Tae Hyung Cho

Novel Effect of Biphasic Electric Current on In Vitro Osteogenesis and Cytokine Production in Human Mesenchymal Stromal Cells

Electrical stimulation (ES) can activate diverse biostimulatory responses in a range of tissues. Of various forms of ES, the application of biphasic electric current (BEC) is a new approach to bone formation. This study is to investigate the effects and mechanism of action of BEC in osteoblast differentiation and cytokine production in human mesenchymal stromal cells (hMSCs). Using an in vitro culture system with a modified version of the BEC stimulator chip used in our previous study, we exposed hMSCs to a 100 Hz ES with a magnitude of 1.5/15 muA/cm(2) for 250/25 mus. hMSCs showed increased proliferation during static BEC stimulation for 5 days. However, alkaline phosphatase activity and calcium deposition were enhanced in hMSCs 7 days after the stimulation, rather than during the period of ES. BEC induced vascular endothelial growth factor (VEGF) and BMP-2 production; the former can enhance the proliferation of human umbilical vein endothelial cells in culture using conditioned media from BEC cultures. Treatment with selective inhibitors of p38 MAPK (SB203580) or Erk (PD98059), as well as calcium channel blockers (verapamil and nifedipine), reduced the BEC-mediated increase of VEGF expression and cell proliferation. These findings reveal that BEC is involved in the osteoblast differentiation of hMSCs through enhancement of cell proliferation and modulation of the local endocrine environment through VEGF and BMP-2 induction through the activation of MAPK (Erk and p38) and the calcium channel. Thus, local stimulation using BEC might be most beneficial in promoting osteogenic differentiation of hMSCs, resulting in enhanced bone formation for bone tissue engineering.

In vivo evaluation of MMP sensitive high-molecular weight HA-based hydrogels for bone tissue engineering.

Hyaluronic acid (170 kDa)-based hydrogel was synthesized using acrylated hyaluronic acid (HA) and matrix metalloproteinase (MMP) sensitive HA-based hydrogels were then prepared by conjugation with two different peptides: cell adhesion peptides containing integrin-binding domains (Arg-Gly-Asp: RGD) and a cross-linker with MMP degradable peptides to mimic the remodeling characteristics of natural extracellular matrices by cell-derived MMPs. Mechanical properties of these hydrogels were evaluated with different weight percentages (2.5 and 3.5 wt %) by measuring elastic modulus, viscous modulus, and swelling ratio. Human mesenchymal stem cells (hMSCs) were then cultured in MMP-sensitive or insensitive HA-based hydrogels and/or immobilized cell adhesive RGD peptides in vitro. Actin staining and image analysis proved that cells cultured in the MMP-sensitive hydrogel with RGD peptides showed extensive cell spreading and sprouting. Gene expression analysis showed that bone specific genes such as alkaline phosphatase, osteocalcin, and osteopontin increased in MMP-sensitive hydrogels as biomolecules such as BMPs and cells were added in the gels. For in vivo calvarial defect regeneration, five different samples (MMP insensitive hydrogel, MMP sensitive hydrogel, MMP sensitive hydrogel with BMP-2, MMP sensitive hydrogel with hMSC, and MMP sensitive hydrogel with BMP-2 and hMSC) were prepared. After 4 weeks of implantation, the Masson-Trichrome staining and micro computed tomography scan results demonstrated that the MMP sensitive hydrogels with BMP-2 and hMSCs have the highest mature bone formation. The MMP sensitive HA-based hydrogel could become useful scaffolds in bone tissue engineering with improvements on tissue remodeling rates and regeneration activity.

In vitro response of primary human bone marrow stromal cells to recombinant human bone morphogenic protein-2 in the early and late stages of osteoblast differentiation.

