Implantable electrical stimulation bioreactor with liquid crystal polymer-based electrodes for enhanced bone regeneration at mandibular large defects in rabbit.

The osseous regeneration of large bone defects is still a major clinical challenge in maxillofacial and orthopedic surgery. Previous studies demonstrated that biphasic electrical stimulation (ES) stimulates bone formation; however, polyimide electrode should be removed after regeneration. This study presents an implantable electrical stimulation bioreactor with electrodes based on liquid crystal polymer (LCP), which can be permanently implanted due to excellent biocompatibility to bone tissue. The bioreactor was implanted into a critical-sized bone defect and subjected to ES for one week, where bone regeneration was evaluated four weeks after surgery using micro-CT. The effect of ES via the bioreactor was compared with a sham control group and a positive control group that received recombinant human bone morphogenetic protein (rhBMP)-2 (20 µg). New bone volume per tissue volume (BV/TV) in the ES and rhBMP-2 groups increased to 132% (p < 0.05) and 174% (p < 0.01), respectively, compared to that in the sham control group. In the histological evaluation, there was no inflammation within the bone defects and adjacent to LCP in all the groups. This study showed that the ES bioreactor with LCP electrodes could enhance bone regeneration at large bone defects, where LCP can act as a mechanically resistant outer box without inflammation. Graphical abstract To enhance bone regeneration, a bioreactor comprising collagen sponge and liquid crystal polymer-based electrode was implanted in the bone defect. Within the defect, electrical current pulses having biphasic waveform were applied from the implanted bioreactor.

Local administration of nuclear factor of activated T cells (NFAT) c1 inhibitor to suppress early resorption and inflammation induced by bone morphogenetic protein-2.

Nuclear factor of activated T cells (NFAT)-c1 is known as a key regulator in osteoclast differentiation and immune response. This study is a follow-up to our previous study showing the antiresorptive activity of VIVIT, a peptide type NFATc1 inhibitor, using absorbable collagen sponge (ACS). This study aimed to investigate the effective concentration range of local VIVIT that suppresses early excessive osteoclast activation and inflammation induced by high-dose recombinant human bone morphogenetic protein (rhBMP)-2 and concomitantly enhances bone healing in a rat critical-sized calvaria defect model. High-dose rhBMP-2 (40 µg/defect) alone significantly increased in vivo osteoclast activation and expression of the inflammatory cytokines interleukin-1ß and transforming necrosis factor-α on the scaffold at 7 days after surgery. However, rhBMP-2 had no direct effect on osteoclast activation in vitro. Osteoclast activation by rhBMP-2 was significantly suppressed by combined treatment with VIVIT at concentrations of 75 and 150 µM, but not at 15 µM, whereas suppression of inflammation occurred at all doses of VIVIT. Microcomputed tomography at 4 and 8 weeks after implantation revealed that the combination of rhBMP-2 and VIVIT at 75 µM VIVIT led to a greater bone fraction at the initial defect area, compared with rhBMP-2 alone. These findings revealed that local administration of VIVIT at certain concentrations has multiple positive effects that weaken early excessive osteoimmunological responses and enhance bone healing after rhBMP-2 administration. VIVIT has the potential to expand the therapeutic area of high-dose rhBMP-2 therapy to inflammatory bone loss. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1299-1310, 2018.

Bone-healing capacity of conditioned medium derived from three-dimensionally cultivated human mesenchymal stem cells and electrical stimulation on collagen sponge.

Continuing from our previous study, we hypothesized that combining electrical stimulation (ES) and three-dimensional (3D) culture would be a useful strategy to obtain more bioactive factors in conditioned medium (CM) derived from human mesenchymal stem cells (hMSC). Our aim in this study was to investigate the bone-healing capacity of CM derived from hMSC after 4 days of culture on a collagen sponge-exposed (CM-ES) or unexposed (CM-control; CM-CON) to ES in comparison with that of hMSC implantation. A cytokine assay of both CMs revealed the presence of cytokines, growth factors, and trophic factors. In vitro evaluation of both CMs showed increased cell growth and alkaline phosphatase activity of the hMSC, with little difference between CMs. We investigated the bone-healing effect using two bone disease models: bone defect and inflammatory bone loss. The calvaria defect was implanted with whole CM or 3D-precultured hMSC unexposed to ES. Microcomputed tomography analysis after 4 weeks indicated a twofold greater bone volume in the CM-CON and CM-ES groups than in the hMSC and vehicle groups, though we found no difference between the CM groups. However, CM-ES enhanced the bone healing of interleukin-1-induced bone loss to a level comparable with hMSC, whereas CM-CON did not. These results show that 3D-cultured CM had a greater or similar capacity for bone healing as treatment using hMSC transplantation, and CM-ES was especially effective against inflammatory bone loss. Thus, 3D-cultured CM with or without ES presents an encouraging alternative to MSC-based bone healing. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 311-320, 2018.

