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1.  Self-Assembling Peptide Hydrogels Modulate In Vitro Chondrogenesis of Bovine Bone Marrow Stromal Cells 
Tissue Engineering. Part A  2009;16(2):465-477.
Our objective was to test the hypothesis that self-assembling peptide hydrogel scaffolds provide cues that enhance the chondrogenic differentiation of bone marrow stromal cells (BMSCs). BMSCs were encapsulated within two unique peptide hydrogel sequences, and chondrogenesis was compared with that in agarose hydrogels. BMSCs in all three hydrogels underwent transforming growth factor-β1-mediated chondrogenesis as demonstrated by comparable gene expression and biosynthesis of extracellular matrix molecules. Expression of an osteogenic marker was unchanged, and an adipogenic marker was suppressed by transforming growth factor-β1 in all hydrogels. Cell proliferation occurred only in the peptide hydrogels, not in agarose, resulting in higher glycosaminoglycan content and more spatially uniform proteoglycan and collagen type II deposition. The G1-positive aggrecan produced in peptide hydrogels was predominantly the full-length species, whereas that in agarose was predominantly the aggrecanase product G1-NITEGE. Unique cell morphologies were observed for BMSCs in each peptide hydrogel sequence, with extensive cell–cell contact present for both, whereas BMSCs in agarose remained rounded over 21 days in culture. Differences in cell morphology within the two peptide scaffolds may be related to sequence-specific cell adhesion. Taken together, this study demonstrates that self-assembling peptide hydrogels enhance chondrogenesis compared with agarose as shown by extracellular matrix production, DNA content, and aggrecan molecular structure.
doi:10.1089/ten.tea.2009.0158
PMCID: PMC2862611  PMID: 19705959
2.  Investigating the Role of FGF18 in the Cultivation and Osteogenic Differentiation of Mesenchymal Stem Cells 
PLoS ONE  2012;7(8):e43982.
Fibroblast growth factor18 (FGF18) belongs to the FGF family and is a pleiotropic protein that stimulates proliferation in several tissues. Bone marrow mesenchymal stem cells (BMSCs) participate in the normal replacement of damaged cells and in disease healing processes within bone and the haematopoietic system. In this study, we constructed FGF18 and investigated its effects on rat BMSCs (rBMSCs). The proliferative effects of FGF18 on rBMSCs were examined using an MTS assay. To validate the osteogenic differentiation effects of FGF18, ALP and mineralization activity were examined as well as osteogenic differentiation-related gene levels. FGF18 significantly enhanced rBMSCs proliferation (p<0.001) and induced the osteogenic differentiation by elevating ALP and mineralization activity of rBMSCs (p<0.001). Furthermore, these osteogenic differentiation effects of FGF18 were confirmed via increasing the mRNA levels of collagen type I (Col I), bone morphogenetic protein 4 (BMP4), and Runt-related transcription factor 2 (Runx2) at 3 and 7 days. These results suggest that FGF18 could be used to improve bone repair and regeneration.
doi:10.1371/journal.pone.0043982
PMCID: PMC3427245  PMID: 22937141
3.  Effect of Cyclic Strain on Cardiomyogenic Differentiation of Rat Bone Marrow Derived Mesenchymal Stem Cells 
PLoS ONE  2012;7(4):e34960.
Mesenchymal stem cells (MSCs) are a potential source of material for the generation of tissue-engineered cardiac grafts because of their ability to transdifferentiate into cardiomyocytes after chemical treatments or co-culture with cardiomyocytes. Cardiomyocytes in the body are subjected to cyclic strain induced by the rhythmic heart beating. Whether cyclic strain could regulate rat bone marrow derived MSC (rBMSC) differentiation into cardiomyocyte-like lineage was investigated in this study. A stretching device was used to generate the cyclic strain for rBMSCs. Cardiomyogenic differentiation was evaluated using quantitative real-time reverse transcription polymerase chain reaction (RT-PCR), immunocytochemistry and western-blotting. The results demonstrated that appropriate cyclic strain treatment alone could induce cardiomyogenic differentiation of rBMSCs, as confirmed by the expression of cardiomyocyte-related markers at both mRNA and protein levels. Furthermore, rBMSCs exposed to the strain stimulation expressed cardiomyocyte-related markers at a higher level than the shear stimulation. In addition, when rBMSCs were exposed to both strain and 5-azacytidine (5-aza), expression levels of cardiomyocyte-related markers significantly increased to a degree suggestive of a synergistic interaction. These results suggest that cyclic strain is an important mechanical stimulus affecting the cardiomyogenic differentiation of rBMSCs. This provides a new avenue for mechanistic studies of stem cell differentiation and a new approach to obtain more committed differentiated cells.
doi:10.1371/journal.pone.0034960
PMCID: PMC3319595  PMID: 22496879
4.  Simulated microgravity facilitates cell migration and neuroprotection after bone marrow stromal cell transplantation in spinal cord injury 
Introduction
Recently, cell-based therapy has gained significant attention for the treatment of central nervous system diseases. Although bone marrow stromal cells (BMSCs) are considered to have good engraftment potential, challenges due to in vitro culturing, such as a decline in their functional potency, have been reported. Here, we investigated the efficacy of rat BMSCs (rBMSCs) cultured under simulated microgravity conditions, for transplantation into a rat model of spinal cord injury (SCI).
