Statin use in patients with type 2 diabetes has lower risk of hip fractures: A Taiwan national population-based study.

AIMS: Type 2 diabetes mellitus (T2DM) frequently co-exists with osteoporosis and dyslipidemia. Statins have been commonly used in the treatment of dyslipidemia. Recent studies have indicated a therapeutic role of statins in decreasing the risk of osteoporosis and fractures, but conflicting results have been reported. This study investigated the association between statin use and hip fracture (HFx) risk among T2DM patients. MATERIALS AND METHODS: A retrospective Taiwan population-based propensity-matched cohort study was performed using the Diabetes Mellitus Health Database from Taiwan National Health Insurance Research Database. Patients with newly diagnosed with T2DM between 2010 and 2014 were identified. Patients who previously used statins and had ever suffered HFx before the index date were excluded. HFx that occurred from 2010 to 2019 was collected to compute the cumulative rate of HFx. Hazard ratios (HRs) were calculated for the HFx risk according to the use or non-use of statins. To evaluate the dose-effect relationship of statins, sensitivity analyses were conducted. RESULTS: After propensity score matching for age and sex, 188,588 patients were identified as statin users and non-statin users. Statin use after T2DM diagnosis was associated with a decreased HFx risk with an adjusted HR (aHR) of 0.69 (P < 0.001). A dose-effect relationship was identified. The aHRs for developing HFx were 1.29, 0.67, and 0.36 for patients who used 28-174, 175-447, and >447 cumulative defined daily doses of statins, respectively (P < 0.001). CONCLUSIONS: Statin use in adults with T2DM showed a lower risk of HFx by demonstrating a dose-response relationship.

Exosomes Derived from Hypoxia-Cultured Human Adipose Stem Cells Alleviate Articular Chondrocyte Inflammaging and Post-Traumatic Osteoarthritis Progression.

Osteoarthritis (OA) is the most common age-related degenerative joint disease. Inflammaging, linking inflammation and aging, is found in senescent cells with the secretions of matrix-degrading proteins and proinflammatory cytokines. The senescence-associated secretory phenotype (SASP) plays a very important role in OA progression. However, there remains no effective way to suppress OA progression, especially by suppressing inflammaging and/or the chondrocyte SASP. Recent studies have shown that exosomes derived from hypoxia-cultured BMSCs can regenerate cartilage in OA animal models. Some reports have further indicated that exosomes secreted from MSCs contribute to the efficacy of MSC therapy in OA. However, whether hypoxia-cultured ADSC-secreted exosomes (hypoxia-ADSC-Exos) can alleviate the chondrocyte SASP or OA progression remains unclear. Accordingly, we hypothesized that hypoxia-ADSC-Exos have a beneficial effect on the normal functions of human articular chondrocytes (HACs), can attenuate the SASP of OA-like HACs in vitro, and further suppress OA progression in rats. Hypoxia-ADSC-Exos were derived from ADSCs cultured in 1% O2 and 10% de-Exo-FBS for 48 h. The molecular and cell biological effects of hypoxia-ADSC-Exos were tested on IL1-ß-induced HACs as OA-like HACs in vitro, and the efficacy of OA treatment was tested in ACLT-induced OA rats. The results showed that hypoxia-ADSC-Exos had the best effect on GAG formation in normal HACs rather than those cultured in normoxia or hypoxia plus 2% de-Exo-FBS. We further found that hypoxia-ADSC-Exos alleviated the harmful effect in OA-like HACs by decreasing markers of normal cartilage (GAG and type II collagen) and increasing markers of fibrous or degenerative cartilage (type I or X collagen), matrix degradation enzymes (MMP13 and ADAMT5), and inflammatory cytokines (TNFα and IL-6). More importantly, intra-articular treatment with hypoxia-ADSC-Exos suppressed OA progression, as evidenced by the weight-bearing function test and cartilage GAG quantification in ACLT rats. Moreover, through NGS and bioinformatic analysis, seven potential miRNAs were found in hypoxia-ADSC-Exos, which may contribute to regulating cellular oxidative stress and attenuating cell senescence. In summary, we demonstrated that hypoxia-ADSC-Exos, carrying potent miRNAs, not only improve normal HAC function but also alleviate HAC inflammaging and OA progression. The results suggest that hypoxia-ADSC-Exo treatment may offer another strategy for future OA therapy.

Chondrocyte Thrombomodulin Protects against Osteoarthritis.