Receptor Activator of Nuclear Factor Kappa-B Ligand-Induced Local Osteoporotic Canine Mandible Model for the Evaluation of Peri-Implant Bone Regeneration.

The canine mandible is useful for studying bone regeneration after dental implant placement. However, it is limited in investigations of peri-implant osteogenesis under osteoporotic conditions due to the insignificant osteoporotic effect of ovariectomy. This study aimed at establishing a local osteoporotic model without ovariectomy by using receptor activator of nuclear factor kappa-B ligand (RANKL) in a canine mandible model. This new model was used to evaluate the effects of injectable ß-tricalcium phosphate (TCP) microsphere bone grafts on peri-implant bone regeneration under osteoporotic conditions with combinations of recombinant human bone morphogenetic protein-2 (rhBMP-2). A local osteoporotic canine mandible model was designed by creating a hole in the mandibular alveolar bone, then implanting a collagen sponge soaked with 20, 40, or 60 µg RANKL into the hole, and leaving it for 2 weeks. After the establishment of the dose for maximum osteoporotic bone loss at 40 µg of RANKL, the main surgery was performed. RANKL-soaked collagen sponges were removed, and dental implants were placed with bone grafts in five groups: implant only, TCP, and TCP + rhBMP-2 at 5, 15, and 45 µg. Peri-implant bone generation was determined by radiologic and histologic evaluations at 6 weeks after dental implant placement. On performing micro-computed tomography analysis, the group with TCP + 5 µg rhBMP-2 showed the highest bone volume than the other groups and a 22% increase (p < 0.05) compared with the implant-only group. In the histologic analysis, the TCP-only and TCP + 5 µg BMP-2 groups showed higher bone areas (14% and 16% increase, respectively) and bone-implant contact (12% and 7% increase, respectively) compared with the implant-only group, but there was no significant difference among the groups. In this study, the RANKL-induced local osteoporotic canine mandible model was useful for peri-implant bone regeneration under osteoporotic conditions such as those found in geriatric patients. The injectable ß-TCP bone grafts used in this study were effective in peri-implant bone generation under osteoporotic conditions, and their efficiency was enhanced at 5 µg BMP-2 compared with higher concentrations of BMP-2.

* Early and Marked Enhancement of New Bone Quality by Alendronate-Loaded Collagen Sponge Combined with Bone Morphogenetic Protein-2 at High Dose: A Long-Term Study in Calvarial Defects in a Rat Model.

Choice of appropriate biomaterial is a key factor for the success of recombinant human bone morphogenetic protein (rhBMP)-2 therapy. Inspired by osteogenic cell-differentiating and osteoclast-suppressing capabilities of alendronate (ALN), we manufactured a composite type of ALN-loaded collagen sponge (ALN-CS), which controls the early detrimental effect of high-dose rhBMP-2. This study aimed to evaluate ALN-CS as a high-dose rhBMP-2 carrier by investigating its initial biomolecular effect and efficacy on intramembranous ossification at 1, 4, 8, and 24 weeks using a rat calvarial defect model compared with nonloaded CS. The in vitro rhBMP-2 release in the ALN-CS showed a low initial burst and steady release phase during the rest period despite lack of calcium compared with that in CS alone. ALN release showed the same tendency as rhBMP-2 release. In vitro characterization showed that osteoblast differentiation and mineralization of mesenchymal stromal cells were more enhanced with ALN-CS. The ALN-CS-BMP group showed higher expression of bone-forming and -resorbing markers in vivo than the CS-BMP group after the first 7 days, which might be attributable to the relatively large amount of rhBMP-2 remaining. However, osteoclast activation in the ALN-CS-BMP group was significantly reduced compared with the CS-BMP group. Radiological and histological analyses revealed that ALN-CS-BMP promoted early and dense ossification at the initial defect, with 100% greater bone mass, 20% greater bone density, and less fatty marrow tissue than CS-BMP, which continued during the whole healing period. However, CS or ALN-CS alone failed to show complete defect closure even at the 24-week healing interval. Our results demonstrate that ALN-CS has remarkable advantages over CS alone in high-dose BMP-2 delivery, with potent suppression of resorption, early and dense ossification at the target area with less fatty marrow formation, and continuation of bone quality over the long term, which highlights its great clinical potential as a rhBMP carrier for bone regeneration at intramembranous ossification sites.

