Characterization and Therapeutic Uses of Exosomes: A New Potential Tool in Orthopedics.

In recent years, regenerative medicine has directed its interests onto the use of stem cells to heal human tissues. One specific class of cells that has been used in this field of research is mesenchymal stem cells (MSCs). Because of difficulties with the usage of whole stem cells, researchers have turned to an alternative, the secretome of the MSCs. In recent years, research has explored numerous aspects of the MSC secretome, especially the most promising aspect, exosomes. This review explores a variety of interests in exosomes including the classification and molecular composition of exosomes, mechanisms for exosome isolation, and the various biological functions of exosomes. As more is discovered about the exosomes, their different diagnostic and therapeutic uses in the medical field have also been explored. A new field attempting to exploit the exosomes in clinical practice is orthopedics. Although a significant deal of research has been carried out, even more is being discovered to allow utilization of the exosomes in clinical practice.

ATP promotes extracellular matrix biosynthesis of intervertebral disc cells.

We have recently found a high accumulation of extracellular adenosine triphosphate (ATP) in the center of healthy porcine intervertebral discs (IVD). Since ATP is a powerful extracellular signaling molecule, extracellular ATP accumulation might regulate biological activities in the IVD. The objective of this study was therefore to investigate the effects of extracellular ATP on the extracellular matrix (ECM) biosynthesis of porcine IVD cells isolated from two distinct anatomical regions: the annulus fibrosus (AF) and nucleus pulposus (NP). ATP treatment significantly promotes ECM deposition and corresponding gene expression (aggrecan and type II collagen) by both cell types in three-dimensional agarose culture. A significant increase in ECM accumulation has been found in AF cells at a lower ATP treatment level (20 µM) compared with NP cells (100 µM), indicating that AF cells are more sensitive to extracellular ATP than NP cells. NP cells also exhibit higher ECM accumulation and intracellular ATP than AF cells under control and treatment conditions, suggesting that NP cells are intrinsically more metabolically active. Moreover, ATP treatment also augments the intracellular ATP level in NP and AF cells. Our findings suggest that extracellular ATP not only promotes ECM biosynthesis via a molecular pathway, but also increases energy supply to fuel that process.

Measurement of ATP-Induced Membrane Potential Changes in IVD cells.

Extracellular adenosine-5'-triphosphate (ATP) triggers biological responses in a wide variety of cells and tissues and activates signaling cascades that affect cell membrane potential and excitability. It has been demonstrated that compressive loading promotes ATP production and release by intervertebral disc (IVD) cells, while a high level of extracellular ATP accumulates in the nucleus pulposus (NP) of the IVD. In this study, a noninvasive system was developed to measure ATP-induced changes in the membrane potential of porcine IVD cells using the potential sensitive dye di-8-butyl-amino-naphthyl-ethylene-pyridinium-propyl-sulfonate (di-8-ANEPPS).The responses of NP and annulus fibrosus (AF) cells to ATP were examined in monolayer and 3-dimensional cultures. It was found that the pattern and magnitude of membrane potential change in IVD cells induced by extracellular ATP depended on cell type, culture condition, and ATP dose. In addition, gene expression of P2X4 purinergic receptor was found in both cell types. Inhibition of the ATP-induced response by pyridoxalphosphate-6-azophenyl-2', 4'-disulfonate (PPADS), a non-competitive inhibitor of P2 receptors, suggests that ATP may modulate the biological activities of IVD cells via P2 purinergic receptors.

Energy metabolism of intervertebral disc under mechanical loading.

Intervertebral disc (IVD) degeneration is closely associated with low back pain (LBP), which is a major health concern in the U.S. Cellular biosynthesis of extracellular matrix (ECM), which is important for maintaining tissue integrity and preventing tissue degeneration, is an energy demanding process. Due to impaired nutrient support in avascular IVD, adenosine triphosphate (ATP) supply could be a limiting factor for maintaining normal ECM synthesis. Therefore, the objective of this study was to investigate the energy metabolism in the annulus fibrosus (AF) and nucleus pulposus (NP) of porcine IVD under static and dynamic compressions. Under compression, pH decreased and the contents of lactate and ATP increased significantly in both AF and NP regions, suggesting that compression can promote ATP production via glycolysis and reduce pH by increasing lactate accumulation. A high level of extracellular ATP content was detected in the NP region and regulated by compressive loading. Since ATP can serve not only as an intra-cellular energy currency, but also as a regulator of a variety of cellular activities extracellularly through the purinergic signaling pathway, our findings suggest that compression-mediated ATP metabolism could be a novel mechanobiological pathway for regulating IVD metabolism.