Osteoarthritis (OA) is a prevalent form of arthritis that affects over 32.5 million adults worldwide, causing significant cartilage damage and disability. Unfortunately, there are currently no effective treatments for OA, highlighting the need for novel therapeutic approaches. Thrombomodulin (TM), a glycoprotein expressed by chondrocytes and other cell types, has an unknown role in OA. Here, we investigated the function of TM in chondrocytes and OA using various methods, including recombinant TM (rTM), transgenic mice lacking the TM lectin-like domain (TMLeD/LeD), and a microRNA (miRNA) antagomir that increased TM expression. Results showed that chondrocyte-expressed TM and soluble TM [sTM, like recombinant TM domain 1 to 3 (rTMD123)] enhanced cell growth and migration, blocked interleukin-1ß (IL-1ß)-mediated signaling and protected against knee function and bone integrity loss in an anterior cruciate ligament transection (ACLT)-induced mouse model of OA. Conversely, TMLeD/LeD mice exhibited accelerated knee function loss, while treatment with rTMD123 protected against cartilage loss even one-week post-surgery. The administration of an miRNA antagomir (miR-up-TM) also increased TM expression and protected against cartilage damage in the OA model. These findings suggested that chondrocyte TM plays a crucial role in counteracting OA, and miR-up-TM may represent a promising therapeutic approach to protect against cartilage-related disorders.

Discoidin domain receptor 1 regulates endochondral ossification through terminal differentiation of chondrocytes.

Chondrocytes in growth plates are responsible for longitudinal growth in long bones during endochondral ossification. Discoidin domain receptor 1 (Ddr1) is expressed in chondrocytes, but the molecular mechanisms by which DDR1 regulates chondrocyte behaviors during the endochondral ossification process remain undefined. To elucidate Ddr1-mediate chondrocyte functions, we generated chondrocyte-specific Ddr1 knockout (CKOΔDdr1) mice in this study. The CKOΔDdr1 mice showed delayed development of the secondary ossification center and increased growth plate length in the hind limbs. In the tibial growth plate in CKOΔDdr1 mice, chondrocyte proliferation was reduced in the proliferation zone, and remarkable downregulation of Ihh, MMP13, and Col-X expression in chondrocytes resulted in decreased terminal differentiation in the hypertrophic zone. Furthermore, apoptotic chondrocytes were reduced in the growth plates of CKOΔDdr1 mice. We concluded that chondrocytes with Ddr1 knockout exhibit decreased proliferation, terminal differentiation, and apoptosis in growth plates, which delays endochondral ossification and results in short stature. We also demonstrated that Ddr1 regulates the Ihh/Gli1/Gli2/Col-X pathway to regulate chondrocyte terminal differentiation. These results indicate that Ddr1 is required for chondrocytes to regulate endochondral ossification in skeletal development.

Effect of Freshly Isolated Bone Marrow Mononuclear Cells and Cultured Bone Marrow Stromal Cells in Graft Cell Repopulation and Tendon-Bone Healing after Allograft Anterior Cruciate Ligament Reconstruction.

Graft cell repopulation and tendon-bone tunnel healing are important after allograft anterior cruciate ligament reconstruction (ACLR). Freshly isolated bone marrow mononuclear cells (BMMNCs) have the advantage of short isolation time during surgery and may enhance tissue regeneration. Thus, we hypothesized that the effect of intra-articular BMMNCs in post-allograft ACLR treatment is comparable to that of cultured bone marrow stromal cells (BMSCs). A rabbit model of hamstring allograft ACLR was used in this study. Animals were randomly assigned to the BMMNC, BMSC, and control groups. Fresh BMMNCs isolated from the iliac crest during surgery and cultured BMSCs at passage four were used in this study. A total of 1 × 107 BMMNCs or BMSCs in 100 µL phosphate-buffered saline were injected into the knee joint immediately after ACLR. The control group was not injected with cells. At two and six weeks post operation, we assessed graft cell repopulation with histological and cell tracking staining (PKH26), and tendon-bone healing with histological micro-computed tomography and immunohistochemical analyses for collagen I and monocyte chemoattractant protein-1 (MCP1). At two weeks post operation, there was no significant difference in the total cell population within the allograft among the three groups. However, the control group showed significantly higher cell population within the allograft than that of BM cell groups at six weeks. Histological examination of proximal tibia revealed that the intra-articular delivered cells infiltrated into the tendon-bone interface. Compared to the control group, the BM cell groups showed broader gaps with interfacial fibrocartilage healing, similar collagen I level, and higher MCP1 expression in the early stage. Micro-CT did not reveal any significant difference among the three groups. BMMNCs and BMSCs had comparable effects on cell repopulation and interfacial allograft-bone healing. Intra-articular BM cells delivery had limited benefits on graft cell repopulation and caused higher inflammation than that in the control group in the early stage, with fibrocartilage formation in the tendon-bone interface after allograft ACLR.

