Local Suppression Effect of Paclitaxel-Impregnated Hydroxyapatite/Collagen on Breast Cancer Bone Metastasis in a Rat Model.

Introduction: Breast cancer is one of the most frequent primary tumors that cause spinal metastases. Metastasis consequences impair both the patient's overall prognosis and quality of life. We previously developed a porous hydroxyapatite collagen composite (HAp/Col) as an osteoconductive scaffold. HAp/Col is a commercially available artificial bone that is frequently utilized in spinal fusion. Because of its high absorbance capacity, HAp/Col is regarded as a good chemical carrier. Methods: This study investigated the effect of local administration of paclitaxel combined with HAp/Col scaffold on breast cancer metastasis. High-performance liquid chromatography was used to assess the in vitro release of paclitaxel from HAp/Col. In an in vivo rat model, the inhibitory effects of paclitaxel-impregnated scaffolds on local osteogenesis was examined, and then the local suppression effects on metastatic cancer were investigated. Results: In vitro testing revealed that roughly 30% of the paclitaxel was released within 96 hours. Paclitaxel-impregnated HAp/Col inhibited local osteogenesis for the first 8 weeks in a rat femur. However, at 12 weeks following surgery, this negative effect appeared to have subsided. In the metastatic model, all rats in the control group reached the humane endpoint 14 days after surgery. On the other hand, the average time to the endpoint in the paclitaxel group was 26.5 days, which was substantially longer than that in the control group. Long-term survivors treated with paclitaxel had no remaining tumor cells in the femoral bone, and osteogenesis was seen surrounding the HAp/Col. Conclusions: Paclitaxel-impregnated HAp/Col reduced local tumor development and extended the time to the target endpoint in rats with metastases from breast cancer. This study shows that local implantation of paclitaxel-impregnated HAp/Col may be a viable therapeutic option for the management of breast cancer metastases.

Local administration and enhanced release of bone metabolic antibodies from hydroxyapatite/chondroitin sulfate nanocomposite microparticles using zinc cations.

As a local delivery carrier of bone metabolic proteins, we have previously reported hydroxyapatite/chondroitin sulfate composite microparticles (HAp/ChS) and their formulation method using zinc cations (Zn), and the in vitro release properties of proteins from the microparticles. Herein, we report the release properties of model antibodies such as immunoglobulin (IgG), human IgG (hIgG), and denosumab (Dmab) from HAp/ChS using this formulation method. Adding Zn in the formulation of IgG loaded with HAp/ChS microparticles enhanced the release of antibodies from HAp/ChS in phosphate buffer saline. In addition, the biological activity of Dmab released from HAp/ChS formulated with Zn was significantly higher than that without Zn. These results suggest a possible beneficial effect on the treatment for local bone diseases. The sclerostin monoclonal antibody (Sclmab) promotes fracture healing. We prepared HAp/ChS microparticles loaded with Sclmab and locally administered the microparticles into a drilled hole in the distal femoral bone of young rats. After three weeks, the area of the newly formed osteoid around the drilled hole where HAp/ChS loaded with Sclmab and Zn was locally administered was significantly higher than that observed in the control group (normal saline). Thus, HAp/ChS microparticles and the formulation method of monoclonal antibodies using Zn could be useful in the treatment of local bone diseases.

Efficacy of Antibiotic-Loaded Hydroxyapatite/Collagen Composites Is Dependent on Adsorbability for Treating Staphylococcus aureus Osteomyelitis in Rats.

Osteomyelitis remains one of the most challenging disorders for orthopedic doctors despite the advancement of therapeutic techniques. The purpose of this study was to investigate the feasibility of local antibiotic administration using hydroxyapatite/collagen (HAp/Col) as a drug delivery system. We hypothesized that higher adsorbability of antibiotics onto HAp/Col will result in more efficacious activity and therefore, treatment of osteomyelitis. Eight antibiotics were examined in this study: amikacin, cefazolin, cefotiam, daptomycin, minocycline, piperacillin, teicoplanin, and vancomycin. Aligning with their adsorbability onto HAp/Col, minocycline, teicoplanin, and vancomycin showed antibacterial effects up to 14 days after subcutaneous implantation in Wistar rats; while antibiotics with reduced adsorbability (cefazolin, cefotiam, piperacillin) had diminished antibacterial effects. Furthermore, when implanted into a rat femur, vancomycin levels from the Hap/Col were detected in the medullary space above the minimum inhibitory concentration for Staphylococcus aureus for 7 days, while cefazolin levels were undetectable. Aligning with these results, implantation of Hap/Col impregnated with vancomycin to the femur in an acute osteomyelitis rat model had a greater therapeutic effect than cefazolin, as measured by the number of bacteria, the extent of bone destruction, and bone regeneration. These results indicated that the adsorbability of antibiotics onto their carrier is important when locally administered and that HAp/Col scaffolds might be a useful antibiotic delivery system for osteomyelitis. © 2019 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society J Orthop Res 38:843-851, 2020.