Effect of sustained release of rhBMP-2 from dried and wet hyaluronic acid hydrogel carriers compared with direct dip coating of rhBMP-2 on peri-implant osteogenesis of dental implants in canine mandibles.

Hyaluronic acid (HA) hydrogel has been used as a carrier of recombinant human bone morphogenetic protein (rhBMP)-2 for sustained delivery. To enhance peri-implant osteogenesis, a dried coating of rhBMP-2 HA hydrogel (BMP-HAH) on dental implants was designed; this approach provides the advantage of omitting in situ preparation of wet HA hydrogel. Sustained release of rhBMP-2 was more efficient for dried hydrogel over wet hydrogel. For both types, the released rhBMP-2 consistently led to enhanced alkaline phosphatase activity and osterix expression in human mesenchymal stromal cells. Histomorphometric analysis 4 weeks after placement of a dental implant in canine mandibles showed that the dried coating of BMP-HAH (10 µg/ml, n = 6) resulted in a significantly greater bone area (BA) than the wet BMP-HAH (10 µg/ml, n = 6) (p = 0.006) and implants without any coating (n = 6) (p = 0.022), while simple dip coating with rhBMP-2 (10 µg/ml, n = 6) resulted in significantly greater BA than the other three groups (p < 0.0005). Bone-to-implant contact (BIC) was significantly different only between the dried and wet coating of BMP-HAH (p = 0.014). Our results suggest that a simple dip coating of rhBMP-2 is more effective for increased peri-implant osteogenesis compared to a coating of BMP-HAH with sustained release.

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.

High-intensity Nd:YAG laser accelerates bone regeneration in calvarial defect models.

High-power pulsed lasers have been recently regarded to be anabolic to bone, but in vivo evidence is still lacking. This study aimed to investigate the capacity of bone repair using a high-power, Q-switched, pulsed, neodymium-doped yttrium aluminium garnet (Nd:YAG) laser, using bilateral calvarial defect models having non-critical sized, 5 mm (rat) or 8 mm (rabbit) diameter. One of the bilateral defects, which were all filled with collagen sponge or left empty, was irradiated with a Nd:YAG laser once every 2 days for 2 weeks at a constant total fluence rate (344 J/cm(2) ), output power (0.75 W), pulse repetition rate (15 pps) and wavelength (1064 nm) and examined for the laser effect. The same experimental scheme was designed using a rabbit calvarial defect model implanted with sponge, which was explored for the dose effect of output power at 0.75 and 3 W with the same quantities of the other parameters. New bone formation was evaluated by micro-computed tomography-based analysis and histological observation at 4 weeks after surgery. Laser irradiation significantly increased new bone formation by approximately 45%, not only in the sponge-filled defects of rats but also when the defects were left empty, compared to the non-irradiated group. Consistently, both doses of output power (0.75 and 3 W) enhanced new bone formation, but there was no significant difference between the two doses. This study is one of the first to demonstrate the beneficial effect of Nd:YAG lasers on the regeneration of bone defects which were left empty or filled with collagen sponge, suggesting its great potential in postoperative treatment targeting local bone healing.

In vivo gene activity of human mesenchymal stem cells after scaffold-mediated local transplantation.

Functional activation of stem cells after transplantation is a main concern in stem cell therapy. For local transplantation, mesenchymal stem cells (MSCs) are usually administered via scaffolds, either by direct implantation or after preculturing of cells, and it is unclear which is better for the activation of transplanted cells. In this study, we investigated the in vivo gene expression activity of human MSCs (hMSCs) transplanted into calvarial defects either directly post-seeding on collagen sponges (Group 1) or after overnight in vitro culturing post-seeding (Group 2). Real-time reverse transcription-polymerase chain reaction at days 7 and 14 after transplantation identified a time-dependent, rapid decrease in gene expression by the hMSCs, which in Group 1 was slightly more attenuated than in Group 2. Both groups exhibited a limited range of human-specific gene expression, which involved type I collagen (ColI), fibronectin, stromal cell-derived factor (SDF-1), and osteoprotegerin. Among these, ColI expression was the most efficient, with higher levels in Group 1 than Group 2. There was a lack of evidence for the expression of osteoblast differentiation-related markers or trophic factors, while resident cells showed clear expression of those genes. Rat-specific ß-actin expression in Group 2 was least among the scaffold control, Group 1, and Group 2, and this pattern was repeated in the expression of other rat osteogenic genes. Group 1 transplants positively influenced the osteogenic process of the defect tissue in part, and rat IGF-1 expression was significantly increased in Group 1. This tendency of gene expression by hMSCs in a rat model was very similar to what was observed in transplantations using immunodeficient mice. The current study showed that a main gene expressed by transplanted hMSCs during the initial weeks following transplantation is ColI, with a lack of differentiation-related markers or growth factor expression by hMSCs. Our data suggest that direct transplantation of hMSCs loaded on a collagen sponge is more efficient for gene activation in transplanted hMSCs, and more favorable to the local host tissue than transplantation after preculturing of cells.

