Journal of Tissue Engineering and Regenerative Medicine最新文献

Background. Periodontitis is characterized by bone resorption and periodontal tissue destruction owing to oral microbiota, mechanical stress, and systemic diseases such as diabetes mellitus. Human dental pulp mesenchymal stem cells (hDPMSCs) were analyzed as potential candidates for periodontal tissue regeneration. Acetylsalicylic acid (ASA), also known as aspirin, has been shown to promote osteogenic differentiation of mesenchymal stem cells. We investigated the effect of ASA pretreatment on periodontitis in order to achieve a more appealing prognosis of bone resorption. Methods. The effect of ASA on cell proliferation was detected by the CCK-8 assay, and alkaline phosphatase (ALP) staining, alizarin red staining (ARS), and western blot were used to investigate the effect of different ASA concentrations on hDPMSCs’ osteogenic differentiation and possible signaling pathways. Periodontitis was induced for 4 weeks. Stem cells pretreated with 50 µg/mL of ASA were transplanted into six-week-old male Sprague-Dawley rats by local and systemic injection once a week for two weeks. Four weeks after cell therapy, the rats were sacrificed for sampling to complete the molecular and morphological experiments. Results. In vitro experiments revealed that 50 µg/mL of ASA had a significant effect on cell osteogenic differentiation. That is, when ASA was administered, the MAPK signaling pathway was activated. Notably, further vivo experiments revealed that ASA-hDPMSCs increased the area of bone regeneration and the OPG/RANKL ratio, suppressed TNF-α and IL-1 expression, and promote alveolar bone repair. Conclusion. Our study extends the findings of previous research, firstly demonstrating that the use of ASA-pretreated hDPMSCs offers a novel therapy for the treatment of periodontitis for future clinical application.

Proliferative vitreoretinopathy (PVR) as a rare fibrotic ocular disease is the main reason for failure of retinal detachment surgery and a reduced prognosis following surgery. Amniotic membrane (AM) is a versatile surgical tool for tissue stabilization, antifibrotic properties, and regeneration. Initial clinical case studies now demonstrated intravitreal tolerance as well as good anatomical and functional results for degenerative retinal diseases. Due to its diverse wound healing properties, AM could have promoting, suppressive, or no effects on PVR. To illuminate the potential of epiretinal AM transplantation in complex retinal detachment cases, we investigated its influence on human primary PVR (hPVR) cells in vitro. In our cell culture study, hPVR cells were isolated from surgically removed PVR membranes. Following incubation with AM for 48 h, AM-incubated hPVR showed significantly reduced proliferation (BrdU-ELISA; p < 0.001), migration (Boyden chamber, scratch assay; p = 0.003 and p < 0.001), and cell adhesion (p = 0.005). Collagen contraction was nearly unaffected (p = 0.04), and toxicity (histone-complexed DNA ELISA, WST-1 assay, and life/dead staining) was excluded. Next, immunofluorescence showed a myofibroblastic phenotype with reduced expression of fibrosis markers in AM-incubated cells, which was confirmed by Western blot analysis. In the proteomics assay, AM significantly regulated proteins by a more than 2-fold increase in expression which were related to the cytoskeleton, lipid metabolism, cell-matrix contraction, and protein folding. In conclusion, this in vitro work suggests no induction of fibrosis and other key steps in the pathogenesis of PVR through AM but rather inhibiting properties of profibrotic cell behavior, making it a possible candidate for suppression of PVR. Further clinical studies are necessary to evaluate the therapeutic relevance.

Mesenchymal stem cell (MSC)-based therapies for articular cartilage regeneration are effective mostly due to paracrine signals mediated by extracellular vesicles, especially small extracellular vesicles (sEV). However, it is unknown whether dental follicle cell-derived sEV (DFC-sEV) affect cartilage regeneration in temporomandibular joint osteoarthritis (TMJ-OA). In this study, the effects of DFC-sEV on IL-1β-induced mandibular condylar chondrocytes (MCCs) were determined using CCK8 assays, scratch assays, flow cytometry, and Western blot analysis of matrix synthesis and catabolic proteins. Furthermore, we used an abnormal occlusion-induced rat model and intra-articular injection of DFC-sEV, the pathological changes of which were observed by HE staining, safranin O staining, immunohistochemistry, and micro-CT analysis of subchondral bone loss. Gene set enrichment analysis (GSEA) was used to determine the related mechanism involved in the effect of DFC-sEV. Immunofluorescence analysis and Western blotting were used to evaluate the expression of HIF-1α, HIF-2α, MMP13, and VEGF in MCCs. Then, lentivirus-induced Epas1 overexpression and Western blot analysis of the downstream regulators of HIF-2α were performed. We found that DFC-sEV promoted MCCs proliferation and migration and protected against cartilage matrix destruction induced by IL-1β. In addition, DFC-sEV prevented cartilage destruction in an abnormal occlusion rat model. Furthermore, we found that DFC-sEV reduced the expression of HIF-1α and HIF-2α in vitro and in vivo and decreased the downstream regulators of HIF-2α, including MMP13 and VEGF. Our study indicated that DFC-sEV attenuated TMJ cartilage damage in vitro and in vivo, which might be involved in the regulation of HIF-2α.

