of cell migration, an early process necessary for tissue regeneration and wound healing. Incorporating PDGF-BB into the fibers did not change the overall morphology of the scaffold, with the exception of a slight increase (approximately 12%) in the number of fibers with diameters ranging from 1-100 nm. Following a strong burst of release during the initial 24 hours, approximately 20% of the total incorporated PDGF-BB was released from the scaffold over 5 days, as determined by ELISA. The presence of the released PDGF-BB was also confirmed via SDS-PAGE. Using an in vitro agarose-cell migration assay with MC3T3 pre-osteoblastic cells, the preserved bioactivity of the released PDGF-BB was demonstrated via its ability to stimulate robust cell migration, equivalent to that of pure unincorporated (control) PDGF-BB. Overall, this study demonstrates that it is feasible to incorporate and deliver bioactive PDGF-BB via an electrospun scaffold for potential tissue repair applications, especially as a potent inducer of cell migration.