Methods
rBMSCs were cultured under two different conditions: standard gravity (1G) and simulated microgravity attained by using the 3D-clinostat. After 7 days of culture, the rBMSCs were analyzed morphologically, with RT-PCR and immunostaining, and were used for grafting. Adult rats were used for constructing SCI models by using a weight-dropping method and were grouped into three experimental groups for comparison. rBMSCs cultured under 1 g and simulated microgravity were transplanted intravenously immediately after SCI. We evaluated the hindlimb functional improvement for 3 weeks. Tissue repair after SCI was examined by calculating the cavity area ratio and immunohistochemistry.
Results
rBMSCs cultured under simulated microgravity expressed Oct-4 and CXCR4, in contrast to those cultured under 1 g conditions. Therefore, rBMSCs cultured under simulated microgravity were considered to be in an undifferentiated state and thus to possess high migration ability. After transplantation, grafted rBMSCs cultured under microgravity exhibited greater survival at the periphery of the lesion, and the motor functions of the rats that received these grafts improved significantly compared with the rats that received rBMSCs cultured in 1 g. In addition, rBMSCs cultured under microgravity were thought to have greater trophic effects on reestablishment and survival of host spinal neural tissues because cavity formations were reduced, and apoptosis-inhibiting factor expression was high at the periphery of the SCI lesion.
Conclusions
Here we show that transplantation of rBMSCs cultured under simulated microgravity facilitates functional recovery from SCI rather than those cultured under 1 g conditions.
doi:10.1186/scrt184
PMCID: PMC3706926  PMID: 23548163
Bone marrow stromal cell; Migration; Simulated microgravity; Spinal cord injury; Survival; Trophic factor
5.  Gravity, a regulation factor in the differentiation of rat bone marrow mesenchymal stem cells 
Background
Stem cell therapy has emerged as a potential therapeutic option for tissue engineering and regenerative medicine, but many issues remain to be resolved, such as the amount of seed cells, committed differentiation and the efficiency. Several previous studies have focused on the study of chemical inducement microenvironments. In the present study, we investigated the effects of gravity on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into force-sensitive or force-insensitive cells.
Methods and results
Rat BMSCs (rBMSCs) were cultured under hypergravity or simulated microgravity (SMG) conditions with or without inducement medium. The expression levels of the characteristic proteins were measured and analyzed using immunocytochemical, RT-PCR and Western-blot analyses. After treatment with 5-azacytidine and hypergravity, rBMSCs expressed more characteristic proteins of cardiomyocytes such as cTnT, GATA4 and β-MHC; however, fewer such proteins were seen with SMG. After treating rBMSCs with osteogenic inducer and hypergravity, there were marked increases in the expression levels of ColIA1, Cbfa1 and ALP. Reverse results were obtained with SMG. rBMSCs treated with adipogenic inducer and SMG expressed greater levels of PPARgamma. Greater levels of Cbfa1- or cTnT-positive cells were observed under hypergravity without inducer, as shown by FACS analysis. These results indicate that hypergravity induces differentiation of rBMSCs into force-sensitive cells (cardiomyocytes and osteoblasts), whereas SMG induces force-insensitive cells (adipocytes).
Conclusion
Taken together, we conclude that gravity is an important factor affecting the differentiation of rBMSCs; this provides a new avenue for mechanistic studies of stem cell differentiation and a new approach to obtain more committed differentiated or undifferentiated cells.
doi:10.1186/1423-0127-16-87
PMCID: PMC2754420  PMID: 19772591
6.  Osterix Overexpression in Mesenchymal Stem cells Stimulates Healing of Critical-Sized Defects in Murine Calvarial Bone 
Tissue engineering  2007;13(10):2431-2440.
Osterix (Osx) is a zinc-finger-containing transcription factor that is expressed in osteoblasts of all endochondral and membranous bones. In Osx null mice, osteoblast differentiation is impaired, and bone formation is absent. We hypothesized that overexpression of Osx in bone marrow–derived mesenchymal stem cells (BMSCs) would enhance osteogenic differentiation during bone regeneration in vivo. Overexpression of Osx in mouse BMSCs was achieved using retroviral infection together with a green fluorescent protein (GFP) vector to monitor transduction efficiency and determine the source of regenerative cells in implantation studies. Bone regeneration in vivo was evaluated by implanting BMSCs overexpressing Osx into 4-mm calvarial bone defects in adult mice using type I collagen sponge as a carrier. New bone formation in the defects was quantified using radiological and histological procedures 5 weeks after implantation. The results showed that implantation of Osx-transduced BMSCs resulted in 85% healing of calvarial bone defects as detected using radiological analyses. Histological examination of the implants demonstrated that the Osx-transduced group exhibited amounts of newly formed bone that was five times as high as in a group transduced with the empty vector. Immunohistochemistry for GFP showed positive immunoreaction localized to areas of newly engineered bone in the Osx-transduced group. Immunohistochemistry with antibodies against the extracellular matrix protein bone sialoprotein resulted in strong staining in areas of new bone formation. In addition, the clonal BMSCs showed an osteogenic potential similar to that of primary cultures of BMSCs, suggesting the usefulness of this model in bone tissue engineering. These results indicate that ex vivo gene therapy of Osx is a useful therapeutic approach in regenerating adult bone tissue.
doi:10.1089/ten.2006.0406
PMCID: PMC2835465  PMID: 17630878
7.  Runx2 Overexpression in Bone Marrow Stromal Cells Accelerates Bone Formation in Critical-Sized Femoral Defects 
Tissue Engineering. Part A  2010;16(9):2795-2808.
