scholarly journals Effect of Pulse Frequency on the Osteogenic Differentiation of Mesenchymal Stem Cells in a Pulsatile Perfusion Bioreactor

2011 ◽  
Vol 133 (9) ◽  
Author(s):  
Katherine D. Kavlock ◽  
Aaron S. Goldstein
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Three Dimensional ◽  
Intermediate Frequency ◽  
Osteoblastic Differentiation ◽  
Protein Accumulation ◽  
Flow Conditions ◽  
Alp Activity ◽  
Static Conditions

Perfusion bioreactors are a promising in vitro strategy to engineer bone tissue because they supply needed oxygen and nutrients and apply an osteoinductive mechanical stimulus to osteoblasts within large porous three-dimensional scaffolds. Model two-dimensional studies have shown that dynamic flow conditions (e.g., pulsatile oscillatory waveforms) elicit an enhanced mechanotransductive response and elevated expression of osteoblastic proteins relative to steady flow. However, dynamic perfusion of three-dimensional scaffolds has been primarily examined in short term cultures to probe for early markers of mechanotransduction. Therefore, the objective of this study was to investigate the effect of extended dynamic perfusion culture on osteoblastic differentiation of primary mesenchymal stem cells (MSCs). To accomplish this, rat bone marrow-derived MSCs were seeded into porous foam scaffolds and cultured for 15 days in osteogenic medium under pulsatile regimens of 0.083, 0.050, and 0.017 Hz. Concurrently, MSCs seeded in scaffolds were also maintained under static conditions or cultured under steady perfusion. Analysis of the cells after 15 days of culture indicated that alkaline phosphatase (ALP) activity, mRNA expression of osteopontin (OPN), and accumulation of OPN and prostaglandin E2 were enhanced for all four perfusion conditions relative to static culture. ALP activity, OPN and OC mRNA, and OPN protein accumulation were slightly higher for the intermediate frequency (0.05 Hz) as compared with the other flow conditions, but the differences were not statistically significant. Nevertheless, these results demonstrate that dynamic perfusion of MSCs may be a useful strategy for stimulating osteoblastic differentiation in vitro.

scholarly journals Comparison of Immunosuppressive and Angiogenic Properties of Human Amnion-Derived Mesenchymal Stem Cells between 2D and 3D Culture Systems

2019 ◽  
Vol 2019 ◽  
pp. 1-16 ◽  
Author(s):  
Vitale Miceli ◽  
Mariangela Pampalone ◽  
Serena Vella ◽  
Anna Paola Carreca ◽  
Giandomenico Amico ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Three Dimensional ◽  
3D Culture ◽  
Culture Method ◽  
Paracrine Factors ◽  
Human Amnion ◽  
Tissue Repair And Regeneration ◽  
3D Culture System

The secretion of potential therapeutic factors by mesenchymal stem cells (MSCs) has aroused much interest given the benefits that it can bring in the field of regenerative medicine. Indeed, the in vitro multipotency of these cells and the secretive capacity of both angiogenic and immunomodulatory factors suggest a role in tissue repair and regeneration. However, during culture, MSCs rapidly lose the expression of key transcription factors associated with multipotency and self-renewal, as well as the ability to produce functional paracrine factors. In our study, we show that a three-dimensional (3D) culture method is effective to induce MSC spheroid formation, to maintain the multipotency and to improve the paracrine activity of a specific population of human amnion-derived MSCs (hAMSCs). The regenerative potential of both 3D culture-derived conditioned medium (3D CM) and their exosomes (EXO) was assessed against 2D culture products. In particular, tubulogenesis assays revealed increased capillary maturation in the presence of 3D CM compared with both 2D CM and 2D EXO. Furthermore, 3D CM had a greater effect on inhibition of PBMC proliferation than both 2D CM and 2D EXO. To support this data, hAMSC spheroids kept in our 3D culture system remained viable and multipotent and secreted considerable amounts of both angiogenic and immunosuppressive factors, which were detected at lower levels in 2D cultures. This work reveals the placenta as an important source of MSCs that can be used for eventual clinical applications as cell-free therapies.

scholarly journals Three-Dimensional Aggregates Enhance the Therapeutic Effects of Adipose Mesenchymal Stem Cells for Ischemia-Reperfusion Induced Kidney Injury in Rats

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Xiaozhi Zhao ◽  
Xuefeng Qiu ◽  
Yanting Zhang ◽  
Shiwei Zhang ◽  
Xiaoping Gu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Ischemia Reperfusion ◽  
Three Dimensional ◽  
Kidney Injury ◽  
Therapeutic Effects ◽  
Adipose Mesenchymal Stem Cells ◽  
In Vitro Data

