Rapid human-derived iPSC osteogenesis combined with three-dimensionally printed Ti6Al4V scaffolds for the repair of bone defects

Human-induced pluripotent stem cells (iPSCs) are an alternative source of mesenchymal stem cells used for bone regeneration. However, the current osteogenically induced methods for iPSCs are slow and complex. We have used retinoic acid (RA) to induce osteogenic iPSCs within 10 days and assess whether a rapid differentiation could improve the osteogenic potential of the three-dimensionally printed Ti6Al4V (3DTi) scaffolds. First, the osteogenic differentiation of iPSCs was induced with RA, and the osteogenic potential of iPSCs was evaluated using standard assays. In addition, a 5-mm mandibular bone defect was generated in rats and was repaired with 3DTi scaffolds that were seeded with iPSC-induced osteoblasts. The capacity of seeded scaffolds for the enhancement of bone regeneration in vivo was assessed. Finally, we tested the potential mechanisms of RA-dependent iPSC bone induction and its effect on the Wnt/β-catenin pathway. The results showed that iPSCs could form osteocytes within 10 days. Animal experiments confirmed that rapid osteo-induced iPSCs could enhance the bone regeneration and osteointegration capacity of the 3DTi scaffolds. Mechanistically, RA could activate the AKT/GSK3β/β-catenin pathway during the process of iPSCs osteogenesis. The rapid osteoinduction of iPSCs combined with 3DTi scaffolds is a safe, effective, and reproducible method for repairing mandibular bone defects.     

CircRNA-vgll3 promotes osteogenic differentiation of adipose-derived mesenchymal stem cells via modulating miRNA-dependent integrin α5 expression

Adipose-derived mesenchymal stem cells (ADSCs) are promising candidate for regenerative medicine to repair non-healing bone defects due to their high and easy availability. However, the limited osteogenic differentiation potential greatly hinders the clinical application of ADSCs in bone repair. Accumulating evidences demonstrate that circular RNAs (circRNAs) are involved in stem/progenitor cell fate determination, but their specific role in stem/progenitor cell osteogenesis, remains mostly undescribed. Here, we show that circRNA-vgll3 originating from the vgll3 locus markedly enhances osteogenic differentiation of ADSCs; nevertheless, silencing of circRNA-vgll3 dramatically attenuates ADSC osteogenesis. Furthermore, we validate that circRNA-vgll3 functions in ADSC osteogenesis through a circRNA-vgll3/miR-326-5p/integrin α5 (Itga5) pathway. Itga5 promotes ADSC osteogenic differentiation and miR-326-5p suppresses Itga5 translation. CircRNA-vgll3 directly sequesters miR-326-5p in the cytoplasm and inhibits its activity to promote osteogenic differentiation. Moreover, the therapeutic potential of circRNA-vgll3-modified ADSCs with calcium phosphate cement (CPC) scaffolds was systematically evaluated in a critical-sized defect model in rats. Our results demonstrate that circRNA-vgll3 markedly enhances new bone formation with upregulated bone mineral density, bone volume/tissue volume, trabeculae number, and increased new bone generation. This study reveals the important role of circRNA-vgll3 during new bone biogenesis. Thus, circRNA-vgll3 engineered ADSCs may be effective potential therapeutic targets for bone regenerative medicine.Subject terms: Epigenetics, Stem-cell research     

Cell kinetics,DNA integrity,differentiation, and lipid fingerprinting analysis of rabbit adipose-derived stem cells

Human adipose tissue has been described as a potential alternative reservoir for stem cells. Although studies have been performed in rabbits using autologous adipose-derived stem cells (ADSC), these cells have not been well characterized. The primary objectives of this study were to demonstrate the presence of adipose-derived stem cells isolated from rabbit inguinal fat pads and to characterize them through osteogenic and adipogenic in vitro differentiation and lipid fingerprinting analysis. The secondary objective was to evaluate cell behavior through growth kinetics, cell viability, and DNA integrity. Rabbit ADSCs were isolated to determine the in vitro growth kinetics and cell viability. DNA integrity was assessed by an alkaline Comet assay in passages 0 and 5. The osteogenic differentiation was evaluated by Von Kossa, and Alizarin Red S staining and adipogenic differentiation were assessed by Oil Red O staining. Lipid fingerprinting analyses of control, adipogenic, and osteogenic differentiated cells were performed by MALDI-TOF/MS. We demonstrate that rabbit ADSC have a constant growth rate at the early passages, with increased DNA fragmentation at or after passage 5. Rabbit ADSC viability was similar in passages 2 and 5 (90.7% and 86.6%, respectively), but there was a tendency to decreased cellular growth rate after passage 3. The ADSC were characterized by the expression of surface markers such as CD29 (67.4%) and CD44 (89.4%), using CD 45 (0.77%) as a negative control. ADSC from rabbits were successfully isolated form the inguinal region. These cells were capable to differentiate into osteogenic and adipogenic tissue when they were placed in inductive media. After each passage, there was a trend towards decreased cell growth. On the other hand, DNA fragmentation increased at each passage. ADSC had a different lipid profile when placed in control, adipogenic, or osteogenic media.     