The repair of large nonunions in long bones remains a significant clinical problem due to high failure rates and limited tissue availability for auto- and allografts. Many cell-based strategies for healing bone defects deliver bone marrow stromal cells (BMSCs) to the defect site to take advantage of the inherent osteogenic capacity of this cell type. However, many factors, including donor age and ex vivo expansion of the cells, cause BMSCs to lose their differentiation ability. To overcome these limitations, we have genetically engineered BMSCs to constitutively overexpress the osteoblast-specific transcription factor Runx2. In the present study, we examined Runx2-modified BMSCs, delivered via polycaprolactone scaffolds loaded with type I collagen meshes, in critical-sized segmental defects in rats compared to unmodified cells, cell-free scaffolds, and empty defects. Runx2 expression in BMSCs accelerated healing of critical-sized defects compared to unmodified BMSCs and defects receiving cell-free treatments. These findings provide an accelerated method for healing large bone defects, which may reduce recovery time and the need for external fixation of critical-sized defects.
doi:10.1089/ten.tea.2010.0025
PMCID: PMC2928046  PMID: 20412027
8.  Temporal Expression of Pelp1 during Proliferation and Osteogenic Differentiation of Rat Bone Marrow Mesenchymal Stem Cells 
PLoS ONE  2013;8(10):e75477.
Background
Osteogenic induction and bone formation are heavily affected by environmental factors, including estrogen, estrogen receptors, and coregulatory proteins, such as the recently reported proline-, glutamic acid-, and leucine-rich protein 1(Pelp1).
Objective
To investigate Pelp1 expression in rat bone mesenchymal stem cells (rBMSCs) during cell proliferation and osteogenic differentiation.
Methods
rBMSCs were cultured in routine and osteogenic differentiation media. Cell proliferation was assessed at days 1, 3, 5, 7, 9, 11, 14, and 21. Pelp1 protein expression in the nucleus and cytoplasm were detected by immunocytochemical analysis. Real-time RT-PCR and western blot were used to detect mRNA and protein expressions of Pelp1, osteocalcin (OCN), and alkaline phosphatase (ALP).
Results
Over 21 days, rBMSCs in routine culture exhibited a 1-2 day lag phase and exponential growth from day 3 to 9, plateauing at day 9, and correlated with temporal mRNA expression of Pelp1, which almost reached baseline levels at day 21. In osteogenic induction cultures, Pelp1 mRNA levels rose at day 9 and steadily increased until day 21, reaching 6.8-fold greater value compared with day 1. Interestingly, Pelp1 mRNA expression in osteogenic cultures exhibited a trend similar to that of OCN expression. Pelp1 knockdown by siRNA transfection inhibited undifferentiated rBMSC proliferation, and bone markers OCN and ALP expressions in rBMSCs cultured in routine and osteogenic differentiation media.
Conclusions
Pelp1 may be a key player in BMSCs proliferation and osteogenic differentiation, meriting further consideration as a target for development of therapies for pathological bone loss conditions, such as menopausal bone loss.
doi:10.1371/journal.pone.0075477
PMCID: PMC3797710  PMID: 24146754
9.  Modulus-Driven Differentiation of Marrow Stromal Cells in 3D Scaffolds That Is Independent of Myosin-based Cytoskeletal Tension 
Biomaterials  2010;32(9):2256-2264.
Proliferation and differentiation of cells are known to be influenced by the physical properties of the extracellular environment. Previous studies examining biophysics underlying cell response to matrix stiffness utilized a two-dimensional (2D) culture format, which is not representative of the three-dimensional (3D) tissue environment in vivo. We report on the effect of 3D matrix modulus on human bone marrow stromal cell (hBMSC) differentiation. hBMSCs underwent osteogenic differentiation in poly(ethylene glycol) hydrogels of all modulus (300-fold modulus range, from 0.2 kPa to 59 kPa) in the absence of osteogenic differentiation supplements. This osteogenic differentiation was modulus-dependent and was enhanced in stiffer gels. Osteogenesis in these matrices required integrin-protein ligation since osteogenesis was inhibited by soluble Arginine-Glycine-Aspartate-Serine peptide, which blocks integrin receptors. Immunostained images revealed lack of well-defined actin filaments and microtubules in the encapsulated cells. Disruption of mechanosensing elements downstream of integrin-binding that have been identified from 2D culture such as actin filaments, myosin II contraction, and RhoA kinase did not abrogate hBMSC material-driven osteogenic differentiation in 3D. These data show that increased hydrogel modulus enhanced osteogenic differentiation of hBMSCs in 3D scaffolds but that hBMSCs did not use the same mechanosensing pathways that have been identified in 2D culture.
doi:10.1016/j.biomaterials.2010.11.065
PMCID: PMC3381351  PMID: 21176956
three-dimensional culture; bone marrow stromal cell; cytoskeleton; hydrogels; matrix modulus
10.  Effect of Hydrogel Porosity on Marrow Stromal Cell Phenotypic Expression 
Biomaterials  2008;29(14):2193-2202.