Enhanced regeneration of rabbit mandibular defects through a combined treatment of electrical stimulation and rhBMP-2 application.

We evaluated the new bone regeneration of a rabbit mandibular defect using hBMSCs under electrical stimulation combined with rhBMP-2 in this study. An inner scaffold prepared by setting a collagen sponge with hBMSCs and hydrogel was placed into a polycaprolactone (PCL) outer box, and an electrical stimulation device was installed between the inner scaffold and the outer box. There were three experimental groups depending on electrical stimulation and application of rhBMP-2. The experimental group was divided into the following three groups. Group 1, in which rhBMP-2 (5 µg/defect) was added to hydrogel and electrical stimulation was not applied; Group 2, in which rhBMP-2 (5 µg/defect) was added as in Group 1 and electrical stimulation was applied; and Group 3, in which electrical stimulation was applied and rhBMP-2 (5 µg/defect) was injected directly into defect site. The delivered electrical stimulation was charge-balanced bi-phasic electric current pulses, and electrical stimulation was conducted for 7 days. The stimulation parameters of the bi-phasic electrical current set at an amplitude of 20 µA, a duration of 100 µs and a frequency of 100 Hz. Four weeks after surgery, new bone formation in each group was evaluated using radiography, histology, and micro-computed tomography (µCT). Groups 2 and 3 exhibited a significant increase in new bone formation compared to Group 1, while Group 3 showed the highest level of new bone regeneration. In a comparison between two groups, Group 2 showed a higher bone volume (BV) by 260 % (p < 0.01) compared with Group 1, and Group 3 showed a higher BV by 442 % (p < 0.01) compared with Group 1. The trend of the bone surface density (ratio of new bone to the real defect volume, BS/TV), trabecular number, and connectivity was identical to that of the BV. The total bone mineral density (BMD) of Groups 2 and 3 showed values higher by the ratios of 103 % (p < 0.01) and 107.5 % (p < 0.01) compared with Group 1, respectively. Part BMD for Groups 2 and 3 showed higher values by the ratios of 104.9 % (p < 0.01) and 122.4 % (p < 0.01) compared with Group 1, respectively. These results suggest that the combined treatment of electrical stimulation, hBMSCs, a collagen sponge, hydrogel, and rhBMP-2 was effective for bone regeneration of large-size mandibular defects. The application of rhBMP-2 with an injection following electrical stimulation demonstrated better efficiency as regards bone regeneration.

Bone regeneration by transplantation of human mesenchymal stromal cells in a rabbit mandibular distraction osteogenesis model.

Ex vivo expanded mesenchymal stromal cells (MSCs) represent a potential cell population for tissue regeneration strategy. Xenogeneic transplantation using human MSCs (hMSCs) can be an approach to reveal what hMSCs guide in bone regeneration with distinguishable gene expression from a host animal. In this study, we investigated the regenerating effect of hMSCs varying injection time point in a rabbit distraction osteogenesis model. Undifferentiated hMSCs (2×10(6) cells) were injected transcutaneously into the osteotomy site of one side of the mandible 1 day before the onset of distraction (Group 1) or after distraction (Group 2). The contralateral side of the mandible, which was subjected to distraction, but no hMSC injection, was used as the control in each group. hMSCs showed lack of major histocompatibility complex class II expression and suppression of xenogeneic lymphocyte proliferation stimulated by a proinflammatory cytokine. A microcomputed tomography-based evaluation showed a significant increase in new bone volume in the distracted callus in Group 1 compared to the contralateral side. Injection of hMSCs increased the bone mineral density (BMD) of the regenerated bone in both Group 1 and 2, although the former had a higher BMD than the latter. hMSCs of Group 1 subjected to distraction after injection expressed insulin-like growth factor-1 (IGF-1) and fibronectin (FN), while the expression of most osteoblast differentiation-related markers and growth factors was negligible. These results demonstrated that hMSCs exerted immune suppressive behavior in rabbit T cells in vitro, and hMSC transplantation into the distracted callus of a rabbit model provided osteogenic benefits that were more pronounced when the hMSCs were injected just before distraction than at the end of distraction. The beneficial effect of hMSCs might be mediated, partly by the expression of matrix proteins or IGF-1, which are known to favor bone formation.

