Effect of 3D scaffold and dynamic culture condition on the global gene expression profile of mouse embryonic stem cells

We have previously demonstrated that mouse embryonic stem (ES) cells differentiated on three-dimensional (3D), highly porous, tantalum-based scaffolds (Cytomatrix™) have significantly higher hematopoietic differentiation efficiency than those cultured under conventional two-dimensional (2D) tissue culture conditions. In addition, ES cell-seeded scaffolds cultured inside spinner bioreactors showed further enhancement in hematopoiesis compared to static conditions. In the present study, we evaluated how these various biomaterial-based culture conditions, e.g. 2D vs. 3D scaffolds and static vs. dynamic, influence the global gene expression profile of differentiated ES cells. We report that compared to 2D tissue culture plates, cells differentiated on porous, Cytomatrix™ scaffolds possess significantly higher expression levels of extracellular matrix (ECM)-related genes, as well as genes that regulate cell growth, proliferation and differentiation. In addition, these differences in gene expression were more pronounced in 3D dynamic culture compared to 3D static culture. We report specific genes that are either uniquely expressed under each condition or are quantitatively regulated, i.e. over expressed or inhibited by a specific culture environment. We conclude that that biomaterial-based 3D cultures, especially under dynamic conditions, might favor efficient hematopoietic differentiation of ES cells by stimulating increased expression of specific ECM proteins, growth factors and cell adhesion related genes while significantly down-regulating genes that act to inhibit expression of these molecules.     

Adipogenesis of murine embryonic stem cells in a three-dimensional culture system using electrospun polymer scaffolds

A mechanistic understanding of adipose tissue differentiation is critical for the treatment and prevention of obesity and type 2 diabetes. Conventional in vitro models of adipogenesis are preadipocytes or freshly isolated adipocytes grown in two-dimensional (2D) cultures. Optimal results using in vitro tissue culture models can be expected only when adipocyte models closely resemble adipose tissue in vivo. Thus the design of an in vitro three-dimensional (3D) model which faithfully mimics the in vivo environment is needed to effectively study adipogenesis. Pluripotent embryonic stem (ES) cells are a self-renewing cell type that can readily be differentiated into adipocytes. In this study, a 3D culture system was developed to mimic the geometry of adipose tissue in vivo. Murine ES cells were seeded into electrospun polycaprolactone scaffolds and differentiated into adipocytes in situ by hormone induction as demonstrated using a battery of gene and protein expression markers along with the accumulation of neutral lipid droplets. Insulin-responsive Akt phosphorylation, and beta-adrenergic stimulation of cyclic AMP synthesis were demonstrated in ES cell-derived adipocytes. Morphologically, ES cell-derived adipocytes resembled native fat cells by scanning electron and phase contrast microscopy. This tissue engineered ES cell-matrix model has potential uses in drug screening and other therapeutic developments.     

Differentiation of Mouse Embryonic Stem Cells to Lympho-Hematopoietic Lineagesin Vitro

Mouse embryonic stem (ES) cells can differentiate in culture to late stages of many cell lineages. have found culture conditions that are favorable for development in vitro of ES cells into hematopoietic cells at a stage equivalent to day 11-14 of fetal liver development. describe here: (1) the growth conditions necessary for maintenance of ES cells in an undifferentiated state, and the conditions that allow differentiation of cystic embryoid bodies that contain precursors of most hematopoietic cell lineages, including lymphoid cells; (2) the development of lymphoid vessels from ES fetusesin vivo; (3) the characterization of lymphoid, erythroid, megakaryoid, and myeloid cells from ES fetuses; and (4) the cloning of cell lines representing lymphoid, myeloid lineage cells from differentiated ES cells.     

Engineering three-dimensional bone tissue in vitro using biodegradable scaffolds: investigating initial cell-seeding density and culture period