Augmentation of fracture healing by hydroxyapatite/collagen paste and bone morphogenetic protein-2 evaluated using a rat femur osteotomy model.

In fracture treatment, biological bone union generally depends on the bone's natural fracture healing capacity, even in surgically treated cases. Hydroxyapatite/collagen composite (HAp/Col) has high osteoconductivity and stimulates osteogenic progenitors. Furthermore, it has the potent capacity to adsorb bone morphogenetic proteins (BMPs). In this study, we prepared an injectable HAp/Col paste and evaluated its augmentation of bone union. Furthermore, the effect of HAp/Col paste combined with BMP-2 was also evaluated. We used a rat femur osteotomy model with a defect size of 1 mm. Male Wistar rats were assigned to one of the following four groups; a control group without any implant, a HAp/Col implant group, a group that received an absorbable collagen sponge (ACS) implant impregnated with BMP-2 (1 µg), and a group that received a HAp/Col implant impregnated with BMP-2 implant. Micro-CT analysis, three-point bending tests, and histological evaluation were performed. Bone union was achieved in two of eight cases in the HAp/Col group, five of eight cases in the ACS + BMP-2 group, and all cases in the HAp/Col + BMP-2 group at 8 weeks post-surgery. The control group did not achieve bone union. In addition, in the HAp/Col + BMP-2 group, the biomechanical strength of the fused femurs was comparable to that of the contralateral intact femur; the ratio of the mechanical load at the breaking point of the osteotomy side relative to that of the contralateral side was 1.00 ± 0.151 (SD). These results indicate that HAp/Col paste with or without BMP-2 augments bone union. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:129-137, 2018.

Transplantation of autologous synovial mesenchymal stem cells promotes meniscus regeneration in aged primates.

Transplantation of aggregates of synovial mesenchymal stem cells (MSCs) enhanced meniscus regeneration in rats. Anatomy and biological properties of the meniscus depend on animal species. To apply this technique clinically, it is valuable to investigate the use of animals genetically close to humans. We investigated whether transplantation of aggregates of autologous synovial MSCs promoted meniscal regeneration in aged primates. Chynomolgus primates between 12 and 13 years old were used. After the anterior halves of the medial menisci in both knees were removed, an average of 14 aggregates consisting of 250,000 synovial MSCs were transplanted onto the meniscus defect. No aggregates were transplanted to the opposite knee for the control. Meniscus and articular cartilage were analyzed macroscopically, histologically, and by MRI T1rho mapping at 8 (n = 3) and 16 weeks (n = 4). The medial meniscus was larger and the modified Pauli's histological score for the regenerated meniscus was better in the MSC group than in the control group in each primate at 8 and 16 weeks. Mankin's score for the medial femoral condyle cartilage was better in the MSC group than in the control group in all primates at 16 weeks. T1rho value for both the regenerated meniscus and adjacent articular cartilage in the MSC group was closer to the normal meniscus than in the control group in all primates at 16 weeks. Transplantation of aggregates of autologous synovial MSCs promoted meniscus regeneration and delayed progression of degeneration of articular cartilage in aged primates. This is the first report dealing with meniscus regeneration in primates. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1274-1282, 2017.

Anterior Cervical Corpectomy and Fusion Using a Synthetic Hydroxyapatite Graft for Ossification of the Posterior Longitudinal Ligament.