Magnetic field exposure is a well-established diagnostic tool. However, its use as a therapeutic in regenerative medicine is relatively new. To better understand how magnetic fields affect neural repair in vitro, we started by performing a systematic review of publications that studied neural repair responses to magnetic fields. The 38 included articles were highly heterogeneous, representing 13 cell types, magnetic field magnitudes of 0.0002–10,000 mT with frequencies of 0–150 Hz, and exposure times ranging from one hour to several weeks. Mathematical modeling based on data from the included manuscripts revealed higher magnetic field magnitudes enhance neural progenitor cell (NPC) viability. Finally, for those regenerative processes not influenced by magnitude, frequency, or time, we integrated the data by meta-analyses. Results revealed that magnetic field exposure increases NPC proliferation while decreasing astrocytic differentiation. Collectively, our approach identified neural repair processes that may be most responsive to magnetic field exposure.

The rehabilitation of bone defects after radiotherapy requires the development of osteoinductive bone substitutes. MicroRNA could be used as an osteogenic factor to fabricate functional materials for bone regeneration. In this study, we used miR-34a to enhance bone regeneration after irradiation. We lyophilized lipofectamine-agomiR-34a lipoplexes on hydroxyapatite (HA) to develop miR-34a-functionalized hydroxyapatite (HA-agomiR-34a). The morphology was observed by scanning electron microscope and atomic force microscope. Fluorescence microscopy confirmed the retention of agomiR-34a on the surface of HA. HA-agomiR-34a showed high transfection efficiency and good biocompatibility. HA-agomiR-34a enhanced the osteoblastic differentiation of radiation-impaired bone marrow stromal cells (BMSCs). Implantation of HA-agomiR-34a promoted bone regeneration in irradiated bone defects. HA-agomiR-34a may be a novel and safe bone substitute to promote the reconstruction of bone defects after radiotherapy.

Studying the crosstalk between the muscle and tendon tissue is an important yet understudied area in musculoskeletal research. In vitro models can help elucidate the function and repair of the myotendinous junction (MTJ) under static and dynamic culture conditions using engineered muscle tissues. The goal of this study was to culture engineered muscle tissues in a novel bioreactor in both static and mechanically stimulated cultures and evaluate the expression of MTJ-specific proteins within the muscle-tendon unit(paxillin and type XXII collagen). C2C12 myoblasts were seeded in hydrogels made from type I collagen ortendon-derived extracellular matrix (tECM) and allowed to form around movable anchors. Engineered tissues were allowed to form and stabilize for 10 days. After 10 days in the culture, stimulated cultures were cyclically stimulated for 3 hours per day for 2 and 4 weeks alongside static cultures. Strain values at the maximum displacement of the anchors averaged about 0.10, a target that has been shown to induce myogenic phenotype in C2C12s. Protein expression of paxillin after 2 weeks did not differ between hydrogel materials in static cultures but increased by 62% in tECM when mechanically stimulated. These differences continued after 4 weeks, with 31% and 57% increases in tECM tissues relative to type I collagen. Expression of type XXII collagen was similarly influenced by hydrogel material and culture conditions. Overall, this research combined a relevant microenvironment to study muscle and tendon interactions with a novel bioreactor to apply mechanical strain, an important regulator of the formation and maintenance of the native MTJ.