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 Masson's 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.

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.

Biphasic electrical targeting plays a significant role in schwann cell activation.

Electrical stimulation (ES) is a promising technique for axonal regeneration of peripheral nerve injuries. However, long-term, continuous ES in the form of biphasic electric current (BEC) to stimulate axonal regeneration has rarely been attempted and the effects of BEC on Schwann cells are unknown. We hypothesized that long-term, continuous ES would trigger the activation of Schwann cells, and we therefore investigated the effect of BEC on the functional differentiation of primary human mesenchymal stromal cells (hMSCs) into Schwann cells, as well as the activity of primary Schwann cells. Differentiation of hMSCs into Schwann cells was determined by coculture with rat pheochromocytoma cells (PC12 cell line). We also investigated the in vivo effects of long-term ES (4 weeks) on axonal outgrowth of a severed sciatic nerve with a 7-mm gap after retraction of the nerve ends in rats by implanting an electronic device to serve as a neural conduit. PC12 cells cocultured with hMSCs electrically stimulated during culture in Schwann cell differentiation medium (Group I) had longer neurites and a greater percentage of PC12 cells were neurite-sprouting than when cocultured with hMSCs cultured in growth medium (control group) or unstimulated hMSCs in the same culture conditions as used for Group I (Group II). Group I cells showed significant upregulation of Schwann cell-related neurotrophic factors such as nerve growth factor and glial-derived neurotrophic factor compared to Group II cells at both the mRNA and protein levels. Primary Schwann cells responded to continuous BEC with increased proliferation and the induction of nerve growth factor and glial-derived neurotrophic factor, similar to Group I cells, and in addition, induction of brain-derived neurotrophic factor was observed. Immunohistochemical investigation of sciatic nerve regenerates revealed that BEC increased axonal outgrowth significantly. These results demonstrate that BEC enhanced the functional activity of Schwann cells via the induction of neurotrophic factor release and guide-increased axonal outgrowth in vivo. The effectiveness of long-term ES highlights the feasibility of a BEC-based therapeutic device to accelerate nerve regeneration of severed peripheral nerve injuries with a gap.

Promising efficacy of Escherichia coli recombinant human bone morphogenetic protein-2 in collagen sponge for ectopic and orthotopic bone formation and comparison with mammalian cell recombinant human bone morphogenetic protein-2.

Nonglycosylated recombinant human bone morphogenetic protein (rhBMP)-2 prepared in Escherichia coli (E. coli rhBMP-2) has recently been considered as an alternative to mammalian cell rhBMP-2. However, its clinical use is still limited owing to lack of evidence for osteogenic activity comparable with that of mammalian cell rhBMP-2 via microcomputed tomography-based analysis. Therefore, this study aimed to evaluate the ability of E. coli rhBMP-2 in absorbable collagen sponge to form ectopic and orthotopic bone and to compare it to that of mammalian rhBMP-2. In vitro investigation was performed to study osteoblast differentiation of human mesenchymal stromal cells. Both types of rhBMP-2 enhanced proliferation, alkaline phosphatase activity, and matrix mineralization of human mesenchymal stromal cells at similar levels. Similar tendencies were observed in microcomputed tomography analysis, which determined bone volume, fractional bone volume, trabecular thickness, trabecular separation, bone mineral density, and other characteristics. Histology from an in vivo osteoinductivity test and from a rat calvarial defect model demonstrated a dose-dependent increase in local bone formation. The E. coli rhBMP-2 group (5 µg) not only induced complete regeneration of an 8-mm critical-sized defect at 4 weeks, but also led to new bone with the same bone mineral density as normal bone at 8 weeks, with the same efficiency as that of mammalian cell rhBMP-2 (5 µg). These uniformly favorable results provide evidence that the osteogenic activity of E. coli rhBMP-2 is not inferior to that of mammalian cell rhBMP-2 despite its low solubility and lack of gylcosylation. These results suggest that the application of E. coli rhBMP-2 in absorbable collagen sponge may be a promising equivalent to mammalian cell rhBMP-2 in bone tissue engineering.