The significant complication rate associated with harvesting autologous iliac bone or fibula has encouraged development of alternative graft substitutes. In this study, the authors investigated the efficacy and safety of synthetic porous hydroxyapatite (HA) combined with local vertebral bone graft for use in anterior cervical corpectomy and fusion (ACCF) for the treatment of patients with ossification of the posterior longitudinal ligament (OPLL). Since 2006, twenty-five OPLL patients underwent ACCF using HA blocks (HA group). Hydroxyapatite blocks with 40% porosity were used for the 1-level ACCFs, and HA blocks with 15% porosity were used for the 2-level ACCFs. Clinical and radiological evaluation was performed with a minimum of 2-year follow-up. Outcomes were compared with those of 25 OPLL patients who underwent ACCFs using auto-fibula grafts at the authors' institution before 2006 (FBG group). Patients' demographic data were similar in the HA and FBG groups. Both groups demonstrated significant neurological improvements postoperatively. No difference was observed in operating time, whereas the intraoperative blood loss was significantly less in the HA group. The fusion rates in the HA group were comparable to those in the FBG group. The incidences of general complications were similar in the 2 groups; however, prolonged donor-site pain was observed in 9 (36.0%) cases in the FBG group. Based on the results of this study, ACCF using HA is a safe and efficacious method for the treatment of patients with OPLL as an alternative to conventional ACCF using autologous fibula bone grafting. [Orthopedics. 2017; 40(2):e334-e339.].

Dexamethasone Regulates EphA5, a Potential Inhibitory Factor with Osteogenic Capability of Human Bone Marrow Stromal Cells.

We previously demonstrated the importance of quality management procedures for the handling of human bone marrow stromal cells (hBMSCs) and provided evidence for the existence of osteogenic inhibitor molecules in BMSCs. One candidate inhibitor is the ephrin type-A receptor 5 (EphA5), which is expressed in hBMSCs and upregulated during long-term culture. In this study, forced expression of EphA5 diminished the expression of osteoblast phenotypic markers. Downregulation of endogenous EphA5 by dexamethasone treatment promoted osteoblast marker expression. EphA5 could be involved in the normal growth regulation of BMSCs and could be a potential marker for replicative senescence. Although Eph forward signaling stimulated by ephrin-B-Fc promoted the expression of ALP mRNA in BMSCs, exogenous addition of EphA5-Fc did not affect the ALP level. The mechanism underlying the silencing of EphA5 in early cultures remains unclear. EphA5 promoter was barely methylated in hBMSCs while histone deacetylation could partially suppress EphA5 expression in early-passage cultures. In repeatedly passaged cultures, the upregulation of EphA5 independent of methylation could competitively inhibit osteogenic signal transduction pathways such as EphB forward signaling. Elucidation of the potential inhibitory function of EphA5 in hBMSCs may provide an alternative approach for lineage differentiation in cell therapy strategies and regenerative medicine.

Bone Defect Regeneration by a Combination of a ß-Tricalcium Phosphate Scaffold and Bone Marrow Stromal Cells in a Non-Human Primate Model.

BACKGROUND: Reconstruction of large bone defects is a great challenge in orthopedic research. In the present study, we prepared composites of bone marrow-derived stromal cells (BMSCs) and ß-tricalcium phosphate (ß-TCP) with three novel aspects: proliferation of BMSCs with continuous dexamethasone treatment, cell loading under low pressure, and use of autologous plasma as the cell loading medium. The effectiveness of the resulting composite for large bone-defect reconstruction was tested in a non-human primate model, and the bone union capability of the regenerated bones was examined. MATERIALS AND METHODS: Primary surgery: Bone defects (5 cm long) were created in the left femurs of nine cynomolgus monkeys with resection of the periosteum (five cases) or without resection (four cases), and porous ß-TCP blocks were transplanted into the defects. Secondary surgery: Bone marrow aspirates harvested from seven of the nine monkeys were cultured with dexamethasone, and BMSCs were obtained. BMSCs were suspended in autologous plasma and introduced into a porous ß-TCP block under low-pressure conditions. The BMSC/ß-TCP composites were transplanted into bone defects created at the same sites as the primary surgery. Bone union evaluation: Five regenerated femurs were shortened by osteotomy surgery 8 to 15 months after transplantation of the ß-TCP/BMSC composites, and bone union was evaluated radiographically. RESULTS: After the primary surgery and treatment with ß-TCP alone, one of the five periosteum-resected monkeys and two of the four periosteum-preserved monkeys exhibited successful bone reconstruction. In contrast, five of the seven cases treated with the ß-TCP/MSC composite showed successful bone regeneration. In four of the five osteotomy cases, bone union was confirmed. CONCLUSION: We validated the effectiveness of a novel ß-TCP/BMSC composite for large bone defect regeneration and confirmed the bone union capability of the regenerated bone.