Isolation of pluripotent neural crest-derived stem cells from adult human tissues by connexin-43 enrichment.

Identification and isolation of pluripotent stem cells in adult tissues represent an important advancement in the fields of stem cell biology and regenerative medicine. For several years, research has been performed on the identification of biomarkers that can isolate stem cells residing in neural crest (NC)-derived adult tissues. The NC is considered a good model in stem cell biology as cells from it migrate extensively and contribute to the formation of diverse tissues in the body during organogenesis. Migration of these cells is modulated, in part, by gap junction communication among the cell sheets. Here we present a study in which, selection of connexin 43 (Cx43) expressing cells from human adult periodontal ligament yields a novel pluripotent stem cell population. Cx43⁺ periodontal ligament stem cells express pluripotency-associated transcription factors OCT4, Nanog, and Sox2, as well as NC-specific markers Sox10, p75, and Nestin. When injected in vivo into an immunodeficient mouse model, these cells were capable of generating teratomas with tissues from the three embryological germ layers: endoderm, mesoderm, and ectoderm. Furthermore, the cells formed mature structures of tissues normally arising from the NC during embryogenesis such as eccrine sweat glands of the human skin, muscle, neuronal tissues, cartilage, and bone. Immunohistochemical analysis confirmed the human origin of the neoplastic cells as well as the ectodermal and endodermal nature of some of the structures found in the tumors. These results suggest that Cx43 may be used as a biomarker to select and isolate the remnant NC pluripotent stem cells from adult human tissues arising from this embryological structure. The isolation of these cells through routine medical procedures such as wisdom teeth extraction further enhances their applicability to the regenerative medicine field.

Mechanical loading affects the energy metabolism of intervertebral disc cells.

Research has shown that mechanical loading affects matrix biosynthesis of intervertebral disc (IVD) cells; however, the pathway(s) to this effect is currently unknown. Cellular matrix biosynthesis is an energy demanding process. The objective of this study was to investigate the effects of static and dynamic compressive loading on energy metabolism of IVD cells. Porcine annulus fibrosus (AF) and nucleus pulposus (NP) cells seeded in 2% agarose were used in this experiment. Experimental groups included 15% static compression and 0.1 and 1 Hz dynamic compression at 15% strain magnitude for 4 h. ATP, lactate, glucose, and nitric oxide (NO) contents in culture media, and ATP content in cell-agarose construct were measured using biochemical assays. While the total ATP content of AF cells was promoted by static and dynamic loading, only 1 Hz dynamic loading increased total ATP content of NP cells. Increases in lactate production and glucose consumption of AF cells suggest that ATP production via glycolysis is promoted by dynamic compression. ATP release and NO production of AF and NP cells were significantly increased by dynamic loading. Thus, this study clearly illustrates that static and dynamic compressive loading affect IVD cell energy production while cellular responses to mechanical loading were both cell type and compression type dependent.

Sox-9 facilitates differentiation of adipose tissue-derived stem cells into a chondrocyte-like phenotype in vitro.