Retinal degeneration is an escalating public health challenge, as diseases such as age-related macular degeneration, diabetic retinopathy, and retinitis pigmentosa cause irreversible vision loss in millions of adults each year. Regenerative medicine has pioneered the development of stem cell replacement therapies, which treat degeneration by replacing damaged retinal neurons with transplanted stem-like cells (SCs). While the collective migration of SCs plays critical roles during retinal development, our understanding of collective SC behaviors within biomaterials transplanted into adult tissue remains understudied. This project examines the potential therapeutic impacts of collective SC migration during transplantation by correlating the expression of cadherin, cell-cell cohesion molecules that maintain intercellular communication during development, with receptor proteins of chemoattractant molecules prevalent in degenerated adult tissue. Experiments examine these well-conserved biomechanisms by using two different model organisms: Drosophila melanogaster, a seminal model for retinal development, and Mus, an important preclinical model for transplantation. Results indicate that SCs from both animal models significantly upregulate cadherin expression to achieve more directed collective migration towards species-specific chemoattractants and exhibit longer distance motility upon different extracellular matrix substrates.

It is generally accepted that the application of hydrostatic pressure (HP) is beneficial for MSC chondrogenesis. There is, however, evidence to suggest that the timing of application might determine its impact on cell fate and tissue development. Furthermore, understanding how the maturity of engineered cartilage affects its response to the application of HP can provide critical insight into determining when such a graft is ready for in vivo implantation into a mechanically loaded environment. In this study, we systematically examined chondrogenic maturation of hMSCs over 35 days in the presence of TGF-β3 in vitro. At specific timepoints, the response of hMSCs to the application of HP following the removal of TGF-β3 was assessed; this partially models conditions such grafts will experience in vivo upon implantation. In free swelling culture, the expression of chondrogenic (COL2A1 and ACAN) and hypertrophic (COL10A1) markers increased with time. At early timepoints, the expression of such markers continued to increase following TGF-β3 withdrawal; however, this was not observed after prolonged periods of chondrogenic priming (35 days). Interestingly, the application of HP was only beneficial after 35 days of chondrogenic priming, where it enhanced sGAG synthesis and improved key chondrogenic gene ratios. It was also found that HP can facilitate a metabolic shift towards oxidative phosphorylation, which can be viewed as a hallmark of successfully differentiating MSCs. These results point to the importance of mechanical loading as a key stimulus for maintaining a chondrogenic phenotype once MSCs are removed from chemically defined culture conditions.

Recombinant adeno-associated virus (rAAV) vectors have a strong potential to promote the healing of traumatic cartilage defects and osteoarthritic lesions upon delivery and overexpression of therapeutic genes from suitable biomaterials that support a controlled release of the candidate constructs. The goal of the present work is to examine whether the administration of chondrogenic rAAV sox9 and rAAV TGF-ß gene vehicles via alginate hydrogel-guided vector delivery stimulates the biological and chondroreparative activities of human bone marrow-derived mesenchymal stromal cells (hMSCs) as a source of improved reparative cells for future implantation in sites of cartilage damage. The delivery of rAAV using an alginate (AlgPH155) hydrogel system is successfully achieved in hMSCs over time (21 days), leading to the effective overexpression of sox9 and TGF-ß that significantly increases the proliferation and chondrogenic differentiation activities of the cells relative to control (marker lacZ) gene transfer while advantageously preventing premature hypertrophy, osteogenesis, and mineralization. This study reveals the potential of alginate hydrogel-based systems to control the delivery of rAAV (sox9 and TGF-ß) gene vectors to adeptly trigger the chondroreparative activities of hMSCs for future applications that aim at improving cartilage repair.

Novel treatment strategies for segmental bone loss in orthopaedic surgery remain under investigation. Regional gene therapy that involves transduction of mesenchymal stem cells with a lentiviral vector that expresses BMP-2 has gained particular interest as this strategy provides osteogenic and osteoinductive factors for bone growth. In particular, transduced adipose-derived stems cells (ASCs) and bone marrow-derived stem cells (BMSCs) have emerged as the leading candidates for the treatment of segmental defects in preclinical models. The aim of the present study was to evaluate the influence of demographic information on in vitro growth characteristics and bone morphogenetic protein-2 production following lentiviral transduction in a large cohort of human donors. We further sought to assess the effects of ASC harvest site on cell yield and growth characteristics. We evaluated a total of 187 human donors (124 adipose harvests and 63 bone marrow aspirates) in our cohort. We found that across all donors, ASCs demonstrated favorable growth characteristics and could be cultured in vitro more reliably than BMSCs regardless of patient-related factors. Furthermore, we noted that following lentiviral transduction, ASCs produced significantly higher levels of BMP-2 compared to BMSCs. Lastly, despite higher initial cell yields from lipoaspirate, posttransduction BMP-2 production was less than that of infrapatellar fat pad samples. These results support the continued investigation of ASCs as a cellular delivery vehicle for regional gene therapy to deliver osteoinductive proteins to specific anatomic bone repair sites.