This study describes investigation of porous photocrosslinked oligo[(polyethylene glycol) fumarate] (OPF) hydrogels as potential matrix for osteoblastic differentiation of marrow stromal cells (MSCs). The porosity and interconnectivity of porous hydrogels were assessed using magnetic resonance microscopy (MRM) as a noninvasive investigative tool that could image the water construct inside the hydrogels at a high spatial resolution. MSCs were cultured onto the porous hydrogels and cell number was assessed using PicoGreen DNA assay. Our results showed 10% of cells initially attached to the surface of scaffolds. However, cells did not show significant proliferation over a time period of 14 days. MSCs cultured on porous hydrogels had increased alkaline phosphatase activity as well as deposition of calcium, suggesting successful differentiation and maturation to the osteoblastic phenotype. Moreover, continued expression of type I collagen and osteonectin over 14 days confirmed osteoblastic differentiation of MSCs. MRM was also applied to monitor osteogenesis of MSCs on porous hydrogels. MRM images showed porous scaffolds became consolidated with osteogenic progression of cell differentiation. These findings indicate that porous OPF scaffolds enhanced MSC differentiation leading to development of bone-like mineralized tissue.
doi:10.1016/j.biomaterials.2008.01.006
PMCID: PMC2386206  PMID: 18262642
Hydrogel; oligo[(polyethylene glycol) fumarate] (OPF); Marrow stromal cells; Magnetic resonance microscopy; Osteogenesis
11.  Improvement of intertrochanteric bone quality in osteoporotic female rats after injection of polylactic acid-polyglycolic acid copolymer/collagen type I microspheres combined with bone mesenchymal stem cells 
International Orthopaedics  2012;36(10):2163-2171.
Purpose
Osteoporosis mainly involves cancellous bone, and the spine and hip, with their relatively high cancellous bone to cortical bone ratio, are severely affected. Studies of bone mesenchymal stem cells (BMSCs) from osteoporotic patients and animal models have revealed that osteoporosis is often associated with reduction of BMSCs’ proliferation and osteogenic differentiation. Our aim was to test whether polylactic acid-polyglycolic acid copolymer(PLGA)/collagen type I(CoI) microspheres combined with BMSCs could be used as injectable scaffolds to improve bone quality in osteoporotic female rats.
Methods
PLGA microspheres were coated with CoI. BMSCs of the third passage and were cultured with PLGA/CoI microspheres for seven days. Forty three-month-old female non-pregnant SD rats were ovariectomized to establish osteoporotic animal models. Three months after being ovariectomized, the osteoporotic rats were randomly divided into five groups: SHAM group, PBS group, cell group, microsphere (MS) group, and cell+MS group. Varying materials were injected into the intertrochanters of each group’s rats. Twenty rats were sacrificed at one month and three months post-op, respectively. The femora were harvested in order to measure the intertrochanteric bone mineral density (BMD) with DEXA and trabecular thickness (Tb.Th), percentage of trabecular area (%Tb.Ar), bone volume fraction (BV/TV) and trabecular spacing (Tb.Sp) with Micro CT. One-way ANOVA and Kruskal-Wallis tests were used.
Results
BMSCs seeded on PLGA/CoI microspheres had a nice adhesion and proliferation. At one month post-op, the BMD (0.33 ± 0.01 g/cm2), Tb.Th (459.65 ± 28.31 μm), %Tb.Ar (9.61 ± 0.29 %) and Tb.Sp (2645.81 ± 94.91 μm) of the cell+ MS group were better than those of the SHAM group and the cell group. At three months post-op, the BMD (0.32 ± 0.01 g/cm2), Tb.Th (372.81 ± 38.45 μm), %Tb.Ar (6.65 ± 0.25 %), BV/TV (6.62 ± 0.25 %) and Tb.Sp (1559.03 ± 57.06 μm) of the cell + MS group were also better than those of the SHAM group and the cell group.
Conclusion
The PLGA/CoI microspheres combined with BMSCs can repair bone defects more quickly. This means that PLGA/CoI microspheres combined with BMSCs can promote trabecular reconstruction and improve bone quality in osteoporotic rats. This scaffold can provide a promising minimally invasive surgical tool for enhancement of bone fracture healing or prevention of fracture occurrence which will in turn minimize complications endemic to patients with osteoporosis.
doi:10.1007/s00264-012-1543-4
PMCID: PMC3460074  PMID: 22539160
12.  Osteogenic Potential of Mandibular vs. Long-bone Marrow Stromal Cells 
Journal of dental research  2010;89(11):1293-1298.
Although fundamentally similar to other bones, the jaws demonstrate discrete responses to developmental, mechanical, and homeostatic regulatory signals. Here, we hypothesized that rat mandible vs. long-bone marrow-derived cells possess different osteogenic potential. We established a protocol for rat mandible and long-bone marrow stromal cell (BMSC) isolation and culture. Mandible BMSC cultures formed more colonies, suggesting an increased CFU-F population. Both mandible and long-bone BMSCs differentiated into osteoblasts. However, mandible BMSCs demonstrated augmented alkaline phosphatase activity, mineralization, and osteoblast gene expression. Importantly, upon implantation into nude mice, mandible BMSCs formed 70% larger bone nodules containing three-fold more mineralized bone compared with long-bone BMSCs. Analysis of these data demonstrates an increased osteogenic potential and augmented capacity of mandible BMSCs to induce bone formation in vitro and in vivo. Our findings support differences in the mechanisms underlying mandible homeostasis and the pathophysiology of diseases unique to the jaws.
doi:10.1177/0022034510378427
PMCID: PMC3113466  PMID: 20811069
marrow stromal cells; BMSCs; mandible; long bone; osteogenic
13.  Osteogenic Potential of Mandibular vs. Long-bone Marrow Stromal Cells 
Journal of Dental Research  2010;89(11):1293-1298.