New three-dimensional (3D) scaffolds for bone tissue engineering have been developed throughout which bone cells grow, differentiate, and produce mineralized matrix. In this study, the percentage of cells anchoring to our polymer scaffolds as a function of initial cell seeding density was established; we then investigated bone tissue formation throughout our scaffolds as a function of initial cell seeding density and time in culture. Initial cell seeding densities ranging from 0.5 to 10 x 10(6) cells/cm(3) were seeded onto 3D scaffolds. After 1 h in culture, we determined that 25% of initial seeded cells had adhered to the scaffolds in static culture conditions. The cell-seeded scaffolds remained in culture for 3 and 6 weeks, to investigate the effect of initial cell seeding density on bone tissue formation in vitro. Further cultures using 1 x 10(6) cells/cm(3) were maintained for 1 h and 1, 2, 4, and 6 weeks to study bone tissue formation as a function of culture period. After 3 and 6 weeks in culture, scaffolds seeded with 1 x 10(6) cells/cm(3) showed similar tissue formation as those seeded with higher initial cell seeding densities. When initial cell seeding densities of 1 x 10(6) cells/cm(3) were used, osteocalcin immunolabeling indicative of osteoblast differentiation was seen throughout the scaffolds after only 2 weeks of culture. Von Kossa and tetracycline labeling, indicative of mineralization, occurred after 3 weeks. These results demonstrated that differentiated bone tissue was formed throughout 3D scaffolds after 2 weeks in culture using an optimized initial cell density, whereas mineralization of the tissue only occurred after 3 weeks. Furthermore, after 6 weeks in culture, newly formed bone tissue had replaced degrading polymer.     

The implications of the response of human mesenchymal stromal cells in three-dimensional culture to electrical stimulation for tissue regeneration

Earlier, we demonstrated that local electrical stimulation (ES) improved bone and peripheral nerve regeneration. To determine how ES induces the regeneration of different kinds of tissues, we studied the initial ES-induced regeneration process by investigating the expression of chemokines and growth factors from human mesenchymal stromal cells (hMSCs). In particular, we assessed the responses of hMSCs grown in three-dimensional (3D) culture on a collagen sponge, as 3D culture techniques induced cell behavior that was similar to in vivo cell behavior. We also compared the gene expression patterns of monolayer hMSCs with those of 3D hMSCs under the condition that cells in either culture are exposed to the same type of ES. Biphasic pulses did not affect the proliferation of hMSCs in 3D culture significantly at the magnitude applied in previous animal studies showing improved bone and peripheral nerve regeneration. However, ES enhanced the gene expression of growth factors (BMP-2, IGF-1, and VEGF), chemokines (CXCL2, interleukin (IL)-8), and chemokine receptors (CXCR4 and IL-8RB) from hMSCs grown in 3D culture. A particular difference between the 3D and monolayer cultures was found in the expression of chemokine receptors, CXCR4 and IL-8RB, which is related to the homing capabilities of mesenchymal stromal cells. These genes were expressed by cells in 3D cultures, but were not or expressed at extremely low levels by cells grown in monolayer cultures. ES led to a significant increase in the expression of CXCR4 and IL-8RB in both monolayer and 3D hMSCs, but the increase in the monolayer culture was detected at an extremely low level. These results demonstrate that ES increased the expression of a variety of growth factors and chemokine genes from 3D hMSCs, which may explain increased tissue regeneration in vivo, independent of the tissue type. A culture-dependent expression of the CXCR4 gene suggested that cell response to external stimulus in 3D systems may be more accurately reflected in in vivo findings than in monolayer cultures.     

Mesodermal and Hematopoietic Differentiation from ES and iPS Cells

Embryonic stem (ES) and induced pluripotent stem (iPS) cells can differentiate into any type of tissue when grown in a suitable culture environment and are considered valuable tools for regenerative medicine. In the field of hematology, generation of hematopoietic stem cells (HSCs) and mature hematopoietic cells (HCs) from ES and iPS cells through mesodermal cells, the ancestors of HCs, can facilitate transplantation and transfusion therapy. Several studies report generation of functional HCs from both mouse and human ES and iPS cells. This approach will likely be applied to individual patient-derived iPS cells for regenerative medicine approaches and drug screening in the future. Here, we summarize current studies of HC-generation from ES and iPS cells.     

Optimization of fibrin scaffolds for differentiation of murine embryonic stem cells into neural lineage cells