Efficacy and safety of porous hydroxyapatite/type 1 collagen composite implantation for bone regeneration: A randomized controlled study.

BACKGROUND: Porous hydroxyapatite/collagen composite (HAp/Col) is a bioresorbable bone substitute composed of nano-scale HAp and porcine type 1 collagen. In this study, the efficacy and safety were assessed in comparison to commercially available porous ß-tricalcium phosphate (ß-TCP). METHODS: Patients with bone defects caused by benign bone tumors, fractures, or harvesting of autografts were randomly allocated for implantation of porous HAp/Col (n = 63) or porous ß-TCP (n = 63). X-ray images were scored and used to evaluate the efficacy of the implantation until 24 weeks after surgery. Blood tests and observation of the surgical site were also performed to evaluate the safety of the implants. In total, 59 and 60 cases were analyzed in the porous HAp/Col and ß-TCP groups, respectively. RESULTS: At 18 and 24 weeks after surgery, the highest grade of bone regeneration was more frequent in the porous HAp/Col group than in the porous ß-TCP group (p = 0.0004 and 0.0254 respectively). Wilcoxon's rank sum test confirmed the superiority of porous HAp/Col from early time points onward (p = 0.0084, 4 w; p = 0.0037, 8 w; p = 0.0030, 12 w; p < 0.0001, 18 w; and p = 0.0316, 24 w). The incidence of adverse effects was higher in the porous HAp/Col group than in the ß-TCP group. However, no serious adverse events were reported and no cases needed to drop out of the clinical trial. CONCLUSIONS: The superiority of porous HAp/Col for bone regeneration in comparison to an established porous ß-TCP was confirmed. Although the incidence of side effects associated with the porous HAp/Col implant was higher than that in the ß-TCP group, no serious adverse events occurred that resulted in rejection of the implants.

Biomechanical evaluation of the rabbit tibia after implantation of porous hydroxyapatite/collagen in a rabbit model.

PURPOSE: Porous hydroxyapatite/collagen composite (HAp/Col) is an artificial bone substitute with excellent osteoconduction and sponge-like elasticity. However, the porosity of porous HAp/Col is as high as 95% and its mechanical strength is very poor. The aim of this study was to biomechanically analyze sites implanted with porous HAp/Col. METHODS: Rectangular cortical bone defects (3 × 8 mm) were made in the tibia of rabbits and filled with porous HAp/Col or porous ß-tricalcium phosphate or left vacant. The tibia was harvested at 4 or 12 weeks after surgery. The harvested specimens were analyzed using a micro-CT system, and the mechanical strength of the specimens was examined by torsion testing. RESULTS: Quantitative micro-CT analysis of the regenerated bone revealed that both bone substitutes equally facilitated bone regeneration. Biomechanical testing demonstrated that the torsional strength of HAp/Col-implanted sites was higher than that of the control (vs control: p = 0.030 and vs ß-TCP: p = 0.056). CONCLUSIONS: The results indicate that porous HAp/Col implantation is an effective strategy for recovery of the mechanical strength of bone defects.

Repair of osteochondral defects in a rabbit model using a porous hydroxyapatite collagen composite impregnated with bone morphogenetic protein-2.

Articular cartilage has a limited capacity for spontaneous repair, and an effective method to repair damaged articular cartilage has not yet been established. The purpose of this study was to evaluate the effect of transplantation of porous hydroxyapatite collagen (HAp/Col) impregnated with bone morphogenetic protein-2 (BMP-2). To evaluate the characteristics of porous HAp/Col as a drug delivery carrier of recombinant human BMP-2 (rhBMP-2), the rhBMP-2 adsorption capacity and release kinetics of porous HAp/Col were analyzed. Porous HAp/Col impregnated with different amounts of rhBMP-2 (0, 5, and 25 µg) was implanted into osteochondral defects generated in the patellar groove of Japanese white rabbits to evaluate the effect on osteochondral defect regeneration. At 3, 6, 12, and 24 weeks after operation, samples were harvested and subjected to micro-computed tomography analysis and histological evaluation of articular cartilage and subchondral bone repair. The adsorption capacity was 329.4 µg of rhBMP-2 per cm(3) of porous HAp/Col. Although 36% of rhBMP-2 was released within 24 h, more than 50% of the rhBMP-2 was retained in the porous HAp/Col through the course of the experiment. Defects treated with 5 µg of rhBMP-2 showed the most extensive subchondral bone repair and the highest histological regeneration score, and differences against the untreated defect group were significant. The histological regeneration score of defects treated with 25 µg of rhBMP-2 increased up to 6 weeks after implantation, but then decreased. Porous HAp/Col, therefore, is an appropriate carrier for rhBMP-2. Implantation of porous HAp/Col impregnated with rhBMP-2 is effective for rigid subchondral bone repair, which is important for the repair of the smooth articular surface.