Although fundamentally similar to other bones, the jaws demonstrate discrete responses to developmental, mechanical, and homeostatic regulatory signals. Here, we hypothesized that rat mandible vs. long-bone marrow-derived cells possess different osteogenic potential. We established a protocol for rat mandible and long-bone marrow stromal cell (BMSC) isolation and culture. Mandible BMSC cultures formed more colonies, suggesting an increased CFU-F population. Both mandible and long-bone BMSCs differentiated into osteoblasts. However, mandible BMSCs demonstrated augmented alkaline phosphatase activity, mineralization, and osteoblast gene expression. Importantly, upon implantation into nude mice, mandible BMSCs formed 70% larger bone nodules containing three-fold more mineralized bone compared with long-bone BMSCs. Analysis of these data demonstrates an increased osteogenic potential and augmented capacity of mandible BMSCs to induce bone formation in vitro and in vivo. Our findings support differences in the mechanisms underlying mandible homeostasis and the pathophysiology of diseases unique to the jaws.
doi:10.1177/0022034510378427
PMCID: PMC3113466  PMID: 20811069
marrow stromal cells; BMSCs; mandible; long bone; osteogenic
14.  An Oligodeoxynucleotide That Induces Differentiation of Bone Marrow Mesenchymal Stem Cells to Osteoblasts in Vitro and Reduces Alveolar Bone Loss in Rats with Periodontitis 
To investigate the effect of oligodeoxynucleotides (ODNs) on the differentiation of rat bone marrow mesenchymal stem cells (BMSCs) to osteoblasts, in order to find a candidate ODN with potential for the treatment of periodontitis, a series of ODNs were designed and selected to test their effect on the promotion of the differentiation of BMSCs to osteoblasts in vitro and on the repair of periodontal tissue in rats with periodontitis. It was found that MT01, one of the ODNs with the sequences of human mitochondrial DNA, stimulated the proliferation of BMSCs, the differentiation of BMSCs to osteoblasts and mRNA expression of bone-associated factors including Runx2, Osterix, OPG, RANKL and collagen I in vitro. In vivo study showed that MT01 prevented the loss of alveolar bone in the rats with periodontitis and induced the production of proteins of OPG and Osterix in the bone tissue. These results indicated that MT01 could induce differentiation of BMSCs to osteoblasts and inhibit the alveolar bone absorption in rats with periodontitis.
doi:10.3390/ijms13032877
PMCID: PMC3317693  PMID: 22489131
oligodeoxynucleotide; bone marrow mesenchymal stem cells; osteoblasts; differentiation; periodontitis
15.  Construction of Mesenchymal Stem Cell–Containing Collagen Gel with a Macrochanneled Polycaprolactone Scaffold and the Flow Perfusion Culturing for Bone Tissue Engineering 
BioResearch Open Access  2012;1(3):124-136.
Abstract
A novel bone tissue-engineering construct was developed by using poly(ɛ-caprolactone) (PCL)-macrochanneled scaffolds combined with stem cell-seeded collagen hydrogels and then applying flow perfusion culture. Rat mesenchymal stem cells (MSCs) were loaded into collagen hydrogels, which were then combined with macrochanneled PCL scaffolds. Collagen hydrogels were demonstrated to provide favorable growth environments for MSCs and to foster proliferation. Cell number determination identified retention of substantially fewer (50–60%) cells when they were seeded directly onto macrochanneled PCL than of cells engineered within collagen hydrogels. Additionally, the cells actively proliferated within the combined scaffold for up to 7 days. MSC-loaded collagen–PCL scaffolds were subsequently cultured under flow perfusion to promote proliferation and osteogenic differentiation. Cells proliferated to levels significantly higher in flow perfusion culture than that under static conditions during 21 days. A quantitative polymerase chain reaction (QPCR) assay revealed significant alterations in the transcription of bone-related genes such as osteopontin (OPN), osteocalcin (OCN), and bone sialoprotein (BSP), such as 8-, 2.5-, and 3-fold induction, respectively, after 10 days of flow perfusion relative to those in static culture. OPN and OCN protein levels, as determined by Western blot, increased under flow perfusion. Cellular mineralization was significantly enhanced by the flow perfusion during 21 and 28 days. Analyses of mechanosensitive gene expression induced by flow perfusion shear stress revealed significant upregulation of c-fos and cyclooxygenase-2 (COX-2) during the initial culture period (3–5 days), suggesting that osteogenic stimulation was possible as a result of mechanical force-driven transduction. These results provide valuable information for the design of a new bone tissue-engineering system by combining stem cell-loaded collagen hydrogels with macrochanneled scaffolds in flow perfusion culture.
doi:10.1089/biores.2012.0234
PMCID: PMC3559226  PMID: 23515189
3D scaffolds; bone tissue engineering; collagen gel; flow perfusion; osteogenic differentiation
16.  Evaluating osteochondral defect repair potential of autologous rabbit bone marrow cells on type II collagen scaffold 
Cytotechnology  2010;63(1):13-23.