The objective of this research was to determine the appropriate cell culture conditions for embryonic stem (ES) cell proliferation and differentiation in fibrin scaffolds by examining cell seeding density, location, and the optimal concentrations of fibrinogen, thrombin, and aprotinin (protease inhibitor). Mouse ES cells were induced to become neural progenitors by adding retinoic acid for 4 days to embryoid body (EB) cultures. For dissociated EBs, the optimal cell seeding density and location was determined to be 250,000 cells/cm² seeded on top of fibrin scaffolds. For intact EBs, three-dimensional (3D) cultures with one EB per 400 μL fibrin scaffold resulted in greater cell proliferation and differentiation than two-dimensional (2D) cultures. Optimal concentrations for scaffold polymerization were 10 mg/mL of fibrinogen and 2 NIH units/mL of thrombin. The optimal aprotinin concentration was determined to be 50 μg/mL for dissociated EBs (2D) and 5 μg/mL for intact EBs in 3D fibrin scaffolds. Additionally, after 14 days in 3D culture EBs differentiated into neurons and astrocytes as indicated by immunohistochemisty. These conditions provide an optimal fibrin scaffold for evaluating ES cell differentiation and proliferation in culture, and for use as a platform for neural tissue engineering applications, such as the treatment for spinal cord injury.     

Paper-based bioactive scaffolds for stem cell-mediated bone tissue engineering

Bioactive, functional scaffolds are required to improve the regenerative potential of stem cells for tissue reconstruction and functional recovery of damaged tissues. Here, we report a paper-based bioactive scaffold platform for stem cell culture and transplantation for bone reconstruction. The paper scaffolds are surface-engineered by an initiated chemical vapor deposition process for serial coating of a water-repellent and cell-adhesive polymer film, which ensures the long-term stability in cell culture medium and induces efficient cell attachment. The prepared paper scaffolds are compatible with general stem cell culture and manipulation techniques. An optimal paper type is found to provide structural, physical, and mechanical cues to enhance the osteogenic differentiation of human adipose-derived stem cells (hADSCs). A bioactive paper scaffold significantly enhances in vivo bone regeneration of hADSCs in a critical-sized calvarial bone defect. Stacking the paper scaffolds with osteogenically differentiated hADSCs and human endothelial cells resulted in vascularized bone formation in vivo. Our study suggests that paper possesses great potential as a bioactive, functional, and cost-effective scaffold platform for stem cell-mediated bone tissue engineering. To the best of our knowledge, this is the first study reporting the feasibility of a paper material for stem cell application to repair tissue defects.     

Osteogenic and angiogenic potentials of monocultured and co-cultured human-bone-marrow-derived mesenchymal stem cells and human-umbilical-vein endothelial cells on three-dimensional porous beta-tricalcium phosphate scaffold

The use of biodegradable beta-tricalcium phosphate (β-TCP) scaffolds holds great promise for bone tissue engineering. However, the effects of β-TCP on bone and endothelial cells are not fully understood. This study aimed to investigate cell proliferation and differentiation of mono- or co-cultured human-bone-marrow-derived mesenchymal stem cells (hBMSCs) and human-umbilical-vein endothelial cells (HUVECs) on a three-dimensional porous, biodegradable β-TCP scaffold. In co-culture studies, the ratios of hBMSCs:HUVECs were 5:1, 1:1 and 1:5. Cellular morphologies of HUVECs, hBMSCs and co-cultured HUVECs/hBMSCs on the β-TCP scaffolds were monitored using confocal and scanning electron microscopy. Cell proliferation was monitored by measuring the amount of double-stranded DNA (dsDNA) whereas hBMSC and HUVEC differentiation was assessed using the osteogenic and angiogenic markers, alkaline phosphatase (ALP) and PECAM-1 (CD31), respectively. Results show that HUVECs, hBMSCs and hBMSCs/HUVECs adhered to and proliferated well on the β-TCP scaffolds. In monoculture, hBMSCs grew faster than HUVECs on the β-TCP scaffolds after 7 days, but HUVECs reached similar levels of proliferation after 14 days. In monoculture, β-TCP scaffolds promoted ALP activities of both hBMSCs and HUVECs when compared to those grown on tissue culture well plates. ALP activity of cells in co-culture was higher than that of hBMSCs in monoculture. Real-time polymerase chain reaction results indicate that runx2 and alp gene expression in monocultured hBMSCs remained unchanged at days 7 and 14, but alp gene expression was significantly increased in hBMSC co-cultures when the contribution of individual cell types was not distinguished.     

