Fan He

Synovium‐derived stem cell‐based chondrogenesis

Synovium is considered a candidate source of cells for cartilage tissue engineering. Compared with mesenchymal stem cells (MSCs) from other sources, synovium-derived stem cells (SDSCs) have a higher capacity for chondrogenic differentiation. Our objective was to define cocktails of growth factors that support the growth and chondrogenic differentiation of SDSCs in chemically defined medium. We established a fast and highly selective technique of negative isolation of SDSC populations. The individual and combined effects of three growth factors-transforming growth factor-beta1 (TGF-beta1), insulin-like growth factor I (IGF-I), and basic fibroblast growth factor (FGF-2)-were evaluated in serum-free pellet cultures of SDSCs for the chondrogenesis of SDSCs using histology, biochemical analysis, and real-time RT-PCR. In vitro studies identified TGF-beta1 as the key factor for both the growth and chondrogenesis of SDSCs. The highest rates of SDSC growth were observed with the synergistic interaction of all three factors. With respect to chondrogenic differentiation of SDSCs, the interaction of TGF-beta1 and IGF-I applied simultaneously was superior to the sequential application of these two factors or any other combination of growth factors studied. Based on these findings, we propose a two-step protocol for the derivation of chondrogenic SDSCs: a cocktail of TGF-beta1, IGF-I, and FGF-2 is applied first to induce cell growth followed by a cocktail of TGF-beta1 and IGF-I applied to induce chondrogenesis.

Repair of full-thickness femoral condyle cartilage defects using allogeneic synovial cell-engineered tissue constructs.

OBJECTIVE Synovium-derived stem cells (SDSCs) have proven to be superior in cartilage regeneration compared with other sources of mesenchymal stem cells. We hypothesized that conventionally passaged SDSCs can be engineered in vitro into cartilage tissue constructs and the engineered premature tissue can be implanted to repair allogeneic full-thickness femoral condyle cartilage defects without immune rejection. METHODS Synovial tissue was harvested from rabbit knee joints. Passage 3 SDSCs were mixed with fibrin glue and seeded into non-woven polyglycolic acid (PGA) mesh. After 1-month incubation with growth factor cocktails, the premature tissue was implanted into rabbit knees to repair osteochondral defects with Collagraft as a bone substitute in the Construct group. Fibrin glue-saturated PGA/Collagraft composites were used as a Scaffold group. The defect was left untreated as an Empty group. RESULTS SDSCs were engineered in rotating bioreactor systems into premature cartilage, which displayed the expression of sulfated glycosaminoglycan (GAG), collagen II, collagen I, and macrophages. Six months after implantation with premature tissue, cartilage defects were full of smooth hyaline-like cartilage with no detectable collagen I and macrophages but a high expression of collagen II and GAG, which were also integrated with the surrounding native cartilage. The Scaffold and Empty groups were resurfaced with fibrous-like and fibrocartilage tissue, respectively. CONCLUSION Allogeneic SDSC-based premature tissue constructs are a promising stem cell-based approach for cartilage defects. Although in vitro data suggest that contaminated macrophages affected the quality of SDSC-based premature cartilage, effects of macrophages on in vivo tissue regeneration and integration necessitate further investigation.

Melatonin reverses H2O2‐induced premature senescence in mesenchymal stem cells via the SIRT1‐dependent pathway

Mesenchymal stem cells (MSCs) represent an attractive source for stem cell‐based regenerative therapy, but they are vulnerable to oxidative stress‐induced premature senescence in pathological conditions. We previously reported antioxidant and antiarthritic effects of melatonin on MSCs against proinflammatory cytokines. In this study, we hypothesized that melatonin could protect MSCs from premature senescence induced by hydrogen peroxide (H2O2) via the silent information regulator type 1 (SIRT1)‐dependent pathway. In response to H2O2 at a sublethal concentration of 200 μm, human bone marrow‐derived MSCs (BM‐MSCs) underwent growth arrest and cellular senescence. Treatment with melatonin before H2O2 exposure cannot significantly prevent premature senescence; however, treatment with melatonin subsequent to H2O2 exposure successfully reversed the senescent phenotypes of BM‐MSCs in a dose‐dependent manner. This result was made evident by improved cell proliferation, decreased senescence‐associated β‐galactosidase activity, and the improved entry of proliferating cells into the S phase. In addition, treatment with 100 μm melatonin restored the osteogenic differentiation potential of BM‐MSCs that was inhibited by H2O2‐induced premature senescence. We also found that melatonin attenuated the H2O2‐stimulated phosphorylation of p38 mitogen‐activated protein kinase, decreased expression of the senescence‐associated protein p16INK4α, and increased SIRT1. Further molecular experiments revealed that luzindole, a nonselective antagonist of melatonin receptors, blocked melatonin‐mediated antisenescence effects. Inhibition of SIRT1 by sirtinol counteracted the protective effects of melatonin, suggesting that melatonin reversed the senescence in cells through the SIRT1‐dependent pathway. Together, these findings lay new ground for understanding oxidative stress‐induced premature senescence and open perspectives for therapeutic applications of melatonin in stem cell‐based regenerative medicine.