A number of factors must be added to human bone marrow stromal cells (hBMSCs) in vitro to induce osteogenesis, including ascorbic acid (AA), β‐glycerophosphate (GP), and dexamethasone (Dex). Bone morphogenic protein (BMP)‐2 is an osteoinductive factor that can commit stromal cells to differentiate into osteoblasts. However, it is still not clear whether the addition of BMP‐2 alone in vitro can induce hBMSCs to complete osteoblast differentiation, resulting in matrix mineralization. This study compares the effects of BMP‐2 and Dex, alone and combined, on the early and late stages of hBMSC differentiation. We found that BMP‐2 causes a significant induction of alkaline phosphatase (ALP) activity in hBMSCs, with a transcriptional upregulation of known BMP‐2‐responsive genes, and the stable expression of cbfa1 in the nucleus and the regions surrounding the nucleus in the early phase of osteoblast differentiation. However, continuous treatment with BMP‐2 alone at doses ranging from 100 to 300 ng/mL results in a less efficient enhancement of in vitro matrix mineralization, despite a significant induction of ALP activity at a concentration of 100 ng/mL. Our results reflect how the effects of BMP‐2 on hBMSCs can vary depending on the stage of osteoblast differentiation, and highlight the need to understand the role of BMP‐2 in primary hBMSCs derived from diverse sources in order to increase the efficiency of using BMP‐2 in osteoinductive therapies.

Effects of recombinant human bone morphogenic protein‐2 and human bone marrow‐derived stromal cells on in vivo bone regeneration of chitosan–poly(ethylene oxide) hydrogel

In vivo bone regeneration of chitosan-poly(ethylene oxide) (PEO) hydrogel in rat carlvarial defects was evaluated by using both human bone marrow-derived stromal cells (hMSCs) and recombinant human bone marrow protein-2 (rhBMP-2) for 4 and 8 weeks. In situ chitosan-PEO hydrogel was fabricated by mixing the precursor solutions of both chitosan-acrylate and PEO-thiol. Fabrication of the injectable hydrogels was modulated from within a minute to hours by controlling the temperature and pHs of the precursor solution. Gel swellings were dependent on the conditions of pHs and temperatures of the precursor solutions, showing higher gel swelling in basic water than in either acidic or neutral water. The compression strengths and in vitro degradation of hydrogels were also evaluated by controlling the concentrations of both precursor solutions and lysozyme, respectively, by referencing to the morphology of the control hydrogel with no enzyme added. Hydrogels showed sustained release of rhodamine-B over time. After implantation of the injectable hydrogels in rat calvarial defects for 4 and 8 weeks, in vivo bone regenerations were compared with by evaluating the degrees of new bone formations with Soft X-ray, microcomputed tomography, and histological stainings of hematoxylin and eosine Y and Massons trichrome. Degrees of in vivo bone regeneration were controlled by encapsulating in advance either hMSCs, rhBMP-2, or both in the precursor solutions of the hydrogel. The defect implanted with hydrogel only showed higher amount of bone tissue regeneration than that of the control defect site. The defect sites with hydrogel containing both hMSCs and rhBMP-2 demonstrated highest amount of bone tissue regeneration among the samples.

High power-pulsed Nd:YAG laser as a new stimulus to induce BMP-2 expression in MC3T3-E1 osteoblasts.

High‐power laser has recently become a physical stimulus for bone regeneration. Little is known about how high‐power laser irradiation affects osteoblast differentiation. This study investigated osteoblast responses to high‐power laser and combined irradiation with BMP‐2 treatment.

An electronic device for accelerating bone formation in tissues surrounding a dental implant

A dental implant is a unique structure which can be used with a noninvasive method because it is inserted into the bone in part and extended extracorporally. This study presents an electronic device that is temporarily connected with the dental implant, and reports its effect on accelerating bone formation in the surrounding tissues in a canine mandibular model. A small sized and low power consumption biphasic electrical current (BEC) stimulator ASIC was developed and the surrounding tissue was exposed to continuous BEC stimulation for 7 days with the parameters of 20 microA/cm(2), 125 micros duration, and 100 pulses/s. After 2 (n = 5) and 5 weeks (n = 5), animals were sacrificed and the specimens were histomorphometrically evaluated. The newly formed bone area (BA) was 1.30 times (3 weeks, P < 0.05) and 1.35 times (5 weeks, P < 0.05) higher in the experimental group compared to the control group, respectively. Bone-implant contact (BIC) in 3-week specimens was 1.62 times (P < 0.05) greater in the experimental group, while there was no statistically significant difference in 5-week specimens. Based on these results showing accelerated bone formation on and around the dental implant, it could be suggested that the latent time for osseointegration in dental implants can be reduced, and the success rate of implants in poor quality bone can be increased by using our device with BEC.