The feasibility of using genipin cross-linked type II collagen scaffold with rabbit bone marrow mesenchymal stem cells (RBMSCs) to repair cartilage defect was herein studied. Induction of RBMSCs into chondrocytic phenotype on type II collagen scaffold in vitro was conducted using TGF-β 3 containing medium. After 3-weeks of induction, chondrocytic behavior, including marker genes expression and specific extracellular matrix (ECM) secretion, was observed. In the in vivo evaluation experiment, the scaffolds containing RBMSCs without prior induction were autologous implanted into the articular cartilage defects made by subchondral drilling. The repairing ability was evaluated. After 2 months, chondrocyte-like cells with lacuna structure and corresponding ECM were found in the repaired sites without apparent inflammation. After 24 weeks, we could easily find cartilage structure the same with normal cartilage in the repair site. In conclusion, it was shown that the scaffolds in combination of in vivo conditions can induce RBMSCs into chondrocytes in repaired area and would be a possible method for articular cartilage repair in clinic and cartilage tissue engineering.
doi:10.1007/s10616-010-9314-9
PMCID: PMC3021150  PMID: 20972620
Cartilage defect; Type II collagen scaffold; Rabbit bone marrow mesenchymal stem cells; Chondrocyte-like cells; In vivo
17.  Osteogenic Differentiation of Human Bone Marrow Stromal Cells in Hydroxyapatite-Loaded Microsphere-Based Scaffolds 
Tissue Engineering. Part A  2011;18(7-8):757-767.
Calcium-based minerals have consistently been shown to stimulate osteoblastic behavior in vitro and in vivo. Thus, use of such minerals in biomaterial applications has become an effective method to enhance bone tissue engineered constructs. In the present study, for the first time, human bone marrow stromal cells (hBMSC) were osteogenically differentiated on scaffolds consisting only of hydroxyapatite (HAp)-loaded poly(d,l-lactic acid-co-glycolic acid) (PLGA) microspheres of high monodispersity. Scaffold formulations included 0, 5, 10, and 20 wt% Hap, and the hBMSC were cultured for 6 weeks. Results demonstrated suppression of some osteogenic genes during differentiation in the HAp group, but higher end-point glycosaminoglycan and collagen content in 10% and 20% HAp samples, as evidenced by biochemical tests, histology, and immunohistochemistry. After 6 weeks of culture, constructs with 0% and 5% HAp had average compressive moduli of 0.7±0.2 and 1.5±0.9 kPa, respectively, whereas constructs with 10% and 20% HAp had higher average moduli of 17.6±4.6 and 18.9±8.1 kPa, respectively. The results of this study indicate that HAp inclusion in microsphere-based scaffolds could be implemented as a physical gradient in combination with bioactive signal gradients seen in previous iterations of these microsphere-based scaffolds to enhance osteoconduction and mechanical integrity of a healing site.
doi:10.1089/ten.tea.2011.0176
PMCID: PMC3313610  PMID: 21992088
18.  A novel, visible light-induced, rapidly cross-linkable gelatin scaffold for osteochondral tissue engineering 
Scientific Reports  2014;4:4457.
Osteochondral injuries remain difficult to repair. We developed a novel photo-cross-linkable furfurylamine-conjugated gelatin (gelatin-FA). Gelatin-FA was rapidly cross-linked by visible light with Rose Bengal, a light sensitizer, and was kept gelled for 3 weeks submerged in saline at 37°C. When bone marrow-derived stromal cells (BMSCs) were suspended in gelatin-FA with 0.05% Rose Bengal, approximately 87% of the cells were viable in the hydrogel at 24 h after photo-cross-linking, and the chondrogenic differentiation of BMSCs was maintained for up to 3 weeks. BMP4 fusion protein with a collagen binding domain (CBD) was retained in the hydrogels at higher levels than unmodified BMP4. Gelatin-FA was subsequently employed as a scaffold for BMSCs and CBD-BMP4 in a rabbit osteochondral defect model. In both cases, the defect was repaired with articular cartilage-like tissue and regenerated subchondral bone. This novel, photo-cross-linkable gelatin appears to be a promising scaffold for the treatment of osteochondral injury.
doi:10.1038/srep04457
PMCID: PMC3964514  PMID: 24662725
19.  Pleiotrophin Commits Human Bone Marrow Mesenchymal Stromal Cells towards Hypertrophy during Chondrogenesis 
PLoS ONE  2014;9(2):e88287.
Pleiotrophin (PTN) is a growth factor present in the extracellular matrix of the growth plate during bone development and in the callus during bone healing. Bone healing is a complicated process that recapitulates endochondral bone development and involves many cell types. Among those cells, mesenchymal stromal cells (MSC) are able to differentiate toward chondrogenic and osteoblastic lineages. We aimed to determine PTN effects on differentiation properties of human bone marrow stromal cells (hBMSC) under chondrogenic induction using histological analysis and quantitative reverse transcription polymerase chain reaction. PTN dramatically potentiated chondrogenic differentiation as indicated by a strong increase of collagen 2 protein, and cartilage-related gene expression. Moreover, PTN increased transcription of hypertrophic chondrocyte markers such as MMP13, collagen 10 and alkaline phosphatase and enhanced calcification and the content of collagen 10 protein. These effects are dependent on PTN receptors signaling and PI3 K pathway activation. These data suggest a new role of PTN in bone regeneration as an inducer of hypertrophy during chondrogenic differentiation of hBMSC.
doi:10.1371/journal.pone.0088287
PMCID: PMC3917886  PMID: 24516627
20.  Potential of Hydrogels Based on Poly(Ethylene Glycol) and Sebacic Acid as Orthopedic Tissue Engineering Scaffolds 
Tissue Engineering. Part A  2009;15(8):2299-2307.