The purpose of this study is to test whether ectopic expression of Sox-9 can induce adipose tissue-derived stem cells (ASCs) to function as real nucleus pulposus (NP) cells in vitro. Adenoviral vectors expressing Sox-9 were reported to infect the chondroblastic and human disc cells, which resulted in increased Sox-9 and type II collagen production. ASCs were isolated from rat inguinal adipose pad, characterized, and transduced in vitro with a retroviral vector encoding the Sox-9 gene. Sox-9-engineered ASCs (ASCs/Sox-9) were induced for the chondrocyte-like cell differentiation by 3D cultured in alginate beads and TGF-ß3 for 2 weeks. Expression of exogenous Sox-9 protein was detected. Type II collagen and Aggrecan gene expressions of induced ASCs/Sox-9 were measured using real-time PCR; proteoglycans expressions were measured by checking the glycosaminoglycan content and type II collagen production by enzyme-linked immunosorbent assay. Isolated ASCs were CD 29(+) /CD44(+) /C-Kit(-) /Lin(-) /CD34(-) /CD45(-) . ASCs/Sox-9 expressed marked increase in exogenous Sox-9 protein. After induction, type II collagen gene expression was sevenfold higher in mRNA levels, with an approximately twofold increase in protein levels of ASCs/Sox-9 compared to ASCs. Type II collagen and proteoglycan productions were significantly increased in the ASCs/Sox-9 compared to the ASCs. In addition, co-culture of induced ASCs/Sox-9 with matured NP cells resulted in enhanced increase in proteoglycan and type II collagen production. Constitutive retroviral expression of Sox-9 could efficiently induce ASCs differentiation into chondrocyte-like cells. This novel approach may provide a practicable system for a simple and rapid differentiation of ASCs into chondrocyte-like cells which may be potentially used as a stem cell-based therapeutic tool for the treatment of degenerative disc diseases.

Osteogenic differentiation of stem cells derived from human periodontal ligaments and pulp of human exfoliated deciduous teeth.

Multipotent stem cells derived from periodontal ligaments (PDLSC) and pulp of human exfoliated deciduous teeth (SHED) represent promising cell sources for bone regeneration. Recent studies have demonstrated that retinoic acid (RA) and dexamethasone (Dex) induce osteogenesis of postnatal stem cells. The objective of this study was to examine the effects of RA and Dex on the proliferation and osteogenic differentiation of SHED and PDLSC and to compare the osteogenic characteristics of SHED and PDLSC under RA treatment. SHED and PDLSC were treated with serum-free medium either alone or supplemented with RA or Dex for 21 days. The proliferation of SHED and PDLSC was significantly inhibited by both RA and Dex. RA significantly upregulated gene expression and the activity of alkaline phosphatase in SHED and PDLSC. Positive Alizarin red and von Kossa staining of calcium deposition was seen on the RA-treated SHED and PDLSC after 21 days of culture. The influences of RA on the osteogenic differentiation of SHED and PDLSC were significantly stronger than with Dex. Supplementation with insulin enhanced RA-induced osteogenic differentiation of SHED. Thus, RA is an effective inducer of osteogenic differentiation of SHED and PDLSC, whereas RA treatment in combination with insulin supplementation might be a better option for inducing osteogenic differentiation. Significantly higher cell proliferation of PDLSC results in greater calcium deposition after 3-week culture, suggesting that PDLSC is a better osteogenic stem cell source. This study provides valuable information for efficiently producing osteogenically differentiated SHED or PDLSC for in vivo bone regeneration.

Cyclic compression maintains viability and induces chondrogenesis of human mesenchymal stem cells in fibrin gel scaffolds.

Mechanical loading has long been shown to modulate cartilage-specific extracellular matrix synthesis. With joint motion, cartilage can experience mechanical loading in the form of compressive, tensile or shearing load, and hydrostatic pressure. Recent studies have demonstrated the capacity of unconfined cyclic compression to induce chondrogenic differentiation of human mesenchymal stem cell (hMSC) in agarose culture. However, the use of a nonbiodegradable material such as agarose limits the applicability of these constructs. Of the possible biocompatible materials available for tissue engineering, fibrin is a natural regenerative scaffold, which possesses several desired characteristics including a controllable degradation rate and low immunogenicity. The objective of the present study was to determine the capability of fibrin gels for supporting chondrogenesis of hMSCs under cyclic compression. To optimize the system, three concentrations of fibrin gel (40, 60, and 80 mg/mL) and three different stimulus frequencies (0.1, 0.5, and 1.0 Hz) were used to examine the effects of cyclic compression on viability, proliferation and chondrogenic differentiation of hMSCs. Our results show that cyclic compression (10% strain) at frequencies >0.5 Hz and gel concentration of 40 mg/mL fibrinogen appears to maintain cellular viability within scaffolds. Similarly, variations in gel component concentration and stimulus frequency can be modified such that a significant chondrogenic response can be achieved by hMSC in fibrin constructs after 8 h of compression spread out over 2 days. This study demonstrates the suitability of fibrin gel for supporting the cyclic compression-induced chondrogenesis of mesenchymal stem cells.