Extracellular matrix enhances differentiation of adipose stem cells from infrapatellar fat pad toward chondrogenesis

The objective was to improve proliferation and chondrogenic potential of adipose stem cells (ASCs) by expansion on extracellular matrix (ECM) deposited by either ASCs or synovium‐derived stem cells (SDSCs). ASCs isolated from porcine infrapatellar fat pad were separately expanded on conventional plastic flasks, ASC‐deposited ECM and SDSC‐deposited ECM. ASCs were centrifuged to form pellets and cultured in a serum‐free chondrogenic medium with either TGFβ3 or TGFβ3 combined with BMP‐6. Cell number yielded on ECM expansion did not show a significant difference in deposition between ASCs and SDSCs but was 6–10 times that grown on non‐coated flasks. ECM‐expanded ASCs exhibited a lower level of intracellular reactive oxygen species (ROS) compared to those grown on non‐coated flasks. Typical chondrogenic markers, including type II collagen and glycosaminoglycans (GAGs), were intensively distributed in the pellets from ECM‐expanded ASCs instead of those from flask‐grown cells. ASCs expanded on ECM, either from ASCs or SDSCs, exhibited a similar chondrogenic index (GAG:DNA), which was significantly higher than that from ASCs grown on non‐coated flasks. The combination of TGFβ3 and BMP‐6 increased 36% more in ASC chondrogenic index than the treatment with TGFβ3 alone. Interestingly, ECM pretreatment also decreased expanded ASC hypertrophic marker genes. ECM deposited by either ASCs or SDSCs did not exhibit enhanced adipogenic differentiation of ASCs. Our study indicates that the sequential application of ECM for cell expansion and combined TGFβ3 with BMP‐6 for chondrogenic differentiation may be a promising approach for ASC‐based cartilage tissue engineering and regeneration. Copyright

Rejuvenation of Nucleus Pulposus Cells Using Extracellular Matrix Deposited by Synovium-Derived Stem Cells

Study Design. After plating for 6 passages on either plastic flasks or extracellular matrix (ECM) deposited by synovium-derived stem cells (SDSCs), expanded nucleus pulposus (NP) cells were evaluated for redifferentiation capacity. Objective. The aim was to assess the feasibility of using ECM deposited by a tissue-specific stem cell to provide a 3-dimensional microenvironment for NP cell rejuvenation. Summary of Background Data. Autologous disc cell-based therapy is a promising approach for intervertebral disc regeneration. Unfortunately, the current in vitro expansion of NP cells in monolayer results in dedifferentiation of these cells. Methods. Primary NP cells were plated on either plastic flasks or ECM for 6 consecutive passages. At each passage, cell numbers were counted for proliferation rate, cell phenotype was evaluated using flow cytometry, and cell differentiation status was assessed using real-time polymerase chain reaction (PCR). The pellets from expanded NP cells at passages 1, 4, and 6 were incubated in a serum-free defined medium for 14 days. Redifferentiation capacity of the expanded NP cells was evaluated using histology, biochemistry, and real-time PCR. Results. NP cells expanded on ECM grew much faster with a smaller size and fibroblast-like shape compared with those on plastic flasks. ECM-treated NP cells acquired an enhanced CD90 expression and higher mRNA levels of types I, II, and X collagen and aggrecan, as well as a robust redifferentiation capacity, evidenced by dramatically increased type II collagen, aggrecan, and Sox9 and decreased type I collagen for up to 6 passages. Conclusion. SDSC-derived ECM can provide a tissue-specific microenvironment for the rejuvenation of NP cells with a higher proliferation rate and redifferentiation capacity. These characteristics may play a role in improving an autologous disc cell–based minimally invasive therapeutic approach toward physiological reconstruction of a biologically functional disc in the clinical setting.