In vivo bone formation following transplantation of human adipose-derived stromal cells that are not differentiated osteogenically

A number of studies have shown in vivo bone regeneration by transplantation of osteogenic cells differentiated in vitro from adipose-derived stromal cells (ADSCs). However, the in vitro osteogenic differentiation process requires an additional culture period, and the dexamethasone that is generally used in the process may be cytotoxic. Here, we tested the hypothesis that ADSCs that are not differentiated osteogenically in vitro prior to transplantation would extensively regenerate bone in vivo when exogenous bone morphogenetic protein-2 (BMP-2) is delivered to the transplantation site. We fabricated a poly(dl-lactic-co-glycolic acid)/hydroxyapatite (PLGA/HA) composite scaffold with osteoactive HA that is highly exposed on the scaffold surface. This scaffold was able to release BMP-2 over a 4-week period in vitro. Human ADSCs cultured on BMP-2-loaded PLGA/HA scaffolds for 2 weeks differentiated toward osteogenic cells expressing alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OCN) mRNA, while cells on PLGA/HA scaffolds without BMP-2 expressed only ALP. To study in vivo bone formation, PLGA/HA scaffolds (group 1), BMP-2-loaded PLGA/HA scaffolds (group 2), undifferentiated ADSCs seeded on PLGA/HA scaffolds (group 3), and undifferentiated ADSCs seeded on BMP-2-loaded PLGA/HA scaffolds (group 4) were implanted into dorsal, subcutaneous spaces of athymic mice. Eight weeks after implantation, group 4 exhibited a 25-fold greater bone formation area and 5-fold higher calcium deposition than group 3. Bone regeneration by transplanted human ADSCs in group 4 was confirmed by expression of human-specific osteoblastic genes, ALP, collagen type I, OPN, OCN, and bone sialoprotein, while group 3 expressed much lower levels of collagen type I and OPN mRNA only. This study demonstrates the feasibility of extensive in vivo bone regeneration by transplantation of ADSCs without prior in vitro osteogenic differentiation, and that a PLGA/HA composite BMP-2 delivery system stimulates bone regeneration following transplantation of undifferentiated human ADSCs.     

Mesenchymal stem cell differentiation to neuronal cells on electrospun nanofibrous substrates for nerve tissue engineering

Bone marrow Mesenchymal stem cells capable of differentiating into neuronal cells on engineered nanofibrous scaffolds have great potential for bionanomaterial–cell transplantation therapy of neurodegenerative diseases and injuries of the nervous system. MSCs have been the highlight of many tissue engineering studies mainly because of their multipotential properties. We investigated the potential of human bone marrow derived Mesenchymal stem cells (MSCs) for neuronal differentiation in vitro on poly(l-lactic acid)-co-poly-(3-caprolactone)/Collagen (PLCL/Coll) nanofibrous scaffolds. PLCL and PLCL/Coll nanofibrous scaffolds were fabricated by electrospinning process and their chemical and mechanical characterizations were carried out using SEM, contact angle, FTIR, and tensile instrument. The differentiation of MSCs was carried out using neuronal inducing factors including β-mercaptoethanol, epidermal growth factor, nerve growth factor and brain derived growth factor in DMEM/F12 media. The proliferations of MSCs evaluated by MTS assay showed that the cells grown on PLCL/Coll nanofibrous scaffolds were comparatively higher (80%) than those on PLCL. Scanning electron microscopy results showed that MSCs differentiated on PLCL/Coll nanofibrous scaffolds showed neuronal morphology, with multipolar elongations and expressed neurofilament and nestin protein by immuno-fluorescent microscopy. Our studies on the differentiation of MSCs to neuronal cells on nanofibrous scaffolds suggest their potential application towards nerve regeneration.     

Colonization and maintenance of murine embryonic stem cells on poly(alpha-hydroxy esters)

The aim of this study was to determine the ability of various poly(alpha-hydroxy esters) to support the in vitro propagation of murine embryonic stem (ES) cells in an undifferentiated state. To this end, ES cell colonization, growth and Oct-4 immunoreactivity following a 48 h culture period upon poly((D,L)-lactide), poly((L)-lactide), poly(glycolide) and poly((D,L)-lactide-co-glycolide) (PLGA) were assessed. By the analysis of live and dead cell number indices and Oct-4 immunoreactivity, ES cell colonization rate during a 48 h culture period was found to be significantly greater on PLGA compared to all the other unmodified poly(alpha-hydroxy esters) tested. Surface treatment of all polymers with 0.1m potassium hydroxide revealed a significant increase in ES cell live numbers when compared to all unmodified polymers, thus revealing a correlation between polymer content, hydrophilicity and colonization rate. These data suggest that surface treated poly(alpha-hydroxy esters) may be employed for ES cell scale up procedures and in tissue engineering applications requiring the colonization of scaffolds by ES cells in an undifferentiated state. According to such applications, once the designated scaffold has been colonized, ES cell directed differentiation into the desired and fully differentiated, functional adult tissue may then be effected.     