An Intermediate Concentration of Calcium with Antioxidant Supplement in Culture Medium Enhances Proliferation and Decreases the Aging of Bone Marrow Mesenchymal Stem Cells.

Human bone marrow stem cells (HBMSCs) are isolated from the bone marrow. Stem cells can self-renew and differentiate into various types of cells. They are able to regenerate kinds of tissue that are potentially used for tissue engineering. To maintain and expand these cells under culture conditions is difficult-they are easily triggered for differentiation or death. In this study, we describe a new culture formula to culture isolated HBMSCs. This new formula was modified from NCDB 153, a medium with low calcium, supplied with 5% FBS, extra growth factor added to it, and supplemented with N-acetyl-L-cysteine and L-ascorbic acid-2-phosphate to maintain the cells in a steady stage. The cells retain these characteristics as primarily isolated HBMSCs. Moreover, our new formula keeps HBMSCs with high proliferation rate and multiple linage differentiation ability, such as osteoblastogenesis, chondrogenesis, and adipogenesis. It also retains HBMSCs with stable chromosome, DNA, telomere length, and telomerase activity, even after long-term culture. Senescence can be minimized under this new formulation and carcinogenesis of stem cells can also be prevented. These modifications greatly enhance the survival rate, growth rate, and basal characteristics of isolated HBMSCs, which will be very helpful in stem cell research.

G Protein-Coupled Estrogen Receptor Mediates Cell Proliferation through the cAMP/PKA/CREB Pathway in Murine Bone Marrow Mesenchymal Stem Cells.

Estrogen is an important hormone to regulate skeletal physiology via estrogen receptors. The traditional estrogen receptors are ascribed to two nuclear estrogen receptors (ERs), ERα and ERß. Moreover, G protein-coupled estrogen receptor-1 (GPER-1) was reported as a membrane receptor for estrogen in recent years. However, whether GPER-1 regulated osteogenic cell biology on skeletal system is still unclear. GPER-1 is expressed in growth plate abundantly before puberty but decreased abruptly since the very late stage of puberty in humans. It indicates GPER-1 might play an important role in skeletal growth regulation. GPER-1 expression has been confirmed in osteoblasts, osteocytes and chondrocytes, but its expression in mesenchymal stem cells (MSCs) has not been confirmed. In this study, we hypothesized that GPER-1 is expressed in bone MSCs (BMSC) and enhances BMSC proliferation. The cultured tibiae of neonatal rat and murine BMSCs were tested in our study. GPER-1-specific agonist (G-1) and antagonist (G-15), and GPER-1 siRNA (siGPER-1) were used to evaluate the downstream signaling pathway and cell proliferation. Our results revealed BrdU-positive cell counts were higher in cultured tibiae in the G-1 group. The G-1 also enhanced the cell viability and proliferation, whereas G-15 and siGPER-1 reduced these activities. The cAMP and phosphorylation of CREB were enhanced by G-1 but inhibited by G-15. We further demonstrated that GPER-1 mediates BMSC proliferation via the cAMP/PKA/p-CREB pathway and subsequently upregulates cell cycle regulators, cyclin D1/cyclin-dependent kinase (CDK) 6 and cyclin E1/CDK2 complex. The present study is the first to report that GPER-1 mediates BMSC proliferation. This finding indicates that GPER-1 mediated signaling positively regulates BMSC proliferation and may provide novel insights into addressing estrogen-mediated bone development.

Discoidin Domain Receptors 1 Inhibition Alleviates Osteoarthritis via Enhancing Autophagy.

