Analysis of Extracellular ATP Distribution in the Intervertebral Disc.

Progressive loss of proteoglycans (PGs) is the major biochemical change during intervertebral disc (IVD) degeneration. Adenosine triphosphate (ATP) as the primary energy source is not only critical for cell survival but also serves as a building block in PG synthesis. Extracellular ATP can mediate a variety of physiological functions and was shown to promote extracellular matrix (ECM) production in the IVD. Therefore, the objective of this study was to develop a 3D finite element model to predict extracellular ATP distribution in the IVD and evaluate the impact of degeneration on extracellular ATP distribution. A novel 3D finite element model of the IVD was developed by incorporating experimental measurements of ATP metabolism and ATP-PG binding kinetics into the mechano-electrochemical mixture theory. The new model was validated by experimental data of porcine IVD, and then used to analyze the extracellular distribution of ATP in human IVDs. Extracellular ATP was shown to bind specifically with PGs in IVD ECM. It was found that annulus fibrosus cells hydrolyze ATP faster than that of nucleus pulposus (NP) cells whereas NP cells exhibited a higher ATP release. The distribution of extracellular ATP in a porcine model was consistent with experimental data in our previous study. The predictions from a human IVD model showed a high accumulation of extracellular ATP in the NP region, whereas the extracellular ATP level was reduced with tissue degeneration. This study provides an understanding of extracellular ATP metabolism and its potential biological influences on the IVD via purinergic signaling.

Effect of doxycycline doped nanoparticles on osteogenic/cementogenic and anti-inflammatory responses of human cells derived from the periodontal ligament.

OBJECTIVES: This work aimed to evaluate if doxycycline-doped polymeric nanoparticles possessed any anti-inflammatory effect and promote osteogenic/cementogenic differentiation of stem cells from human periodontal ligament (PDLSCs). METHODS: The polymeric nanoparticles (NPs) were produced by a polymerization/precipitation process and doped with doxycycline (Dox-NPs). PDLSCs were cultured in the presence or absence of the NPs under osteogenic medium or IL-1ß treatment. Cells' differentiation was assessed by gene expression analysis of osteogenic/cementogenic markers alkaline phosphatase (ALP) and Runt-related transcription factor 2 (RUNX2). An anti-inflammatory effect was also ascertained by analyzing IL-1ß gene expression. Adipogenic and chondrogenic differentiation was used to confirm the multipotency of PDLSCs. RESULTS: Gene expression of ALP and RUNX2 in PDLSCs was significantly upregulated by the osteogenic medium (ALP: p<0.001; RUNX2: p = 0.005) while Dox-NPs further enhanced ALP gene expression of PDLSCs treated with the osteogenic medium. Furthermore, Dox-NPs suppressed the up-regulation of IL-1ß when cells were subjected to an inflammatory challenge. CONCLUSIONS: Dox-NPs enhanced PDLSCs differentiation into osteoblasts/cementoblasts lineages while providing an anti-inflammatory effect. CLINICAL SIGNIFICANCE: Due to their biocompatibility as well as anti-inflammatory and osteogenic/cementogenic effects, Dox-NPs are potential candidates for being used in periodontal regeneration.

Effects of Anti-Inflammatory Agents on Expression of Early Responsive Inflammatory and Catabolic Genes in Ex Vivo Porcine Model of Acute Knee Cartilage Injury.

Objective Early intervention therapies targeting inflammation and cell death during the acute phase of cartilage injury have the potential to prevent posttraumatic osteoarthritis. The objective of this study was to investigate the effects of interleukin receptor antagonist protein (IRAP), hyaluronan (HA), dexamethasone (DEX), and mesenchymal stem cell (MSC) treatment on the expression of established genetic markers for matrix degradation, apoptosis, and inflammation in articular cartilage during the acute phase of injury. Design A custom impact device was used to create replicable injury ex vivo to intact porcine knee joint. One hour after impact, IRAP, HA, DEX, or MSCs was intra-articularly injected. At 8 hours postinjury, cartilage and meniscus samples were harvested for genetic expression analysis. Expression of miR-27b, miR-140, miR-125b, miR-16, miR-34a, miR-146a, miR-22, ADAMTS-4, ADAMTS-5, MMP-3, IL-1ß, and TNF-α was analyzed by real-time polymerase chain reaction. Results At 8 hours postinjury, expression of ADAMTS-4, ADAMTS-5, MMP-3, IL-1ß, and TNF-α in cartilage was significantly decreased in IRAP- and DEX-treated joints as compared to nontreated injured joints, whereas only IRAP upregulated expression of miR-140, miR-125b, miR-27b, miR-146a, and miR-22 in cartilage. HA and MSC treatments had no significant effects on catabolic and inflammatory gene expression in cartilage. However, HA treatment significantly upregulated expression of all miRNAs except miR-16. In addition, the treatments tested also exhibited significant influences on meniscus. Conclusions This study provides a valuable starting point for further research into potential targets for and efficacy of various early intervention strategies that may delay or prevent the progression of posttraumatic osteoarthritis after acute cartilage injury.