miR-21 regulates osteogenic and adipogenic differentiation of BMSCs by targeting PTEN

Objective:To explore the effects and mechanism of miR-21 on the osteogenic/adipogenic differentiation of mouse BMSCs.Methods:The bilateral ovaries of C57BL/6J mice (n=24) were removed to construct an osteoporosis model. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-21, osteogenic/adipogenic genes, and PTEN. ALP and ARS and ORO staining were used to detect the formation of calcium nodules and lipid droplets in BMSCs. Western blot was used to detect the expression of PTEN.Results:miR-21 was significantly down-regulated in osteoporotic mice. The expression of miR-21 was significantly up-regulated after the osteogenic induction of BMSCs, and the expression of miR-21 was significantly down-regulated after the adipogenic induction. Overexpression of miR-21 significantly promoted the osteogenic differentiation of BMSCs and inhibits the adipogenic differentiation of BMSCs.Conclusion:MiR-21 can promote osteogenic differentiation of BMSCs and inhibit their adipogenic differentiation by negatively regulating PTEN.     

Articular cartilage-derived cells hold a strong osteogenic differentiation potential in comparison to mesenchymal stem cells in vitro

Cartilaginous matrix-degenerative diseases like osteoarthritis (OA) are characterized by gradual cartilage erosion, and also by increased presence of cells with mesenchymal stem cell (MSC) character within the affected tissues. Moreover, primary chondrocytes long since are known to de-differentiate in vitro and to be chondrogenically re-differentiable. Since both findings appear to conflict with each other, we quantitatively assessed the mesenchymal differentiation potential of OA patient cartilage-derived cells (CDC) towards the osteogenic and adipogenic lineage in vitro and compared it to that of MSC isolated from adipose tissue (adMSC) of healthy donors. We analyzed expression of MSC markers CD29, CD44, CD105, and CD166, and, following osteogenic and adipogenic induction in vitro, quantified their expression of osteogenic and adipogenic differentiation markers. Furthermore, CDC phenotype and proliferation were monitored. We found that CDC exhibit an MSC CD marker expression pattern similar to adMSC and a similar increase in proliferation rate during osteogenic differentiation. In contrast, the marked reduction of proliferation observed during adipogenic differentiation of adMSC was absent in CDC. Quantification of differentiation markers revealed a strong osteogenic differentiation potential for CDC, however almost no capacity for adipogenic differentiation. Since in the pathogenesis of OA, cartilage degeneration coincides with high bone turnover rates, the high osteogenic differentiation potential of OA patient-derived CDC may affect clinical therapeutic regimens aiming at autologous cartilage regeneration in these patients.     

The Effect of 3D Nanofibrous Scaffolds on the Chondrogenesis of Induced Pluripotent Stem Cells and Their Application in Restoration of Cartilage Defects

The discovery of induced pluripotent stem cells (iPSCs) rendered the reprogramming of terminally differentiated cells to primary stem cells with pluripotency possible and provided potential for the regeneration and restoration of cartilage defect. Chondrogenic differentiation of iPSCs is crucial for their application in cartilage tissue engineering. In this study we investigated the effect of 3D nanofibrous scaffolds on the chondrogenesis of iPSCs and articular cartilage defect restoration. Super-hydrophilic and durable mechanic polycaprolactone (PCL)/gelatin scaffolds were fabricated using two separate electrospinning processes. The morphological structure and mechanical properties of the scaffolds were characterized. The chondrogenesis of the iPSCs in vitro and the restoration of the cartilage defect was investigated using scanning electron microscopy (SEM), the Cell Counting Kit-8 (CCK-8), histological observation, RT-qPCR, and western blot analysis. iPSCs on the scaffolds expressed higher levels of chondrogenic markers than the control group. In an animal model, cartilage defects implanted with the scaffold-cell complex exhibited an enhanced gross appearance and histological improvements, higher cartilage-specific gene expression and protein levels, as well as subchondral bone regeneration. Therefore, we showed scaffolds with a 3D nanofibrous structure enhanced the chondrogenesis of iPSCs and that iPSC-containing scaffolds improved the restoration of cartilage defects to a greater degree than did scaffolds alone in vivo.     