We recently reported that the chondrocyte-specific knockout of discoidin domain receptors 1 (Ddr1) delayed endochondral ossification (EO) in the growth plate by reducing the chondrocyte hypertrophic terminal differentiation, and apoptosis. The biologic and phenotypic changes in chondrocytes in the articular cartilage with osteoarthritis (OA) are similar to the phenomena observed in the process of EO. Additionally, autophagy can promote chondrocyte survival and prevent articular cartilage from degradation in OA. On this basis, we explored the effect of Ddr1 inhibition on OA prevention and further investigated the roles of autophagy in treating OA with a Ddr1 inhibitor (7 rh). The anterior cruciate ligament transection (ACLT)-OA model was used to investigate the role of 7 rh in vivo. Forty 8-week-old mice were randomly assigned to four groups, including the sham group, ACLT group, and two treated groups (ACLT with 7 rh 6.9 nM or 13.8 nM). According to the study design, normal saline or 7 rh were intra-articular (IA) injected into studied knees 3 times per week for 2 weeks and then once per week for 4 weeks. The results showed that 7 rh treatment significantly improved the functional performances (the weight-bearing ability and the running endurance), decreased cartilage degradation, and also reduced the terminal differentiation markers (collagen type X, Indian hedgehog, and matrix metalloproteinase 13). Moreover, 7 rh decreased chondrocyte apoptosis by regulating chondrocyte autophagy through reducing the expression of the mammalian target of rapamycin and enhancing the light chain 3 and beclin-1 expression. These results demonstrated that the IA injection of 7 rh could reduce the chondrocyte apoptosis and promote chondrocyte autophagy, leading to the attenuation of cartilage degradation. Our observations suggested that the IA injection of 7 rh could represent a potential disease-modifying therapy to prevention OA progression.

The effect of low-level laser irradiation on hyperglycemia-induced inflammation in human gingival fibroblasts.

Hyperglycemia-induced inflammation can greatly increase the risk of periodontal disease in people with diabetes. Low-level laser irradiation (LLLI) has been used for wound healing and anti-inflammation in many cases, and LLLI is known to inhibit the lipopolysaccharide (LPS)-stimulated inflammatory response. However, the therapeutic effect of LLLI in diabetes patients with periodontitis remains unknown. In this study, we cultured human gingival fibroblasts (HGFs) in high-glucose medium (35 mM) to mimic a hyperglycemic environment, and then measured the anti-inflammatory effect of LLLI by assessing the expression of pro-inflammatory genes including tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6, and IL-8 by quantitative real-time polymerase chain reaction. The results demonstrated no significant inflammatory response in HGFs cultured in mannitol medium and in those treated only with LLLI. However, HGFs cultured only in high-glucose medium showed significantly higher expression of pro-inflammatory cytokine than in those treated together with LLLI. We then observed that LLLI reduced the expression of pro-inflammatory cytokines in HGFs cultured in high-glucose medium by modulating cAMP signaling. We also investigated whether antioxidant (vitamin C) treatment reduced the inflammatory effect of oxidative stress in HGFs cultured in high-glucose medium but found no additive effect upon co-treatment with LLLI, suggesting that LLLI may activate cAMP signaling, but not reactive oxygen species (ROS) signaling, to reduce the high glucose-induced inflammation. In conclusion, LLLI may have an anti-inflammatory effect on HGFs in a high glucose environment and may benefit the treatment of periodontal disease in diabetes patients.

Alpha-5 Integrin Mediates Simvastatin-Induced Osteogenesis of Bone Marrow Mesenchymal Stem Cells.

Simvastatin (SVS) promotes the osteogenic differentiation of mesenchymal stem cells (MSCs) and has been studied for MSC-based bone regeneration. However, the mechanism underlying SVS-induced osteogenesis is not well understood. We hypothesize that α5 integrin mediates SVS-induced osteogenic differentiation. Bone marrow MSCs (BMSCs) derived from BALB/C mice, referred to as D1 cells, were used. Alizarin red S (calcium deposition) and alkaline phosphatase (ALP) staining were used to evaluate SVS-induced osteogenesis of D1 cells. The mRNA expression levels of α5 integrin and osteogenic marker genes (bone morphogenetic protein-2 (BMP-2), runt-related transcription factor 2 (Runx2), collagen type I, ALP and osteocalcin (OC)) were detected using quantitative real-time PCR. Surface-expressed α5 integrin was detected using flow cytometry analysis. Protein expression levels of α5 integrin and phosphorylated focal adhesion kinase (p-FAK), which is downstream of α5 integrin, were detected using Western blotting. siRNA was used to deplete the expression of α5 integrin in D1 cells. The results showed that SVS dose-dependently enhanced the gene expression levels of osteogenic marker genes as well as subsequent ALP activity and calcium deposition in D1 cells. Upregulated p-FAK was accompanied by an increased protein expression level of α5 integrin after SVS treatment. Surface-expressed α5 integrin was also upregulated after SVS treatment. Depletion of α5 integrin expression significantly suppressed SVS-induced osteogenic gene expression levels, ALP activity, and calcium deposition in D1 cells. These results identify a critical role of α5 integrin in SVS-induced osteogenic differentiation of BMSCs, which may suggest a therapeutic strategy to modulate α5 integrin/FAK signaling to promote MSC-based bone regeneration.