It has been shown that administration of adipose derived mesenchymal stem cells (AdMSCs) enhanced structural and functional recovery of renal ischemia-reperfusion (IR) injury. Low engraftment of stem cells, however, limits the therapeutic effects of AdMSCs. The present study was designed to enhance the therapeutic effects of AdMSCs by delivering AdMSCs in a three-dimensional (3D) aggregates form. Microwell was used to produce 3D AdMSCs aggregates. In vitro data indicated that AdMSCs in 3D aggregates were less susceptible to oxidative and hypoxia stress induced by 200 μM peroxide and hypoxia/reoxygenation, respectively, compared with those cultured in two-dimensional (2D) monolayer. Furthermore, AdMSCs in 3D aggregates secreted more proangiogenic factors than those cultured in 2D monolayer. 2D AdMSCs or 3D AdMSCs aggregates were injected into renal cortex immediately after induction of renal IR injury. In vivo data revealed that 3D aggregates enhanced the effects of AdMSCs in recovering function and structure after renal IR injury. Improved grafted AdMSCs were observed in kidney injected with 3D aggregates compared with AdMSCs cultured in 2D monolayer. Our results demonstrated that 3D AdMSCs aggregated produced by microwell enhanced the retention and therapeutic effects of AdMSCs for renal IR injury.

scholarly journals Dihydrotestosterone and 17-Estradiol Enhancement of In Vitro Osteogenic Differentiation of Castrated Male Rat Bone Marrow Mesenchymal Stem Cells (rBMMSCs)

2019 ◽  
Author(s):  
FAM Abo-Aziza ◽  
AA Zaki ◽  
AS Amer ◽  
RA Lotfy
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Population Doubling ◽  
Calcium Deposition ◽  
Alp Activity ◽  
Marrow Mesenchymal Stem Cells ◽  
Rat Bone

Background: In vitro impact of dihydrotestosterone (DHT) and 17-estradiol (E2) in osteogenic differentiation of castrated rat bone marrow mesenchymal stem cells (rBMMSC) still need to be clarified. Materials and Methods: The viability, proliferation and density of cultured rBMMSC isolated from sham operated (Sham) and castrated (Cast) male rats were evaluated. rBMMSC were cultured with osteogenic differentiating medium (ODM) in the presence of DHT (5,10 nM) and E2 (10,100 nM). Osteogenesis was evaluated by alizarin red staining and measurement of calcium deposition and bone alkaline phosphatase (BALP) activity. Results: Population doubling (PD) of rBMMSC isolated from Cast rats was significantly lower (P<0.05) compared to that isolated from Sham rats. rBMMSC from Cast rats showed low scattered calcified nodule after culturing in ODM and did not cause a significant increase in calcium deposition and B-ALP activity compared to rBMMSCs from Sham rats. Exposure of rBMMSC isolated from Cast rats to DHT (5 nM) or E2 (10 nM) in ODM showed medium scattered calcified nodules with significantly higher (P<0.05) calcium deposition and B-ALP activity. Moreover, exposure of rBMMSC to DHT (10 nM) or E2 (100 nM) showed high scattered calcified nodules with higher (P<0.01) calcium deposition and B-ALP activity Conclusion: These results indicated that the presence of testes might participate in controlling the in vitro proliferation and osteogenic differentiation capacity of rBMMSCs. DHT and E2 can enhance the osteogenic capacity of rBMMSCs in a dose-dependent manner. Based on these observations, optimum usage of DHT and E2 can overcome the limitations of MSCs and advance the therapeutic bone regeneration potential in the future.

Impaired Osteogenic Differentiation of Mesenchymal Stem Cells Derived From Multiple Myeloma Patients Is Associated with a Failure In the Deactivation of the NOTCH Pathway

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1894-1894
Author(s):  
Song Xu ◽  
Jinsong Hu ◽  
Dehui Xu ◽  
Isabelle Vande Broek ◽  
Xavier Leleu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Notch Pathway ◽  
Notch Signalling ◽  
Matrix Mineralization ◽  
Alp Activity ◽  
Hes1 Expression