Impact of bacteria and bacterial components on osteogenic and adipogenic differentiation of adipose-derived mesenchymal stem cells

Adult mesenchymal stem cells (MSC) are present in several tissues, e.g. bone marrow, heart muscle, brain and subcutaneous adipose tissue. In invasive infections MSC get in contact with bacteria and bacterial components. Not much is known about how bacterial pathogens interact with MSC and how contact to bacteria influences MSC viability and differentiation potential. In this study we investigated the impact of three different wound infection relevant bacteria, Escherichia coli, Staphylococcus aureus, and Streptococcus pyogenes, and the cell wall components lipopolysaccharide (LPS; Gram-negative bacteria) and lipoteichoic acid (LTA; Gram-positive bacteria) on viability, proliferation, and osteogenic as well as adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells (adMSC). We show that all three tested species were able to attach to and internalize into adMSC. The heat-inactivated Gram-negative E. coli as well as LPS were able to induce proliferation and osteogenic differentiation but reduce adipogenic differentiation of adMSC. Conspicuously, the heat-inactivated Gram-positive species showed the same effects on proliferation and adipogenic differentiation, while its cell wall component LTA exhibited no significant impact on adMSC. Therefore, our data demonstrate that osteogenic and adipogenic differentiation of adMSC is influenced in an oppositional fashion by bacterial antigens and that MSC-governed regeneration is not necessarily reduced under infectious conditions.     

Incorporated-bFGF polycaprolactone/polyvinylidene fluoride nanocomposite scaffold promotes human induced pluripotent stem cells osteogenic differentiation

Bioactive scaffolds that can increase transplanted cell survival time at the defect site have a great promising potential to use clinically since tissue regeneration or secretions crucially depend on the transplanted cell survival. In this study embedded basic fibroblast growth factor (bFGF)-polycaprolactone-polyvinylidene fluoride (PCL-PVDF) hybrid was designed and fabricated by electrospinning as a bio-functional nanofibrous scaffold for bone tissue engineering. After morphological characterization of the PCL-PVDF (bFGF) scaffold, nanofibers biocompatibility was investigated by culturing of the human induced pluripotent stem cells (iPSCs). Then, the bone differentiation capacity of the iPSCs was evaluated when grown on the PCL-PVDF and PCL-PVDF (bFGF) scaffolds in comparison with culture plate as a control using evaluating of the common osteogenic markers. The viability assay displayed a significant increase in iPSCs survival rate when grown on the bFGF content scaffold. The highest alkaline phosphatase activity and mineralization were detected in the iPSCs while grown on the PCL-PVDF (bFGF) scaffolds. Obtained results from gene and protein expression were also demonstrated the higher osteoinductive property of the bFGF content scaffold compared with the scaffold without it. According to the results, the release of bFGF from PCL-PVDF nanofibers increased survival and proliferation rate of the iPSCs, which followed by an increase in its osteogenic differentiation potential. Taking together, PCL-PVDF (bFGF) nanofibrous scaffold demonstrated that can be noted as a promising candidate for treating the bone lesions by tissue engineering products.     

SDF-1 Enhances Wound Healing of Critical-Sized Calvarial Defects beyond Self-Repair Capacity

Host blood circulating stem cells are an important cell source that participates in the repair of damaged tissues. The clinical challenge is how to improve the recruitment of circulating stem cells into the local wound area and enhance tissue regeneration. Stromal-derived factor-1 (SDF-1) has been shown to be a potent chemoattractant of blood circulating stem cells into the local wound microenvironment. In order to investigate effects of SDF-1 on bone development and the repair of a large bone defect beyond host self-repair capacity, the BMP-induced subcutaneous ectopic bone formation and calvarial critical-sized defect murine models were used in this preclinical study. A dose escalation of SDF-1 were loaded into collagen scaffolds containing BMP, VEGF, or PDGF, and implanted into subcutaneous sites at mouse dorsa or calvarial critical-sized bone defects for 2 and 4 weeks. The harvested biopsies were examined by microCT and histology. The results demonstrated that while SDF-1 had no effect in the ectopic bone model in promoting de novo osteogenesis, however, in the orthotopic bone model of the critical-sized defects, SDF-1 enhanced calvarial critical-sized bone defect healing similar to VEGF, and PDGF. These results suggest that SDF-1 plays a role in the repair of large critical-sized defect where more cells are needed while not impacting de novo bone formation, which may be associated with the functions of SDF-1 on circulating stem cell recruitment and angiogenesis.     

Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate

Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. Adipose-derived stromal cells (ASCs) have proven to be an abundant source of multipotent stem cells capable of undergoing osteogenic, chondrogenic, adipogenic, and myogenic differentiation. Many studies have explored the osteogenic potential of these cells in vivo with the use of various scaffolding biomaterials for cellular delivery. It has been demonstrated that by utilizing an osteoconductive, hydroxyapatite-coated poly(lactic-co-glycolic acid) (HA-PLGA) scaffold seeded with ASCs, a critical-sized calvarial defect, a defect that is defined by its inability to undergo spontaneous healing over the lifetime of the animal, can be effectively show robust osseous regeneration. This in vivo model demonstrates the basis of translational approaches aimed to regenerate the bone tissue - the cellular component and biological matrix. This method serves as a model for the ultimate clinical application of a progenitor cell towards the repair of a specific tissue defect.     

Comparative evaluation of in vivo osteogenic differentiation of fetal and adult mesenchymal stem cell in rat critical-sized femoral defect model

Mesenchymal stem cells (MSCs) can be obtained from various sources. MSCs from different origins appear to have different preferences for differentiation. In this study, we have compared the in vivo osteogenic potential of adult MSCs from adipose tissue (AT) and bone marrow (BM) with fetal MSCs from umbilical cord (UC) and umbilical cord blood (UCB) by using a rat critical-sized femoral defect model. We have also sought to determine whether pretreatment with an osteogenic medium promotes osteogenesis in MSCs. Study groups were divided as follows: (1) defect only, (2) scaffold only, (3) AT MSCs in scaffolds, (4) BM MSCs in scaffolds, (5) UC MSCs in scaffolds and (6) UCB MSCs in scaffolds. Groups with MSCs were further divided with respect to their pretreatment. At 12 weeks after surgery, in vivo osteogenesis was measured radiographically and by micro-computed tomography (CT). Based on quantitative assessment by micro-CT, no significant difference of the mean bone volume fraction value (BV/TV) was seen between adult MSCs (AT and BM MSCs) and fetal MSCs (UC and UCB MSCs). The mean BV/TVs were significantly higher in non-pretreated BM MSC (14.2±1.4%) and UCB MSC (14.0±1.2%) and pretreated UC MSC (14.8±2.0%) than in those with the scaffold only (11.3±1.3%; P<0.05). In addition, AT (from 10.4±1.2% to 13.1±2.2%) and UC (from 10.3±0.7% to 14.8±2.0%) MSCs from solid tissues showed a significant increase in the mean BV/TV with pretreatment (P<0.05). In contrast, BM MSC (from 14.2±1.4% to 10.9±1.2%) and UCB MSC (from 14.0±1.2% to 11.6±1.0%) from non-solid tissues showed a significant decrease with pretreatment (P<0.05).     

The frequency, growth kinetics, and osteogenic/adipogenic differentiation properties of canine bone marrow stromal cells

Bone marrow stromal cells (BMSCs) have gained considerable attention as a potential source for cell transplantation therapies for a variety of diseases due to their accessibility, proliferative capacity, and multilineage differentiation properties. Canine BMSCs have been shown to contribute to regeneration of osseous tissues, but knowledge about their biology is currently limited. In the present study, we investigated the frequency of adult canine BMSCs in bone marrow, morphological features, growth kinetics, and osteogenic as well as adipogenic differentiation properties in vitro. Our data suggest that adult canine bone marrow contains approximately one BMSC in every 2.38 × 10⁴ bone marrow mononucleated cells (0.0042 ± 0.0019%, n = 5). Primary BMSC cultures consisted of morphologically heterogeneous adherent cell populations from which spindle-shaped cells grew and became the predominant cell type. Growth kinetics patterns were dependent on the initial cell seeding densities, resulting in the highest fold increase at lower cell density. In the presence of osteogenic and adipogenic inducers, primary BMSCs underwent morphological and phenotypic changes characteristic of osteogenic and adipogenic differentiation, respectively. This study provides insights into basic characterization of adult canine BMSCs.     