In this study, the bioactive effects of poly(ethylene glycol) (PEG) sebacic acid diacrylate (PEGSDA) hydrogels with or without RGD peptide modification on osteogenic differentiation and mineralization of marrow stromal cells (MSCs) were examined. In a separate experiment, the ability of PEGSDA hydrogel to serve as a delivery vehicle for bone morphogenetic protein 2 (BMP-2) was also investigated. As a scaffold, the attachment and proliferation of MSCs on PEGSDA hydrogel scaffolds with and without RGD peptide modification was similar to the control, tissue culture polystyrene. In contrast, cells were barely seen on unmodified PEG diacrylate (PEGDA) hydrogel throughout the culture period for up to 21 days. Osteogenic phenotypic expression such as alkaline phosphatase (ALP) of MSCs as well as mineralized calcium content were significantly higher on PEGSDA-based hydrogels than those on the control or PEGDA hydrogels. Potential use of PEGSDA scaffold as a delivery vehicle of osteogenic molecules such as BMP-2 was also evaluated. Initial burst release of BMP-2 from PEGSDA hydrogel scaffold (14.7%) was significantly reduced compared to PEGDA hydrogel scaffold (84.2%) during the first 3 days of a 21-day release period. ALP activity of an osteoblast was significantly higher in the presence of BMP-2 released from PEGSDA hydrogel scaffolds compared to that in the presence of BMP-2 released from PEGDA scaffolds, especially after 6 days of release. Overall, PEGSDA hydrogel scaffolds without further modification may be useful as orthopedic tissue engineering scaffolds as well as local drug carriers for prolonged sustained release of osteoinductive molecules.
doi:10.1089/ten.tea.2008.0326
PMCID: PMC2792107  PMID: 19292677
21.  The role of osteoblast cells in the pathogenesis of unicameral bone cysts 
Purpose
The pathogenesis of unicameral bone cysts (UBCs) remains largely unknown. Osteoclasts have been implicated, but the role of osteoblastic cells has, to date, not been explored. This study investigated the pathophysiology of UBCs by examining the interactions between the cyst fluid and human bone marrow stromal cells (hBMSCs) and the effect of the fluid on osteogenesis.
Methods
Fluid was aspirated from two UBCs and analysed for protein, electrolyte and cytokine levels. Graded concentrations of the fluid were used as culture media for hBMSCs to determine the effects of the fluid on hBMSC proliferation and osteogenic differentiation. The fibrocellular lining was analysed histologically and by electron microscopy.
Results
Alkaline phosphatase (ALP) staining of hBMSCs that were cultured in cyst fluid demonstrated increased cell proliferation and osteogenic differentiation compared to basal media controls. Biochemical analysis of these hBMSCs compared to basal controls confirmed a marked increase in DNA content (as a marker of proliferation) and ALP activity (as a marker of osteogenic differentiation) which was highly significant (p < 0.001). Osteoclasts were demonstrated in abundance in the cyst lining. The cyst fluid cytokine profile revealed levels of the pro-osteoclast cytokines IL-6, MIP-1α and MCP-1 that were 19×, 31× and 35× greater than those in reference serum.
Conclusions
Cyst fluid promoted osteoblastic growth and differentiation. Despite appearing paradoxical that the cyst fluid promoted osteogenesis, osteoblastic cells are required for osteoclastogenesis through RANKL signalling. Three key cytokines in this pathway (IL-6, MIP-1α, MCP-1) were highly elevated in cyst fluid. These findings may hold the key to the pathogenesis of UBCs, with implications for treatment methods.
doi:10.1007/s11832-012-0419-x
PMCID: PMC3425701  PMID: 23904902
Unicameral bone cyst; Osteoblast cell; Osteoclast; Cytokine; RANK ligand
22.  Hydrogel-mediated DNA delivery confers estrogenic response in non-responsive osteoblast cells 
Oligo (polyethylene glycol) fumarate (OPF) hydrogel has been employed in musculoskeletal tissue engineering for photo-encapsulation of chondrocytes and as a matrix for marrow stromal cells differentiation. In this study, we have studied the application of OPF hydrogel for co-encapsulation of DNA and bone cells and examined whether co-encapsulation can enhance gene transfer by maintaining the DNA within the cellular microenvironment. Our results showed that plasmid DNA encoding green fluorescence protein (GFP), co-encapsulated with bone tumor cells, was capable of transfecting the cells and the transfected tumor cells continuously expressed GFP protein over the time course of study (21 days). Furthermore, we have examined the co-encapsulation of estrogen receptor (ER) encoding plasmid DNA and human fetal osteoblast cells (hFOB) that lack endogenous ER. Our results show that the transfected cells responded to estrogen as alkaline phosphatase (ALP), and estrogen response element (ERE)-directed luciferase enzyme activities increased with estrogen-treatment. Taken together, these studies show that OPF hydrogel could be further explored for targeted gene delivery in bone and other tissues encapsulated within the hydrogels.
doi:10.1002/jbm.a.32291
PMCID: PMC2783666  PMID: 19148929
Bone tissue engineering; DNA delivery, Hydrogel; Osteoblast, Estrogen receptor
23.  Bone Morphogenetic Protein Receptor in the Osteogenic Differentiation of Rat Bone Marrow Stromal Cells 
Yonsei Medical Journal  2010;51(5):740-745.