An implantable electrical bioreactor for enhancement of cell viability.

Low survival of injected cells which are prepared by ex-vivo culture is main obstacle in cell-based tissue regeneration. To elevate cell adaptation, we designed an implantable electrical bioreactor where human mesenchymal stromal cells (hMSCs) can be cultured and stimulated electrically. Bioreactor was composed of biocompatible cylindrical Teflon body containing a flexible polyimide electrode and implantable stimulator. The Teflon body has about 300 holes with a diameter of 300 um for effective nutrients supply inside the bioreactor and has a length of 17 mm and a diameter of 8mm for implantation. After hMSCs seeded on the collagen sponge that serves as scaffold to form a bone tissue graft, they are cultured in the bioreactor with biphasic electric current (BEC) stimulation. BEC stimulation with amplitude of 20/40 uA, duration of 100 us and a frequency of 100 Hz was applied for one week in the early stage of cultivation. Subsequently, after hMSCS were cultured for another week without electrical stimulation, cell response such as cell proliferation, cell attachment and gene expression are evaluated. In vitro and In vivo culture of hMSCs showed 19% and 22% increase in cell proliferation at stimulated groups, compared to unstimulated control. The expression of type I collagen increased significantly at stimulated group. These results suggest that the usage of implantable electrical bioreactor can be a good strategy to enhance the efficiency of stem cell-based 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.

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 material's 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.

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

BACKGROUND AND OBJECTIVE: 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. STUDY DESIGN/MATERIALS AND METHODS: MC3T3-E1 pre-osteoblasts were exposed to laser irradiation, 100 ng/ml BMP-2 or both. Cells were irradiated with a Q-switched, pulsed neodymium-doped yttrium aluminum garnet (Nd:YAG) laser, with a 1,064 nm wavelength and 0.75 W output power under 1.5, 3, or 5 J/cm(2) energy densities. Cell proliferation was evaluated using tetrazolium salt, WST-8. To determine the effect of these treatments on in vitro osteogenesis, we examined alkaline phosphatase (ALP) activity, mineral deposition, and expression of genes associated with osteogenesis. Quantitative real time PCR or ELISA was used to examine cytokine expression. In each experiment, either non-irradiated or BMP-2 (100 ng/ml)-treated cells were used as controls. RESULTS: High-power, low-level, Nd:YAG laser irradiation significantly increased ALP activity, when combined with BMP-2 or not. Cell proliferation declined in the irradiation and combined irradiation/BMP-2 groups. Interestingly, Nd:YAG laser stimulation resulted in significant induction of endogenous BMP-2 protein and gene expression. The increased expression of upstream regulators cbfa1 by Nd:YAG laser alone was comparable to exogenous BMP-2 treatment (100 ng/ml). Combined laser/BMP-2 treatment was synergistic in the expression of some genes (IGF-1, cbfa1) and ALP activity, compared to both BMP-2 treatment and laser irradiation alone. In vitro matrix mineralization was significantly accelerated by laser stimulation compared to that of the control, more so than with the combined laser/BMP-2 treatment. CONCLUSIONS: The present in vitro findings demonstrate that high-power, low-level Nd:YAG laser increased osteoblast activity, very efficiently accelerating mineral deposition. Osteoinductive effect of laser is likely mediated by activation of BMP-2-related signaling pathway.

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. MATERIALS AND METHODS: The implantable electrical stimulator was fabricated on a polyimide-based conduit with an integrated electrode, a stimulation chip, and a battery; 3 mg/mL of collagen gel was coated onto the conduit surface and electrical stimulation (20 µ A, 100 µ s, and 100 Hz biphasic current) was continuously applied between the nerve stumps for four weeks. The stimulator was tested on a severed sciatic nerve with a 7-mm gap in rats. The effects of both the electrical stimulation and the collagen application were examined. RESULTS: Functionality was evaluated through walk track assessments and by recording the action potential of the regenerated nerve. Immunohistochemical staining of the regenerated nerve was done using peripheral myelin protein 22. CONCLUSION: The results suggest that the functional recovery of a severed peripheral nerve by the proposed implantable electrical stimulator was achieved through electrical current stimulation along the use of a collagen coating on the conduit surface.