Nuclear receptor profile in calvarial bone cells undergoing osteogenic versus adipogenic differentiation

Nuclear receptors (NRs) are key regulators of cell function and differentiation. We examined NR expression during osteogenic versus adipogenic differentiation of primary mouse calvarial osteoblasts (MOBs). MOBs were cultured for 21 days in osteogenic or adipogenic differentiation media. von Kossa and Oil Red O staining, and qRT-PCR of marker genes and 49 NRs were performed. PCR amplicons were subcloned to establish correct sequences and absolute standard curves. Forty-three NRs were detected at days 0-21. Uncentered average linkage hierarchical clustering identified four expression clusters: NRs (1) upregulated during osteogenic, but not adipogenic, differentiation, (2) upregulated in both conditions, with greater upregulation during adipogenic differentiation, (3) upregulated equally in both conditions, (4) downregulated during adipogenic, but not osteogenic, differentiation. One-way ANOVA with contrast revealed 20 NRs upregulated during osteogenic differentiation and 12 NRs upregulated during adipogenic differentiation. Two-way ANOVA demonstrated that 18 NRs were higher in osteogenic media, while 9 NRs were higher in adipogenic media. The time effect revealed 16 upregulated NRs. The interaction of condition with time revealed 6 NRs with higher expression rate during adipogenic differentiation and 3 NRs with higher expression rate during osteogenic differentiation. Relative NR abundance at days 0 and 21 were ranked. Basal ranking changed at least 5 positions for 13 NRs in osteogenic media and 9 NRs in adipogenic media. Osteogenic and adipogenic differentiation significantly altered NR expression in MOBs. These differences offer a fingerprint of cellular commitment and may provide clues to the underlying mechanisms of osteogenic versus adipogenic differentiation.     

Phenotypic characterization of craniofacial bone marrow stromal cells: unique properties of enhanced osteogenesis,cell recruitment,autophagy, and apoptosis resistance

Previous studies have shown that craniofacial bone marrow stromal cells (MSCs) have greater osteogenic potential than appendicular bone MSCs. However, detailed phenotypic characterization of MSCs from bone marrow in the different sites remains unclear. To investigate bone repair and regeneration of craniofacial MSCs and the regulatory mechanisms underlying their unique properties, we compared osteogenesis, cell recruitment, autophagy, and apoptosis resistance of MSCs from the mandible (M-MSCs) to those from tibia (T-MSCs) in vitro and in vivo. Compared with T-MSCs, M-MSCs formed more colonies, possessed stronger proliferation activity, exhibited higher expression of pluripotency genes such as Oct4 and Nanog, and held stronger osteogenic differentiation in osteogenic medium. Moreover, M-MSCs had greater autophagy and anti-apoptotic capacities than T-MSCs under hypoxia and serum deprivation conditions. M-MSCs were found to be more capable of recruiting more MSCs than T-MSCs. When these MSCs were transplanted into mandible critical-sized defects, more bone formed in the M-MSC-treated animals than in their T-MSC counterparts. Collectively, these findings reveal that MSCs have unique characteristics and bone-repairing properties from the mandible as compared with those from tibia, presumably by enhanced osteogenic potential, cell recruitment, autophagy and apoptosis resistance.     

Canonical Wnt signaling differently modulates osteogenic differentiation of mesenchymal stem cells derived from bone marrow and from periodontal ligament under inflammatory conditions

Background

Cellular plasticity and complex functional requirements of the periodontal ligament (PDL) assume a local stem cell (SC) niche to maintain tissue homeostasis and repair. Here, pathological alterations caused by inflammatory insults might impact the regenerative capacities of these cells. As bone homeostasis is fundamentally controlled by Wnt-mediated signals, it was the aim of this study to characterize the SC-like capacities of cells derived from PDL and to investigate their involvement in bone pathophysiology especially regarding the canonical Wnt pathway.

Methods

PDLSCs were investigated for their SC characteristics via analysis of cell surface marker expression, colony forming unit efficiency, proliferation, osteogenic differentiation and adipogenic differentiation, and compared to bone marrow derived mesenchymal SCs (BMMSCs). To determine the impact of both inflammation and the canonical Wnt pathway on osteogenic differentiation, cells were challenged with TNF-α, maintained with or without Wnt3a or DKK-1 under osteogenic induction conditions and investigated for p-IκBα, p-NF-κB, p-Akt, β-catenin, p-GSK-3β, ALP and Runx2.

Results

PDLSCs exhibit weaker adipogenic and osteogenic differentiation capacities compared to BMMSCs. TNF-α inhibited osteogenic differentiation of PDLSCs more than BMMSCs mainly through regulating canonical Wnt pathway. Blocking the canonical Wnt pathway by DKK-1 reconstituted osteogenic differentiation of PDLSCs under inflammatory conditions, whereas activation by Wnt3a increased osteogenic differentiation of BMMSCs.

Conclusions

Our results suggest a diverse regulation of the inhibitory effect of TNF-α in BMMSCs and PDLSCs via canonical Wnt pathway modulation.

General significance

These findings provide novel insights on PDLSC SC-like capacities and their involvement in bone pathophysiology under the impact of the canonical Wnt pathway.