Electrically Stimulated Adipose Stem Cells on Polypyrrole-Coated Scaffolds for Smooth Muscle Tissue Engineering

We investigated the use of polypyrrole (PPy)-coated polymer scaffolds and electrical stimulation (ES) to differentiate adipose stem cells (ASCs) towards smooth muscle cells (SMCs). Since tissue engineering lacks robust and reusable 3D ES devices we developed a device that can deliver ES in a reliable, repeatable, and cost-efficient way in a 3D environment. Long pulse (1 ms) or short pulse (0.25 ms) biphasic electric current at a frequency of 10 Hz was applied to ASCs to study the effects of ES on ASC viability and differentiation towards SMCs on the PPy-coated scaffolds. PPy-coated scaffolds promoted proliferation and induced stronger calponin, myosin heavy chain (MHC) and smooth muscle actin (SMA) expression in ASCs compared to uncoated scaffolds. ES with 1 ms pulse width increased the number of viable cells by day 7 compared to controls and remained at similar levels to controls by day 14, whereas shorter pulses significantly decreased viability compared to the other groups. Both ES protocols supported smooth muscle expression markers. Our results indicate that electrical stimulation on PPy-coated scaffolds applied through the novel 3D ES device is a valid approach for vascular smooth muscle tissue engineering.     

Flow Perfusion Culture of Marrow Stromal Cells Seeded on Porous Biphasic Calcium Phosphate Ceramics

Calcium phosphate ceramics have been widely used for filling bone defects to aid in the regeneration of new bone tissue. Addition of osteogenic cells to porous ceramic scaffolds may accelerate the bone repair process. This study demonstrates the feasibility of culturing marrow stromal cells (MSCs) on porous biphasic calcium phosphate ceramic scaffolds in a flow perfusion bioreactor. The flow of medium through the scaffold porosity benefits cell differentiation by enhancing nutrient transport to the scaffold interior and by providing mechanical stimulation to cells in the form of fluid shear. Primary rat MSCs were seeded onto porous ceramic (60% hydroxyapatite, 40% β-tricalcium phosphate) scaffolds, cultured for up to 16 days in static or flow perfusion conditions, and assessed for osteoblastic differentiation. Cells were distributed throughout the entire scaffold by 16 days of flow perfusion culture whereas they were located only along the scaffold perimeter in static culture. At all culture times, flow perfused constructs demonstrated greater osteoblastic differentiation than statically cultured constructs as evidenced by alkaline phosphatase activity, osteopontin secretion into the culture medium, and histological evaluation. These results demonstrate the feasibility and benefit of culturing cell/ceramic constructs in a flow perfusion bioreactor for bone tissue engineering applications.     

Critical Steps toward a tissue-engineered cartilage implant using embryonic stem cells

Embryonic stem (ES) cells are a potential source for cartilage tissue engineering because they provide an unlimited supply of cells that can be differentiated into chondrocytes. So far, chondrogenic differentiation of both mouse and human ES cells has only been demonstrated in two-dimensional cultures, in pellet cultures, in a hydrogel, or on thin biomaterials. The next challenge will be to form cartilage on a load-bearing, clinically relevant-sized scaffold in vitro and in vivo, to regenerate defects in patients suffering from articular cartilage disorders. For a successful implant, cells have to be seeded efficiently and homogenously throughout the scaffold. Parameters investigated were the scaffold architecture, seeding method, and cellular condition. Seeding in a three-dimensional fiber-deposited (3DF) scaffold was more homogenous than in a compression-molded scaffold. The seeding efficiency on bare scaffolds was compromised by the absence of serum in the chondrogenic medium, but could be improved by combining the cells with a gel and subsequent injection into the 3DF scaffolds. However, the viability of the cells was unsatisfactory in the interior of the graft. Cell aggregates, the so-called embryoid bodies (EBs), were seeded with increased survival rate. Mouse ES cells readily underwent chondrogenic differentiation in vitro in pellets, on bare scaffolds, in Matrigel, and in agarose, both as single cells and in EBs. The differentiation protocol requires further improvement to achieve homogenous differentiation and abolish teratoma formation in vivo. We conclude that ES cells can be used as a cell source for cartilage tissue engineering, pending further optimization of the strategy.     