Anti-inflammatory effects of low-level laser therapy on human periodontal ligament cells: in vitro study.

Periodontal disease is a chronic inflammatory disease that is commonly treated with surgical and nonsurgical techniques. However, both approaches have limitations. Low-level laser therapy (LLLT) has been widely applied in reducing inflammatory reactions, and research indicates that LLLT induces an anti-inflammatory effect that may enhance periodontal disease therapy. The purpose of this study was to investigate the anti-inflammatory effect of LLLT on human periodontal ligament cells (hPDLCs) in an inflammatory environment and aimed to determine the possible mechanism of action. Cells were cultured and treated with or without lipopolysaccharide (LPS) from Porphryromonas gingivalis or Escherichia coli, followed by irradiation with a gallium-aluminum-arsenide (GaAlAs) laser (660 nm) at an energy density of 8 J/cm2. Quantitative real-time polymerase chain reactions were used to assess the expression of pro-inflammatory genes, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6, and IL-8. The dual-luciferase reporter assay was used to examine nuclear factor-κB (NF-κB) transcriptional activity. An enzyme-linked immunosorbent assay was used to monitor the concentration of intracellular cyclic adenosine monophosphate (cAMP). Both LPS treatments significantly induced the mRNA expression of pro-inflammatory cytokines. However, LLLT inhibited the LPS-induced pro-inflammatory cytokine expression and elevated intracellular levels of cAMP. The LLLT inhibitory effect may function by downregulating NF-κB transcriptional activity and by increasing the intracellular levels of cAMP. LLLT might inhibit LPS-induced inflammation in hPDLCs through cAMP/NF-κB regulation. These results should be further studied to improve periodontal therapy.

Combination of a Bioceramic Scaffold and Simvastatin Nanoparticles as a Synthetic Alternative to Autologous Bone Grafting.

The fragile nature of porous bioceramic substitutes cannot match the toughness of bone, which limits the use of these materials in clinical load-bearing applications. Statins can enhance bone healing, but it could show rhabdomyolysis/inflammatory response after overdosing. In this study, the drug-containing bone grafts were developed from poly(lactic acid-co-glycolic acid)-polyethylene glycol (PLGA-PEG) nanoparticles encapsulating simvastatin (SIM) (SIM-PP NPs) loaded within an appropriately mechanical bioceramic scaffold (BC). The combination bone graft provides dual functions of osteoconduction and osteoinduction. The mechanical properties of the bioceramic are enhanced mainly based on the admixture of a combustible reverse-negative thermoresponsive hydrogel (poly(N-isopropylacrylamide base). We showed that SIM-PP NPs can increase the activity of alkaline phosphatase and osteogenic differentiation of bone marrow stem cells. To verify the bone-healing efficacy of this drug-containing bone grafts, a nonunion radial endochondral ossification bone defect rabbit model (N = 3/group) and a nonunion calvarial intramembranous defect Sprague Dawley (SD) rat model (N = 5/group) were used. The results indicated that SIM-PP NPs combined with BC can improve the healing of nonunion bone defects of the radial bone and calvarial bone. Therefore, the BC containing SIM-PP NPs may be appropriate for clinical use as a synthetic alternative to autologous bone grafting that can overcome the problem of determining the clinical dosage of simvastatin drugs to promote bone healing.

(-)-Epigallocatechin-3-Gallate (EGCG) Enhances Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells.