Dexamethasone enhances osteogenic differentiation of bone marrow- and muscle-derived stromal cells and augments ectopic bone formation induced by bone morphogenetic protein-2.

UNLABELLED: We evaluated whether dexamethasone augments the osteogenic capability of bone marrow-derived stromal cells (BMSCs) and muscle tissue-derived stromal cells (MuSCs), both of which are thought to contribute to ectopic bone formation induced by bone morphogenetic protein-2 (BMP-2), and determined the underlying mechanisms. Rat BMSCs and MuSCs were cultured in growth media with or without 10-7 M dexamethasone and then differentiated under osteogenic conditions with dexamethasone and BMP-2. The effects of dexamethasone on cell proliferation and osteogenic differentiation, and also on ectopic bone formation induced by BMP-2, were analyzed. Dexamethasone affected not only the proliferation rate but also the subpopulation composition of BMSCs and MuSCs, and subsequently augmented their osteogenic capacity during osteogenic differentiation. During osteogenic induction by BMP-2, dexamethasone also markedly affected cell proliferation in both BMSCs and MuSCs. In an in vivo ectopic bone formation model, bone formation in muscle-implanted scaffolds containing dexamethasone and BMP-2 was more than two fold higher than that in scaffolds containing BMP-2 alone. Our results suggest that dexamethasone potently enhances the osteogenic capability of BMP-2 and may thus decrease the quantity of BMP-2 required for clinical application, thereby reducing the complications caused by excessive doses of BMP-2. HIGHLIGHTS: 1. Dexamethasone induced selective proliferation of bone marrow- and muscle-derived cells with higher differentiation potential. 2. Dexamethasone enhanced the osteogenic capability of bone marrow- and muscle-derived cells by altering the subpopulation composition. 3. Dexamethasone augmented ectopic bone formation induced by bone morphogenetic protein-2.

Bone Marrow Stromal Cells Combined With a Honeycomb Collagen Sponge Facilitate Neurite Elongation In Vitro and Neural Restoration in the Hemisected Rat Spinal Cord.

In the last decade, researchers and clinicians have reported that transplantation of bone marrow stromal cells (BMSCs) promotes functional recovery after brain or spinal cord injury (SCI). However, an appropriate scaffold designed for the injured spinal cord is needed to enhance the survival of transplanted BMSCs and to promote nerve regeneration. We previously tested a honeycomb collagen sponge (HC), which when applied to the transected spinal cord allowed bridging of the gap with nerve fibers. In this study, we examined whether the HC implant combined with rat BMSCs increases nerve regeneration in vitro and enhances functional recovery in vivo. We first evaluated the neurite outgrowth of rat dorsal root ganglion (DRG) explants cultured on HC with or without BMSCs in vitro. Regeneration of neurites from the DRGs was increased by BMSCs combined with HC scaffolds. In the in vivo study, 3-mm-long HC scaffolds with or without BMSCs were implanted into the hemisected rat thoracic spinal cord. Four weeks after the procedure, rats implanted with HC scaffolds containing BMSCs displayed better motor and sensory recovery than those implanted with HC scaffolds only. Histologically, more CGRP-positive sensory fibers at the implanted site and 5-HT-positive serotonergic fibers contralateral to the implanted site were observed in spinal cords receiving BMSCs. Furthermore, more rubrospinal neurons projected distally to the HC implant containing BMSCs. Our study indicates that the application of BMSCs in a HC scaffold in the injured spinal cord directly promoted sensory nerve and rubrospinal tract regeneration, thus resulting in functional recovery.

Low-intensity pulsed ultrasound prompts tissue-engineered bone formation after implantation surgery.