Enhancement of Energy Production of the Intervertebral Disc by the Implantation of Polyurethane Mass Transfer Devices.

Insufficient nutrient supply has been suggested to be one of the etiologies for intervertebral disc (IVD) degeneration. We are investigating nutrient transport into the IVD as a potential treatment strategy for disc degeneration. Most cellular activities in the IVD (e.g., cell proliferation and extracellular matrix production) are mainly driven by adenosine-5'-triphosphate (ATP) which is the main energy currency. The objective of this study was to investigate the effect of increased mass transfer on ATP production in the IVD by the implantation of polyurethane (PU) mass transfer devices. In this study, the porcine functional spine units were used and divided into intact, device and surgical groups. For the device and surgical groups, two puncture holes were created bilaterally at the dorsal side of the annulus fibrosus (AF) region and the PU mass transfer devices were only implanted into the holes in the device group. Surgical groups were observed for the effects of placing the holes through the AF only. After 7 days of culture, the surgical group exhibited a significant reduction in the compressive stiffness and disc height compared to the intact and device groups, whereas no significant differences were found in compressive stiffness, disc height and cell viability between the intact and device groups. ATP, lactate and the proteoglycan contents in the device group were significantly higher than the intact group. These results indicated that the implantation of the PU mass transfer device can promote the nutrient transport and enhance energy production without compromising mechanical and cellular functions in the disc. These results also suggested that compromise to the AF has a negative impact on the IVD and must be addressed when treatment strategies are considered. The results of this study will help guide the development of potential strategies for disc degeneration.

Systematic characterization of porosity and mass transport and mechanical properties of porous polyurethane scaffolds.

One of the key challenges in porous scaffold design is to create a porous structure with desired mechanical function and mass transport properties which support delivery of biofactors and development of function tissue substitute. In recent years, polyurethane (PU) has become one of the most popular biomaterials in various tissue engineering fields. However, there are no studies fully investigating the relations between porosity and both mass transport and mechanical properties of PU porous scaffolds. In this paper, we fabricated PU scaffolds by combining phase inversion and salt (sodium chloride) leaching methods. The tensile and compressive moduli were examined on PU scaffolds fabricated with different PU concentrations (25%, 20% and 15% w/v) and salt/PU weight ratios (9/1, 6/1, 3/1 and 0/1). The mass transport properties of PU scaffolds including hydraulic permeability and glucose diffusivity were also measured. Furthermore, the relationships between the porosity and mass transport and mechanical properties of porous PU scaffold were systemically investigated. The results demonstrated that porosity is a key parameter which governs both mass transport and mechanical properties of porous PU scaffolds. With similar pore sizes, the mass transport and mechanical properties of porous PU scaffold can be described as single functions of porosity regardless of initial PU concentration. The relationships between scaffold porosity and properties can be utilized to facilitate porous PU scaffold fabrication with specific mass transport and mechanical properties. The systematic approach established in this study can be applied to characterization of other biomaterials for scaffold design and fabrication.

Regional Differential Genetic Response of Human Articular Cartilage to Impact Injury.

OBJECTIVE: Normal physiological movement creates different weightbearing zones within a human knee: the medial condyle bearing the highest and the trochlea bearing the lowest weight. Adaptation to different physiological loading conditions results in different tissue and cellular properties within a knee. The objective of this study was to use microarray analysis to examine gene expression differences among three anatomical regions of human knee articular cartilage at baseline and following induction of an acute impact injury. DESIGN: Cartilage explants were harvested from 7 cadaveric knees (12 plugs per knee). A drop tower was utilized to introduce injury. Plugs were examined 24 hours after impact for gene expression using microarray. The primary analysis is the comparison of baseline versus impacted samples within each region separately. In addition, pairwise comparisons among the three regions were performed at baseline and after impact. False discovery rate (FDR) was used to evaluate significance of differential gene expression. RESULTS: In the comparison of before and after injury, the trochlear had 130 differentially expressed genes (FDR ≤ 0.05) while the condyles had none. In the comparison among regions, smaller sets of differentially expressed genes (n ≤ 21) were found, with trochlea being more different than the condyles. Most of more frequently expressed genes in trochlea are developmental genes. CONCLUSIONS: Within the experimental setup of this study, only the trochlea was displaying an acute genetic response on injury. Our data demonstrated the regional-specific response to injury in human articular cartilage.

Neural crest-derived dental stem cells--where we are and where we are going.