Development of Feeder-Free Culture Systems for Generation of ckit+sca1+ Progenitors from Mouse iPS Cells

Patient-specific therapeutic cells derived from induced pluripotent stem (iPS) cells may bypass the ethical issues associated with embryonic stem (ES) cells and avoid potential immunological reactions associated with allogenic transplantation. It is critical, for the ultimate clinical applicability of iPS cell-derived therapies, to establish feeder-free cultures that ensure efficient differentiation of iPS cells into therapeutic progenitors. It is also necessary to understand if iPS cell-derived progenitors differ from those derived from ES cells. In this study, we compared the efficiency of three different feeder-free cultures for differentiating mouse iPS cells into ckit+sca1+ hematopoietic progenitor cells (HPCs) and compared how differentiation and functionality varies between ES and iPS cells. Our results indicated that both iPS and ES cells can be efficiently differentiated into HPCs in suspension cultures supplemented with secretion factors from mouse bone marrow stromal cells (OP9-DL1 conditioned medium). The functionality of these cells was demonstrated by differentiation into CD11c+ dendritic cells (DCs). Both ES and iPS-derived DCs expressed activation molecules (CD86, CD80) in response to LPS stimulation and stimulated T cell proliferation in a mixed lymphocyte reaction (MLR). Extensive quantitative RT-PCR studies were used to study the differences in gene expression profiles of ckit+sca1+ cells generated from the various culture systems as well as differences between ES-derived and iPS-derived cells. We conclude that a feeder-free system using stromal conditioned medium can efficiently generate HPCs as well as functional DCs from iPS cells and the generated cells have similar gene expression profile as those from ES cells.     

Construction of tissue-engineered homograft bioprosthetic heart valves in vitro

Human mesenchymal stem cells (MSCs) differentiate into multiple cell-lineages and may serve as an alternative source of seed cells for tissue engineering. We investigated whether MSCs could be induced to differentiate into endothelial cells (ECs) and function as seed cells for the in vitro construction of tissue-engineered heart valves (TEHVs). Aortic or pulmonary valve homografts were decellularized with 0.1% sodium dodecylsulphate and used as scaffolds for TEHVs. The MSCs were isolated from human bone marrow by Percoll gradient centrifugation (1.073 g/ml), differentiated into ECs with vascular endothelial growth factor (10 ng/ml), and seeded onto a decellularized scaffold (high-density seeding, >10(5) cells/cm2) and grown in static culture for 14 days. Over 90% of the differentiated cells from MSCs stained positively for von Willebrand factor and Tie-2-related antigen. Additionally, Weibel-Palade corpuscle was observed in the cytoplasm of these cells. Levels of reendothelialization in static culture on days 7, 14, and 20, were 73%, 85%, and 95%, respectively. These results show that MSCs from human bone marrow can differentiate, in vitro, into ECs that can then be used to construct TEHVs. Reendothelialization in static culture can be used to provide the basic material for pulsatile-flow cultivation.     

Electrical stimulation therapy and rotary jet-spinning scaffold to treat bone defects

The combination of electrical stimulation (ES) and bone tissue engineering (BTE) has been successful in treatments of bone regeneration. This study evaluated the effects of ES combined with PCL + β-TCP 5% scaffolds obtained by rotary jet spinning (RJS) in the regeneration of bone defects in the calvaria of Wistar rats. We used 120 animals with induced bone defects divided into 4 groups (n = 30): (C) without treatment; (S) with PCL+ β-TCP 5% scaffold; (ES) treated with ES (10 μA/5 min); (ES + S) with PCL + β-TCP 5% scaffold. The ES occurred twice a week during the entire experimental period. Cell viability (in vitro: Days 3 and 7) and histomorphometric, histochemical, and immunohistochemical (in vivo; Days 30, 60, and 90) analysis were performed. In vitro, ES + S increased cell viability after Day 7 of incubation. In vivo, it was observed modulation of inflammatory cells in ES therapy, which also promoted blood vessels proliferation, and increase of collagen. Moreover, ES therapy played a role in osteogenesis by decreasing ligand kappa B nuclear factor-TNFSF11 (RANKL), increasing alkaline phosphatase (ALP), and decreasing the tartarate-resistant acid phosphatase. The combination of ES with RJS scaffolds may be a promising strategy for bone defects regeneration, since the therapy controlled inflammation, favored blood vessels proliferation, and osteogenesis, which are important processes in bone remodeling.