Extracellular matrix modulates the biological effects of melatonin in mesenchymal stem cells

Both self-renewal and lineage-specific differentiation of mesenchymal stem cells (MSCs) are triggered by their in vivo microenvironment including the extracellular matrix (ECM) and secreted hormones. The ECM may modulate the physiological functions of hormones by providing binding sites and by regulating downstream signaling pathways. Thus, the purpose of this study was to evaluate the degree of adsorption of melatonin to a natural cell-deposited ECM and the effects of this interaction on the biological functions of melatonin in human bone marrow-derived MSCs (BM-MSCs). The fibrillar microstructure, matrix composition, and melatonin-binding affinity of decellularized ECM were characterized. The cell-deposited ECM improved melatonin-mediated cell proliferation by 31.4%, attenuated accumulation of intracellular reactive oxygen species accumulation, and increased superoxide dismutase (SOD) mRNA and protein expression. Interaction with ECM significantly enhanced the osteogenic effects of melatonin on BM-MSCs by increasing calcium deposition by 30.5%, up-regulating osteoblast-specific gene expression and down-regulating matrix metalloproteinase (MMP) expression. The underlying mechanisms of these changes in expression may involve intracellular antioxidant enzymes, because osteoblast-specific genes were down-regulated, whereas MMP expression was up-regulated, in the presence of SOD-specific inhibitors. Collectively, our findings indicate the importance of native ECM in modulating the osteoinductive and antioxidant effects of melatonin and provide a novel platform for studying the biological actions of growth factors or hormones in a physiologically relevant microenvironment. Moreover, a better understanding of the enhancement of MSC growth and osteogenic differentiation resulting from the combination of ECM and melatonin could improve the design of graft substitutes for skeletal tissue engineering.

Alcohol Induces Cellular Senescence and Impairs Osteogenic Potential in Bone Marrow-Derived Mesenchymal Stem Cells

Aims Chronic and excessive alcohol consumption is a high-risk factor for osteoporosis. Bone marrow-derived mesenchymal stem cells (BM-MSCs) play an important role in bone formation; however, they are vulnerable to ethanol (EtOH). The purpose of this research was to investigate whether EtOH could induce premature senescence in BM-MSCs and subsequently impair their osteogenic potential. Methods Human BM-MSCs were exposed to EtOH ranging from 10 to 250 mM. Senescence-associated β-galactosidase (SA-β-gal) activity, cell cycle distribution, cell proliferation and reactive oxygen species (ROS) were evaluated. Mineralization and osteoblast-specific gene expression were evaluated during osteogenesis in EtOH-treated BM-MSCs. To investigate the role of silent information regulator Type 1 (SIRT1) in EtOH-induced senescence, resveratrol (ResV) was used to activate SIRT1 in EtOH-treated BM-MSCs. Results EtOH treatments resulted in senescence-associated phenotypes in BM-MSCs, such as decreased cell proliferation, increased SA-β-gal activity and G0/G1 cell cycle arrest. EtOH also increased the intracellular ROS and the expression of senescence-related genes, such as p16INK4α and p21. The down-regulated levels of SIRT1 accompanied with suppressed osteogenic differentiation were confirmed in EtOH-treated BM-MSCs. Activation of SIRT1 by ResV partially counteracted the effects of EtOH by decreasing senescence markers and rescuing the inhibited osteogenesis. Conclusion EtOH treatments induced premature senescence in BM-MSCs in a dose-dependent manner that was responsible for EtOH-impaired osteogenic differentiation. Activation of SIRT1 was effective in ameliorating EtOH-induced senescence phenotypes in BMSCs and could potentially lead to a new strategy for clinically preventing or treating alcohol-induced osteoporosis. Short summary Ethanol (EtOH) treatments induce premature senescence in marrow-derived mesenchymal stem cells in a dose-dependent manner that is responsible for EtOH-impaired osteogenic differentiation. Activation of SIRT1 is effective in ameliorating EtOH-induced senescence phenotypes, which potentially leads to a new strategy for clinically treating alcohol-induced osteoporosis.