Osteoporosis is the second most-prevalent epidemiologic disease in the aging population worldwide. Cross-sectional and retrospective evidence indicates that tea consumption can mitigate bone loss and reduce risk of osteoporotic fractures. Tea polyphenols enhance osteoblastogenesis and suppress osteoclastogenesis in vitro. Previously, we showed that (-)-epigallocatechin-3-gallate (EGCG), one of the green tea polyphenols, increased osteogenic differentiation of murine bone marrow mesenchymal stem cells (BMSCs) by increasing the mRNA expression of osteogenesis-related genes, alkaline phosphatase activity and, eventually, mineralization. We also found that EGCG could mitigate bone loss and improve bone microarchitecture in ovariectomy-induced osteopenic rats, as well as enhancing bone defect healing partially via bone morphogenetic protein 2 (BMP2). The present study investigated the effects of EGCG in human BMSCs. We found that EGCG, at concentrations of both 1 and 10 µmol/L, can increase mRNA expression of BMP2, Runx2, alkaline phosphatase (ALP), osteonectin and osteocalcin 48 h after treatment. EGCG increased ALP activity both 7 and 14 days after treatment. Furthermore, EGCG can also enhance mineralization two weeks after treatment. EGCG without antioxidants also can enhance mineralization. In conclusion, EGCG can increase mRNA expression of BMP2 and subsequent osteogenic-related genes including Runx2, ALP, osteonectin and osteocalcin. EGCG further increased ALP activity and mineralization. Loss of antioxidant activity can still enhance mineralization of human BMSCs (hBMSCs).

Liposomal Encapsulation for Systemic Delivery of Propranolol via Transdermal Iontophoresis Improves Bone Microarchitecture in Ovariectomized Rats.

The stimulatory effects of liposomal propranolol (PRP) on proliferation and differentiation of human osteoblastic cells suggested that the prepared liposomes-encapsulated PRP exerts anabolic effects on bone in vivo. Iontophoresis provides merits such as sustained release of drugs and circumvention of first pass metabolism. This study further investigated and evaluated the anti-osteoporotic effects of liposomal PRP in ovariectomized (OVX) rats via iontophoresis. Rats subjected to OVX were administered with pure or liposomal PRP via iontophoresis or subcutaneous injection twice a week for 12 weeks. Changes in the microarchitecture at the proximal tibia and the fourth lumbar spine were assessed between pure or liposomal PRP treated and non-treated groups using micro-computed tomography. Administration of liposomal PRP at low dose (0.05 mg/kg) via iontophoresis over 2-fold elevated ratio between bone volume and total tissue volume (BV/TV) in proximal tibia to 9.0% whereas treatment with liposomal PRP at low and high (0.5 mg/kg) doses via subcutaneous injection resulted in smaller increases in BV/TV. Significant improvement of BV/TV and bone mineral density (BMD) was also found in the fourth lumbar spine when low-dose liposomal PRP was iontophoretically administered. Iontophoretic low-dose liposomal PRP also elevated trabecular numbers in tibia and trabecular thickness in spine. Enhancement of bone microarchitecture volumes has highlighted that liposomal formulation with transdermal iontophoresis is promising for PRP treatment at the lower dose and with longer duration than its clinical therapeutic range and duration to exhibit optimal effects against bone loss in vivo.

Suppression of discoidin domain receptor 1 expression enhances the chondrogenesis of adipose-derived stem cells.

Effectively directing the chondrogenesis of adipose-derived stem cells (ADSCs) to engineer articular cartilage represents an important challenge in ADSC-based articular cartilage tissue engineering. The discoidin domain receptor 1 (DDR1) has been shown to affect cartilage homeostasis; however, little is known about the roles of DDR1 in ADSC chondrogenesis. In this study, we used the three-dimensional culture pellet culture model system with chondrogenic induction to investigate the roles of DDR1 in the chondrogenic differentiation of human ADSCs (hADSCs). Real-time polymerase chain reaction and Western blot were used to detect the expression of DDRs and chondrogenic genes. Sulfated glycosaminoglycan (sGAG) was detected by Alcian blue and dimethylmethylene blue (DMMB) assays. Terminal deoxy-nucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining was used to assess cell death. During the chondrogenesis of hADSCs, the expression of DDR1 but not DDR2 was significantly elevated. The depletion of DDR1 expression in hADSCs using short hairpin RNA increased the expression of chondrogenic genes (SOX-9, collagen type II, and aggrecan) and cartilaginous matrix deposition (collagen type II and sGAG) and only slightly increased cell death (2-8%). DDR1 overexpression in hADSCs decreased the expression of chondrogenic genes (SOX-9, collagen type II, and aggrecan) and sGAG and enhanced hADSC survival. Moreover, DDR1-depleted hADSCs showed decreased expression of the terminal differentiation genes runt-related transcription factor 2 (Runx2) and matrix metalloproteinase 13 (MMP-13). These results suggest that DDR1 suppression may enhance ADSC chondrogenesis by enhancing the expression of chondrogenic genes and cartilaginous matrix deposition. We proposed that the suppression of DDR1 in ADSCs may be a candidate strategy of genetic modification to optimize ADSC-based articular cartilage tissue engineering.