Synergistic action of static stretching and BMP-2 stimulation in the osteoblast differentiation of C2C12 myoblasts

Static stretching is a major type of mechanical stimuli utilized during distraction osteogenesis (DO), a general surgical method for the lengthening of bone. The molecular signals that drive the regenerative process in DO include a variety of cytokines. Among these, bone morphogenic protein (BMP, -2 and -4) has been reported to exhibit strongly enhanced expression following the application of mechanical strain during the distraction phase. We hypothesize that mechanical stretching enhances osteoblast differentiation in DO by means of interaction with BMP-2 induced cytokine stimulation. C2C12 pluripotential myoblasts were exposed to stretching load and the resulting cell proliferation and osteoblast differentiation were then examined. The application of static stretching force resulted in significant cell proliferation at day 3, although with variable intensity according to the magnitude of stretching. A combined treatment of stretching load with BMP-2 stimulation significantly increased alkaline phosphatase (ALP) activity and up-regulated the gene expression of osteogenic markers (ALP, type I collagen, osteopontin, osteocalcin, cbfa1, osterix and dlx5). Results obtained with the combined treatment yielded more activity than just the BMP-2 treatment or stretching alone. These results reveal that specific levels of static stretching force increase cell proliferation and effectively stimulate the osteoblast differentiation of C2C12 cells in conjunction with BMP-2 stimulation, thus indicating a synergistic interaction between mechanical strain and cytokine signaling.

Functional regeneration of a severed peripheral nerve with a 7-mm gap in rats through the use of an implantable electrical stimulator and a conduit electrode with collagen coating.

Objective:  This paper examined the efficacy of an implantable electrical stimulator in rats for the functional regeneration of peripheral nerves.

Comparison of a synthetic bone substitute composed of carbonated apatite with an anorganic bovine xenograft in particulate forms in a canine maxillary augmentation model.

OBJECTIVE To evaluate the effect of a porous geometry in particulate bone on new bone formation by comparison of anorganic bovine carbonate apatite (ABCA) with synthetic carbonated apatite (SCA), which have similar properties but different micro-structures. MATERIAL AND METHODS Porous structures and anorganic components of ABCA and SCA were evaluated using scanning electron microscope and Fourier transform infrared. They were implanted in maxillary augmentation models with the mouth split design in a total of 15 Beagle dogs. The animals were sacrificed 4, 8 and 16 weeks after surgery, and the histomorphometrical results were statistically analyzed for the materials geometrical relationship and new bone formation in relation to the available space and contact surface for osteoconduction. RESULTS Both materials showed a typical infrared pattern of CO(3)(2-) -substituted hydroxyapatite (HA). Porous structures and a bridging effect of osteoconductive bone material were relatively better observed in SCA. The ratio of the material area to the total area was higher (P<0.01) for ABCA (28.03±6.09) than for SCA (20.26±4.23). The ratio of the number of particles possessing a pore structure to the total number and the interparticular space was greater (P<0.001 and 0.01) for SCA (18.12±9.44 and 79.74±4.23) compared with ABCA (1.45±1.74 and 71.63±5.85). The new bone areas and the bone-material contact lengths were greater in SCA than in ABCA (P<0.05). CONCLUSIONS The present study showed that porous structures may have an influence on new bone formation in osteoconductive bone substitutes.

Comparative study of BMP-2 alone and combined with VEGF carried by hydrogel for maxillary alveolar bone regeneration

The effect of vascular endothelial growth factor (VEGF) combined with bone morphogenetic protein-2 (BMP-2) for bone regeneration is still controversial as to whether or not VEGF has a synergistic or additive effect. This study attempted to evaluate the synergistic effect of VEGF and BMP-2 compared to BMP-2 alone for maxillary alveolar bone regeneration using collagen sponge/hydrogel complex sheets in a canine model. After mixing BMP-2 and VEGF with a hyaluronic acid-based hydrogel (HAH), the collagen sponge/hydrogel complex was transplanted into maxillary alveolar bone defects (n=14) after the extraction of canine upper first molars on both sides. Bone regeneration was evaluated in three groups (control group without growth factors, experimental groups I and II with BMP-2 alone and BMP-2 and VEGF, respectively) using micro-computed tomography and histological staining. The total amount of new bone formations and bone mineral density were significantly higher in the group with BMP-2 only and the group with BMP-2 combined with VEGF than it in the control group. The area with positive staining of von Willebrand factor bone defect was significantly greater in the group with BMP-2 only and with dual growth factors than the control. BMP-2 released from the HAH promoted new bone formation. However, the combination of BMP-2 and VEGF did not show a synergistic or additive effect on bone regeneration at canine maxillary alveolar bone defects.