Abstract Abstract 1894 Mesenchymal stem cells (MSCs) give rise to bone marrow (BM) stromal cells and play an essential role in the formation and function of the MM microenvironment. Some recent studies revealed that MSCs from myeloma patients (MM-hMSCs) show an enhanced spontaneous and myeloma cell-induced production of cytokines and a distinctive gene expression profile, when compared to MSCs from normal donors (ND-hMSCs). However, regarding the osteogenic differentiation ability of MM-hMSCs conflicting observations were reported. In this study, we observed that MM-hMSCs, especially for those from MM patients with bone lesions, exhibited in the presence of osteogenic differentiation (OD) medium, significantly decreased alkaline phosphatase (ALP) activity, reduced expression of specific osteogenic markers (OPN, BMP2, OTX and BSP) and impaired matrix mineralization, compared to ND-hMSCs. However, MGUS-hMSCs, did not show a significantly impaired osteogenesis ability. Primary CFU-ALP assay from BM samples of diseased mice in the 5T33MM model also confirmed that the osteogenic differentiation ability of MSCs was impaired. Previous reports indicated that MM cells can suppress MSCs osteogenesis by HGF and DKK1 as observed in vitro (Giuliani et al, Cancer Res. 2007; Standal et al, Blood. 2007). Since MM-hMSCs have been cultured in vitro for several weeks and without any stimulation of MM cells, we believe that the impaired osteogenic differentiation of MM-hMSCs was due to an intrinsic abnormality. Several reports suggested that NOTCH signalling can maintain bone marrow mesenchymal progenitors in a more undifferentiated state by suppressing osteoblast differentiation (Hilton et al, Nat Med. 2008; Zanotti et al, Endocrinology. 2008). Therefore, we postulate that impaired osteogenic ability of MM-hMSCs might be (at least partly) related to abnormal NOTCH activity during osteogenesis. We found by quantitative real time PCR that NOTCH1, NOTCH2, Dll-1, Jagged-1, and NOTCH pathway downstream genes hes1, hey1, hey2, heyL were considerably decreased in ND-hMSCs after shifting them from normal culture medium to OD medium, indicating that NOTCH signalling was gradually suppressed during MSC osteogenesis. However, it was observed that the expression of NOTCH1, Jagged-1, Hes1 and Hes5 in MM-hMSCs did not decrease to the level of ND-hMSC with statistical difference. This implicates that the NOTCH signaling pathway remains in MM-hMSCs over-activated even in the presence of osteogenesis inducing signals. When the NOTCH signalling inhibitor DAPT was added to MM-hMSCs in OD medium, we found that hes1 expression was suppressed while, RUNX2 expression, a key transcription factor for osteoblastogenesis, as well as ALP activity, osteogenic genes expression and mineralization deposition were all increased. In conclusion our data indicate that MM-hMSCs exhibit in vitro lower osteogenic differentiation ability compared to ND-hMSCs, and that this impairement is associated with an inappropriate NOTCH pathway deactivation during the osteogenesis process. Targeting hMSCs in vivo by NOTCH inhibitors might have therapeutical potential to control bone disease in MM patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Three-dimensional printed bone scaffolds: The role of nano/micro-hydroxyapatite particles on the adhesion and differentiation of human mesenchymal stem cells

2017 ◽  
Vol 231 (6) ◽  
pp. 555-564 ◽  
Author(s):  
Marco Domingos ◽  
Antonio Gloria ◽  
Jorge Coelho ◽  
Paulo Bartolo ◽  
Joaquim Ciurana
Keyword(s):  
Tissue Engineering ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Calcium Phosphates ◽  
Three Dimensional ◽  
Human Mesenchymal Stem Cells ◽  
Mechanical Tests ◽  
Engineering Research

Bone tissue engineering is strongly dependent on the use of three-dimensional scaffolds that can act as templates to accommodate cells and support tissue ingrowth. Despite its wide application in tissue engineering research, polycaprolactone presents a very limited ability to induce adhesion, proliferation and osteogenic cell differentiation. To overcome some of these limitations, different calcium phosphates, such as hydroxyapatite and tricalcium phosphate, have been employed with relative success. This work investigates the influence of nano-hydroxyapatite and micro-hydroxyapatite (nHA and mHA, respectively) particles on the in vitro biomechanical performance of polycaprolactone/hydroxyapatite scaffolds. Morphological analysis performed with scanning electron microscopy allowed us to confirm the production of polycaprolactone/hydroxyapatite constructs with square interconnected pores of approximately 350 µm and to assess the distribution of hydroxyapatite particles within the polymer matrix. Compression mechanical tests showed an increase in polycaprolactone compressive modulus ( E) from 105.5 ± 11.2 to 138.8 ± 12.9 MPa (PCL_nHA) and 217.2 ± 21.8 MPa (PCL_mHA). In comparison to PCL_mHA scaffolds, the addition of nano-hydroxyapatite enhanced the adhesion and viability of human mesenchymal stem cells as confirmed by Alamar Blue assay. In addition, after 14 days of incubation, PCL_nHA scaffolds showed higher levels of alkaline phosphatase activity compared to polycaprolactone or PCL_mHA structures.

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