OBJECTIVES: There are five types of post-natal human dental stem cells that have been identified, isolated and characterized. Here, we review the information available on dental stem cells as well as their potential applications in dentistry, regenerative medicine and the development of other therapeutic approaches. DATA: Data pertinent to dental stem cells and their applications, published in peer-reviewed journals from 1982 to 2013 in English were reviewed. SOURCES: Sources were retrieved from PubMed databases as well as related references that the electronic search yielded. STUDY SELECTION: Manuscripts describing the origin, retrieval, characterization and application of dental stem cells were obtained and reviewed. CONCLUSIONS: Dental stem cell populations present properties similar to those of mesenchymal stem cells, such as the ability to self-renew and the potential for multilineage differentiation. While they have greater capacity to give rise to odontogenic cells and regenerate dental pulp and periodontal tissue, they have the capacity to differentiate into all three germ line cells, proving that a population of pluripotent stem cells exists in the dental tissues. CLINICAL SIGNIFICANCE: Dental stem cells have the capacity to differentiate into endoderm, mesoderm and ectoderm tissues. Consequently they do not only have applications in dentistry, but also neurodegenerative and ischemic diseases, diabetes research, bone repair, and other applications in the field of tissue regeneration.

Subphysiological compressive loading reduces apoptosis following acute impact injury in a porcine cartilage model.

BACKGROUND: Acute cartilage injuries induce cell death and are associated with an increased incidence of osteoarthritis development later in life. The objective of this study was to investigate the effect of posttraumatic cyclic compressive loading on chondrocyte viability and apoptosis in porcine articular cartilage plugs. HYPOTHESIS: Compressive loading of acutely injured cartilage can maintain chondrocyte viability by reducing apoptosis after a traumatic impact injury. STUDY DESIGN: In vitro controlled laboratory study. LEVEL OF EVIDENCE: Level 5. METHODS: Each experiment compared 4 test groups: control, impact, impact with compressive loading (either 0.5 or 0.8 MPa), and no impact but compressive loading (n = 15 per group). Flat, full-thickness articular cartilage plugs were harvested from the trochlear region of porcine knees. A drop tower was utilized to introduce an impact injury. The articular plugs were subjected to two 30-minute cycles of either 0.5 or 0.8 MPa of dynamic loading. Cell viability, apoptosis, and gene expression of samples were evaluated 24 hours postimpaction. RESULTS: Cell viability staining showed that 0.5 MPa of dynamic compressive loading increased cell viability compared with the impact group. Apoptotic analysis revealed a decrease in apoptotic expression in the group with 0.5 MPa of dynamic compressive loading compared with the impact group. Significantly higher caspase 3 and lower collagen II expressions were observed in impacted samples without compressive loading, compared with those with. Compressive loading of nonimpacted samples significantly increased collagen II and decreased caspase 3 expressions. CONCLUSION: In this porcine in vitro model, dynamic compressive loading at subphysiological levels immediately following impact injury decreases apoptotic expression, thereby maintaining chondrocyte viability. CLINICAL RELEVANCE: Therapeutic exercises could be designed to deliver subphysiological loading to the injured cartilage, thereby minimizing injury.

Plasticity of stem cells derived from adult periodontal ligament.

BACKGROUND: The neural crest contains pluripotent cells that can give rise to neurons and glial cells of the peripheral nervous system, endocrine cells, connective tissue cells, muscle cells and pigment cells during embryonic development. Stem cells derived from the neural crest may still reside in neural crest derivatives including the periodontal ligament (PDL). However, the pluripotency of PDL-derived stem cells has not been investigated. AIM: To identify subpopulations of stem cells from the adult PDL and study their pluripotency. Human PDLs were harvested from impacted wisdom teeth (patients aged 19-22 years). RESULTS: This study demonstrated that subpopulations of PDL cells expressed embryonic stem cell markers (Oct4, Sox2, Nanog and Klf4) and a subset of neural crest markers (Nestin, Slug, p75 and Sox10). Such PDL cell subpopulations exhibited the potential to differentiate into neurogenic, cardiomyogenic, chondrogenic and osteogenic lineages. Furthermore, preliminary evidence suggesting insulin production of PDL cells might be indicative of the generation of cells of the endodermal lineage. CONCLUSION: These findings suggest that the PDL may contain pluripotent stem cells that originate from the neural crest. Our observations open the door to prospective autologous therapeutic applications for a variety of conditions.

Temporal expression patterns and corresponding protein inductions of early responsive genes in rabbit bone marrow-derived mesenchymal stem cells under cyclic compressive loading.