Characterization and evaluation of the differentiation ability of human adipose-derived stem cells growing in scaffold-free suspension culture.

BACKGROUND AIMS: Human adipose-derived stem cells (hADSCs) have become a popular stem cell source because of their abundant supplies, high differentiation ability and the fact that they present few ethical concerns. Suspension culture, a type of three-dimensional culture, is a more suitable model for mimicking cell-cell and cell-extracellular matrix interactions than is two-dimensional monolayer culture. The aim of this study was to determine the effects of suspension culture on the viability and differentiation potential of hADSCs. METHODS: Different densities of hADSCs were cultured in ultra-low-attachment surface plates. The morphology and mean diameter of the resultant aggregates were determined by means of microscopy. The viability of the aggregates was evaluated with the use of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt, lactate dehydrogenase and live/dead assays. To detect osteogenesis, chondrogenesis and adipogenesis in hADSCs in suspension culture, cell aggregates were stained to determine cell function, and the expression of specific markers was evaluated through the use of real-time reverse transcriptase-polymerase chain reaction. RESULTS: The hADSCs remained viable in suspension culture and formed cell aggregates. The diameter of the majority of the aggregates was in the range of 50-200 µm, regardless of cell density. The aggregation of the hADSCs served to maintain cell survival. In addition, the results of the histomorphometric and gene expression analyses showed that the hADSCs were more efficiently induced to differentiate into osteoblasts, chondrocytes and adipocytes in suspension culture than in two-dimensional monolayer culture. CONCLUSIONS: Suspension culture can be used to maintain cell viability and contributes to the effective differentiation of hADSCs, providing an alternative cell growth strategy for application to stem cell-based regenerative medicine.

PPARγ silencing enhances osteogenic differentiation of human adipose-derived mesenchymal stem cells.

Peroxisome proliferator-activated receptor gamma (PPARγ) is the master regulator of adipogenesis, and has been indicated as a potential therapeutic target to promote osteoblast differentiation. However, recent studies suggest that suppression of PPARγ inhibits adipogenesis, but does not promote osteogenic differentiation in human bone marrow-derived mesenchymal stem cells (hBMSCs). It was reasoned that the osteogenic effect of PPARγ suppression may be masked by the strong osteogenesis-inducing condition commonly used, resulting in a high degree of matrix mineralization in both control and experimental groups. This study investigates the role of PPARγ in the lineage commitment of human adipose-derived mesenchymal stem cells (hADSCs) by interfering with the function of PPARγ mRNA through small interfering RNAs (siRNAs) specific for PPARγ2. By applying an osteogenic induction condition less potent than that used conventionally, we found that PPARγ silencing led to retardation of adipogenesis and stimulated a higher level of matrix mineralization. The mRNA level of PPARγ decreased to 47% of control 2 days after treatment with 50 nmol/l PPARγ2 siRNA, while its protein expression was 60% of mock control. In the meantime, osteogenic marker genes, including bone morphogenic protein 2 (BMP2), runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP) and osteocalcin (OC), were up-regulated under PPARγ silencing. Our results suggest that transient suppression of PPARγ promotes the onset of osteogenesis, and may be considered a new strategy to stimulate bone formation in bone tissue engineering using hADSCs.

Anterior cruciate ligament remnant preservation attenuates apoptosis and enhances the regeneration of hamstring tendon graft.