Purpose
Several signaling pathways have been shown to regulate the lineage commitment and terminal differentiation of bone marrow stromal cells (BMSCs). Bone morphogenetic protein (BMP) signaling has important effects on the process of skeletogenesis. In the present study, we tested the role of bone morphogenetic protein receptor (BMPR) in the osteogenic differentiation of rat bone marrow stromal cells in osteogenic medium (OM) with or without BMP-2.
Materials and Methods
BMSCs were harvested from rats and cultured in OM containing dexamethasone, β-glycerophosphate, and ascorbic acid, with or without BMP-2 in order to induce osteogenic differentiation. The alkaline phosphatase (ALP) activity assay and von kossa staining were used to assess the osteogenic differentiation of the BMSCs. BMPR mRNA expression was assessed using reverse transcription-polymerase chain reaction (RT-PCR).
Results
The BMSCs that underwent osteogenic differentiation in OM showed a higher level of ALP activity and matrix mineralization. BMP-2 alone induced a low level of ALP activity and matrix mineralization in BMSCs, but enhanced the osteogenic differentiation of BMSCs when combined with OM. The OM significantly induced the expression of type IA receptor of BMPR (BMPRIA) and type II receptor of BMPR (BMPRII) in BMSCs after three days of stimulation, while BMP-2 significantly induced BMPRIA and BMPRII in BMSCs after nine or six days of stimulation, respectively.
Conclusion
BMSCs commit to osteoblastic differentiation in OM, which is enhanced by BMP-2. In addition, BMP signaling through BMPRIA and BMPRII regulates the osteogenic differentiation of rat BMSCs in OM with or without BMP-2.
doi:10.3349/ymj.2010.51.5.740
PMCID: PMC2908870  PMID: 20635450
Bone marrow stromal cells; osteogenesis; differentiation; bone morphogenetic protein receptor
24.  Regulation of human bone marrow stromal cell proliferation and differentiation capacity by glucocorticoid receptor and AP-1 crosstalk 
Journal of Bone and Mineral Research  2010;25(10):2115-2125.
Although marrow adipocytes and osteoblasts derive from a common bone marrow stromal cells (BMSCs), the mechanisms that underlie osteoporosis-associated bone loss and marrow adipogenesis during prolonged steroid treatment are unclear. We show in human BMSCs (hBMSCs) that glucocorticoid receptor (GR) signaling in response to high concentrations of glucocorticoid (GC) supports adipogenesis but inhibits osteogenesis by reducing c-Jun expression and hBMSC proliferation. Conversely, significantly lower concentrations of GC, which permit hBMSC proliferation, are necessary for normal bone mineralization. In contrast, platelet-derived growth factor (PDGF) signaling increases both JNK/c-Jun activity and hBMSC expansion, favoring osteogenic differentiation instead of adipogenesis. Indeed, PDGF antagonizes the proadipogenic qualities of GC/GR signaling. Thus our results reveal a novel c-Jun-centered regulatory network of signaling pathways in differentiating hBMSCs that controls the proliferation-dependent balance between osteogenesis and adipogenesis.
doi:10.1002/jbmr.120
PMCID: PMC3607410  PMID: 20499359
osteoblasts and stem cells; bone; adipose; corticosteroid osteoporosis
25.  Adult Bone Marrow Stromal Cell-Based Tissue-Engineered Aggrecan Exhibits Ultrastructure and Nanomechanical Properties Superior to Native Cartilage 
Summary
Objective
To quantify the structural characteristics and nanomechanical properties of aggrecan produced by adult bone marrow stromal cells (BMSCs) in peptide hydrogel scaffolds and compare to aggrecan from adult articular cartilage.
Design
Adult equine BMSCs were encapsulated in 3D-peptide hydrogels and cultured for 21 days with TGF-β1 to induce chondrogenic differentiation. BMSC-aggrecan was extracted and compared with aggrecan from age-matched adult equine articular cartilage. Single molecules of aggrecan were visualized by atomic force microcopy-based imaging and aggrecan nanomechanical stiffness was quantified by high resolution force microscopy. Population-averaged measures of aggrecan hydrodynamic size, core protein structures and CS sulfation compositions were determined by size-exclusion chromatography, Western analysis, and fluorescence-assisted carbohydrate electrophoresis (FACE).
Results
BMSC-aggrecan was primarily full-length while cartilage-aggrecan had many fragments. Single molecule measurements showed that core protein and GAG chains of BMSC-aggrecan were markedly longer than those of cartilage-aggrecan. Comparing full-length aggrecan of both species, BMSC-aggrecan had longer GAG chains, while the core protein trace lengths were similar. FACE analysis detected a ∼1:1 ratio of chondroitin-4-sulfate to chondroitin-6-sulfate in BMSC-GAG, a phenotype consistent with aggrecan from skeletally-immature cartilage. The nanomechanical stiffness of BMSC-aggrecan was demonstrably greater than that of cartilage-aggrecan at the same total sGAG (fixed charge) density.
Conclusions
The higher proportion of full-length monomers, longer GAG chains and greater stiffness of the BMSC-aggrecan makes it biomechanically superior to adult cartilage-aggrecan. Aggrecan stiffness was not solely dependent on fixed charge density, but also on GAG molecular ultrastructure. These results support the use of adult BMSCs for cell-based cartilage repair.
doi:10.1016/j.joca.2010.07.015
PMCID: PMC2975943  PMID: 20692354
Aggrecan; Bone-marrow stromal cell; Cartilage repair; Tissue engineering; Self-assembling peptide; Molecular Nanomechanical properties

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