Ascorbate-dependent impact on cell-derived matrix in modulation of stiffness and rejuvenation of infrapatellar fat derived stem cells toward chondrogenesis

Developing an in vitro microenvironment using cell-derived decellularized extracellular matrix (dECM) is a promising approach to efficiently expand adult stem cells for cartilage engineering and regeneration. Ascorbic acid serves as a critical stimulus for cells to synthesize collagens, which constitute the major component of dECM. In this study, we hypothesized that optimization of ascorbate treatment would maximize the rejuvenation effect of dECM on expanded stem cells from human infrapatellar fat pad in both proliferation and chondrogenic differentiation. In the duration regimen study, we found that dECM without L-ascorbic acid phosphate (AA) treatment, exhibiting lower stiffness measured by atomic force microscopy, yielded expanded cells with higher proliferation capacity but lower chondrogenic potential when compared to those with varied durations of AA treatment. dECM with 250 µM of AA treatment for 10 d had better rejuvenation in chondrogenic capacity if the deposited cells were from passage 2 rather than passage 5, despite no significant difference in matrix stiffness. In the dose regimen study, we found that dECMs deposited by varied concentrations of AA yielded expanded cells with higher proliferation capacity despite lower expression levels of stem cell related surface markers. Compared to cells expanded on tissue culture polystyrene, those on dECM exhibited greater chondrogenic potential, particularly for the dECMs with 50 µM and 250 µM of AA treatment. With the supplementation of ethyl-3,4-dihydroxybenzoate (EDHB), an inhibitor targeting procollagen synthesis, the dECM with 50 µM of AA treatment exhibited a dramatic decrease in the rejuvenation effect of expanded cell chondrogenic potential at both mRNA and protein levels despite no significant difference in matrix stiffness. Defined AA treatments during matrix preparation will benefit dECM-mediated stem cell engineering and future treatments for cartilage defects.

Impact of Wnt signals on human intervertebral disc cell regeneration: WNT SIGNALS AND DISC REGENERATION

Although preconditioning strategies are growing areas of interest for therapies targeting intervertebral discs (IVDs), it is unknown whether the Wnt signals previously implicated in chondrogenesis, Wnt3A, Wnt5A, and Wnt11, play key roles in the promotion of human nucleus pulposus (NP) cell redifferentiation. In this study, NP cells isolated from herniated disc patients were transduced with lentiviral vectors to overexpress the WNT3A, WNT5A, or WNT11 genes, or CRISPR associated protein 9 (Cas9)/single‐guide RNA (sgRNA) vectors to knock out these genes. Following expansion, transduced NP cells were induced for redifferentiation toward the NP phenotype. The overexpression of specific WNT factors led to increases in both glycosaminoglycan (GAG) deposition and expression of redifferentiation genes. These effects were attenuated by knockout of the same WNT genes. These results indicate that specific WNT signals can regulate the expression of redifferentiation genes, unequally impact GAG deposition, and contribute to the redifferentiation of human NP cells.

Incremental temperature cement delivery technique may prevent cement leakage in metastatic vertebral lesions

Objectives: To analyze the clinical efficacy and cement leakage rate of percutaneous kyphoplasty (PKP) for the treatment of metastatic vertebral lesions between the two groups using different cement infusion techniques. Methods: One hundred twenty-nine patients (160 metastatic vertebral fractures) who chose “the incremental temperature cement delivery technique” (ITCDT group) and 105 patients (128 metastatic vertebral fractures) who chose “traditional infusion technique” (TI group) were finally enrolled (nine patients were lost to the follow-up). The visual analog scale (VAS), Oswestry Disability Index (ODI), the local Cobb’s angle, the relative height of the vertebral anterior border, and cement leakage were analyzed to evaluate the clinical efficacy of the two cement infusion techniques within postoperative 12 months. Results: There was no significant difference in the VAS scores, ODI scores, the relative height of the vertebral anterior border, and local Cobb’s angle between the ITCDT group and TI group preoperatively and postoperatively (p > 0.05). The cement leakage occurred in three patients of ITCDT group (2.3%), which was significantly less than TI group (12 patients, 11.4%; p < 0.05). Conclusions: The ITCDT and TI technique in kyphoplasty are effective at relieving pain and improving functional disability, vertebral height, and kyphosis angle; However, kyphoplasty using the ITCDT causes less cement leakage significantly.