Our recent study suggested that cyclic compressive loading may promote chondrogenesis of rabbit bone-marrow mesenchymal stem cells (BM-MSCs) in agarose cultures through the transforming growth factor (TGF)-beta signaling pathway. It has been shown that the activating protein 1 (AP-1) (Jun-Fos) complex mediated autoinduction of TGF-beta1 and its binding activity was essential for promoting chondrogenesis of mesenchymal cells, whereas Sox9 was identified as an essential transcription factor for chondrogenesis of embryonic mesenchymal cells. The objective of this study was to examine temporal expression patterns of early responsive genes (Sox9, c-Fos, c-Jun, and TGF-beta type I and II receptors) and induction of their corresponding proteins in agarose culture of rabbit BM-MSCs subjected to cyclic compressive loading. The rabbit BM-MSCs were obtained from the tibias and femurs of New Zealand White rabbits. Cell-agarose constructs were made by suspending BM-MSCs in 2% agarose gel (10(7) cells/ml) for cyclic, unconfined compression tests performed in a custom-made bioreactor. In the loading experiment, specimens were subjected to sinusoidal loading with a magnitude of 15% strain at a frequency of 1 hertz for 4 hours per day. Experiments were conducted for 2 consecutive days. This study showed that cyclic compressive loading promoted gene expressions of Sox9, c-Jun, and both TGF-beta receptors and productions of their corresponding proteins, whereas those gene expressions exhibited different temporal expression patterns among genes and between 2 days of testing. The gene expression of c-Fos was detected only in the samples subjected to1-hour dynamic compressive loading. These findings suggest that the TGF-beta signal transduction and activities of AP-1 and Sox9 are involved in the early stage of BM-MSC chondrogenesis promoted by dynamic compressive loading.

Effects of cyclic compressive loading on chondrogenesis of rabbit bone-marrow derived mesenchymal stem cells.

The objective of this study was to examine the effects of cyclic compressive loading on chondrogenic differentiation of rabbit bone-marrow mesenchymal stem cells (BM-MSCs) in agarose cultures. Rabbit BM-MSCs were obtained from the tibias and femurs of New Zealand white rabbits. After the chondrogenic potential of BM-MSCs was verified by pellet cultures, cell-agarose constructs were made by suspending BM-MSCs in 2% agarose (10(7) cells/ml) for a cyclic, unconfined compression test performed in a custom-made bioreactor. Specimens were divided into four groups: control; transforming growth factor (TGF-beta) (with TGF-beta1 treatment); loading (with stimulation of cyclic, unconfined compressive loading); and TGF-beta loading (with TGF-beta1 treatment and loading stimulation) groups. In the loading experiment, specimens were subjected to sinusoidal loading with a 10% strain magnitude at a frequency of 1 Hz for 4 hours a day. Experiments were conducted for 3, 7, and 14 consecutive days. While the experimental groups (TGF-beta, loading, and TGF-beta loading) exhibited significantly higher levels of expressions of chondrogenic markers (collagen II and aggrecan) at three time periods, there were no differences among the experimental groups after an extra 5-day culture. This suggests that compressive loading alone induces chondrogenic differentiation of rabbit BM-MSCs as effectively as TGF-beta or TGF-beta plus loading treatment. Moreover, both the compressive loading and the TGF-beta1 treatment were found to promote the TGF-beta1 gene expression of rabbit BM-MSCs. These findings suggest that cyclic compressive loading can promote the chondrogenesis of rabbit BM-MSCs by inducing the synthesis of TGF-beta1, which can stimulate the BM-MSCs to differentiate into chondrocytes.

Chondrogenesis of human bone marrow-derived mesenchymal stem cells in agarose culture.

Mesenchymal stem cells derived from human bone marrow (hBM-MSCs) can differentiate into chondrogenic cells for the potential treatment of injured articular cartilage. To evaluate agarose gels as a supportive material for chondrogenesis of hBM-MSCs, this study examined chondrogenesis of hBM-MSCs in the agarose cultures. Pellet cultures were employed to confirm the chondrogenic potential of the hBM-MSCs that were used in agarose cultures. The hBM-MSCs were seeded in 2% agarose constructs at the initial cell-seeding densities of 3, 6, and 9 x 10(6) cells/ml while each of pellets was formed using 2.5 x 10(5) cells. Chondrogenesis of hBM-MSCs was induced by culturing cell-agarose constructs and pellets for 21 days in the presence of a defined medium containing transforming growth factor beta3 (TGF-beta3). The analysis of reverse transcription-polymerase chain reaction showed that hBM-MSCs of agarose and pellet cultures expressed the chondrogenic markers of collagen type II and aggrecan in the presence of TGF-beta3. The deposition of cartilage-specific macromolecules was detected in both agarose and pellet cultures by histological and immunohistochemical assessments. Chondrogenesis of hBM-MSCs in agarose gels directly correlated with the initial cell-seeding density, with the cell-agarose constructs of higher initial cell-seeding density exhibiting more cartilage-specific gene expressions. This study establishes a basic model for future studies on chondrogenesis of hBM-MSCs using the agarose cultures.