AIMS: The effects of remnant preservation on the anterior cruciate ligament (ACL) and its relationship with the tendon graft remain unclear. We hypothesized that the co-culture of remnant cells and bone marrow stromal cells (BMSCs) decreases apoptosis and enhances the activity of the hamstring tendons and tenocytes, thus aiding ACL reconstruction. METHODS: The ACL remnant, bone marrow, and hamstring tendons were surgically harvested from rabbits. The apoptosis rate, cell proliferation, and expression of types I and III collagen, transforming growth factor-ß (TGF-ß), vascular endothelial growth factor (VEGF), and tenogenic genes (scleraxis (SCX), tenascin C (TNC), and tenomodulin (TNMD)) of the hamstring tendons were compared between the co-culture medium (ACL remnant cells (ACLRCs) and BMSCs co-culture) and control medium (BMSCs-only culture). We also evaluated the apoptosis, cell proliferation, migration, and gene expression of hamstring tenocytes with exposure to co-culture and control media. RESULTS: Compared to BMSCs-only culture medium, the co-culture medium showed substantially decreased early and late apoptosis rates, attenuation of intrinsic and extrinsic apoptotic pathways, and enhanced proliferation of the hamstring tendons and tenocytes. In addition, the expression of collagen synthesis, TGF-ß, VEGF, and tenogenic genes in the hamstring tendons and tenocytes significantly increased in the co-culture medium compared to that in the control medium. CONCLUSION: In the presence of ACLRCs and BMSCs, the hamstring tendons and tenocytes significantly attenuated apoptosis and enhanced the expression of collagen synthesis, TGF-ß, VEGF, and tenogenic genes. This in vitro study suggests that the ACLRCs mixed with BMSCs could aid regeneration of the hamstring tendon graft during ACL reconstruction.Cite this article: Bone Joint Res 2023;12(1):9-21.

Proliferation and differentiation potential of human adipose-derived mesenchymal stem cells isolated from elderly patients with osteoporotic fractures.

Aging has less effect on adipose-derived mesenchymal stem cells (ADSCs) than on bone marrow-derived mesenchymal stem cells (BMSCs), but whether the fact holds true in stem cells from elderly patients with osteoporotic fractures is unknown. In this study, ADSCs and BMSCs of the same donor were harvested and divided into two age groups. Group A consisted of 14 young patients (36.4 ± 11.8 years old), and group B consisted of eight elderly patients (71.4 ± 3.6 years old) with osteoporotic fractures. We found that the doubling time of ADSCs from both age groups was maintained below 70 hrs, while that of BMSCs increased significantly with the number of passage. When ADSCs and BMSCs from the same patient were compared, there was a significant increase in the doubling time of BMSCs in each individual from passages 3 to 6. On osteogenic induction, the level of matrix mineralization of ADSCs from group B was comparable to that of ADSCs from group A, whereas BMSCs from group B produced least amount of mineral deposits and had a lower expression level of osteogenic genes. The p21 gene expression and senescence-associated ß-galactosidase activity were lower in ADSCs compared to BMSCs, which may be partly responsible for the greater proliferation and differentiation potential of ADSCs. It is concluded that the proliferation and osteogenic differentiation of ADSCs were less affected by age and multiple passage than BMSCs, suggesting that ADSCs may become a potentially effective therapeutic option for cell-based therapy, especially in elderly patients with osteoporosis.

Enhancement of Osteoblast Function through Extracellular Vesicles Derived from Adipose-Derived Stem Cells.

Adipose-derived stem cells (ADSCs) are a type of mesenchymal stem cell that is investigated in bone tissue engineering (BTE). Osteoblasts are the main cells responsible for bone formation in vivo and directing ADSCs to form osteoblasts through osteogenesis is a research topic in BTE. In addition to the osteogenesis of ADSCs into osteoblasts, the crosstalk of ADSCs with osteoblasts through the secretion of extracellular vesicles (EVs) may also contribute to bone formation in ADSC-based BTE. We investigated the effect of ADSC-secreted EVs (ADSC-EVs) on osteoblast function. ADSC-EVs (size ≤ 1000 nm) were isolated from the culture supernatant of ADSCs through ultracentrifugation. The ADSC-EVs were observed to be spherical under a transmission electron microscope. The ADSC-EVs were positive for CD9, CD81, and Alix, but ß-actin was not detected. ADSC-EV treatment did not change survival but did increase osteoblast proliferation and activity. The 48 most abundant known microRNAs (miRNAs) identified within the ADSC-EVs were selected and then subjected to gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. The GO analysis revealed that these miRNAs are highly relevant to skeletal system morphogenesis and bone development. The KEGG analysis indicated that these miRNAs may regulate osteoblast function through autophagy or the mitogen-activated protein kinase or Ras-related protein 1 signaling pathway. These results suggest that ADSC-EVs enhance osteoblast function and can contribute to bone regeneration in ADSC-based BTE.