BACKGROUND: A practical problem impeding clinical translation is the limited bone formation seen in artificial bone grafts. Low-pressure/vacuum seeding and dynamic culturing in bioreactors have led to a greater penetration into the scaffolds, enhanced production of bone marrow cells, and improved tissue-engineered bone formation. The goal of this study was to promote more extensive bone formation in the composites of porous ceramics and bone marrow stromal cells (BMSCs). METHODS: BMSCs/ß-tricalcium phosphate (ß-TCP) composites were subcultured for 2 weeks and then subcutaneously implanted into syngeneic rats that were split into a low-intensity pulsed ultrasound (LIPUS) treatment group and a control group. These implants were harvested at 5, 10, 25, and 50 days after implantation. The samples were then biomechanically tested and analyzed for alkaline phosphate (ALP) activity and osteocalcin (OCN) content and were also observed by light microscopy. RESULTS: The levels of ALP activity and OCN content in the composites were significantly higher in the LIPUS group than in the control group. Histomorphometric analysis revealed a greater degree of soft tissue repair, increased blood flow, better angiogenesis, and more extensive bone formation in the LIPUS groups than in the controls. No significant difference in the compressive strength was found between the two groups. CONCLUSION: LIPUS treatment appears to enhance bone formation and angiogenesis in the BMSCs/ß-TCP composites.

Intrathecal AAV serotype 9-mediated delivery of shRNA against TRPV1 attenuates thermal hyperalgesia in a mouse model of peripheral nerve injury.

Gene therapy for neuropathic pain requires efficient gene delivery to both central and peripheral nervous systems. We previously showed that an adenoassociated virus serotype 9 (AAV9) vector expressing short-hairpin RNA (shRNA) could suppress target molecule expression in the dorsal root ganglia (DRG) and spinal cord upon intrathecal injection. To evaluate the therapeutic potential of this approach, we constructed an AAV9 vector encoding shRNA against vanilloid receptor 1 (TRPV1), which is an important target gene for acute pain, but its role in chronic neuropathic pain remains unclear. We intrathecally injected it into the subarachnoid space at the upper lumbar spine of mice 3 weeks after spared nerve injury (SNI). Delivered shTRPV1 effectively suppressed mRNA and protein expression of TRPV1 in the DRG and spinal cord, and it attenuated nerve injury-induced thermal allodynia 10-28 days after treatment. Our study provides important evidence for the contribution of TRPV1 to thermal hypersensitivity in neuropathic pain and thus establishes intrathecal AAV9-mediated gene delivery as an investigative and potentially therapeutic platform for the nervous system.

After repeated division, bone marrow stromal cells express inhibitory factors with osteogenic capabilities, and EphA5 is a primary candidate.

The differentiation capability of human bone marrow stromal cells (hBMSCs) is thought to deteriorate over multiple doubling processes. To clarify the deterioration mechanisms, the multilineage differentiation capabilities of short- and long-term passaged BMSCs were compared. Predictably, long-term passaged BMSCs showed reduced differentiation capacities compared to short-term passaged cells. Furthermore, a non-human primate heterotopic bone formation model demonstrated that long-term passaged BMSCs have bone formation capabilities but also exert inhibitory effects on bone formation. This finding indicated that long-term passaged BMSCs express higher levels of inhibitory factors than short-term passaged BMSCs do. Co-culture assays of short- and long-term passaged BMSCs suggested that the inhibitory signals required cell-cell contact and would therefore be expressed on the cell membrane. A microarray analysis of BMSCs identified ephrin type-A receptor 5 (EphA5) as an inhibitory factor candidate. Quantitative PCR revealed that among all members of the ephrin and Eph receptor families, only the expression of EphA5 was increased by BMSC proliferation. A gene knockdown analysis using siRNAs demonstrated that knockdown of EphA5 gene expression in long-term passaged BMSCs led to an increase in ALP mRNA expression. These results indicate that EphA5 may be a negative regulator of bone formation. A better understanding of the roles of the ephrin and Eph receptor families in hBMSCs may lead to alternative approaches for manipulating hBMSC fate. In addition, this avenue of discovery may provide new therapeutic targets and quality-control markers of the osteogenic differentiation capabilities of hBMSCs.

Massive bone reconstruction with heat-treated bone graft loaded autologous bone marrow-derived stromal cells and ß-tricalcium phosphate composites in canine models.

Bone marrow-derived stromal cells (BMSCs) contain mesenchymal stem cells that are capable of forming various mesenchymal tissues. We hypothesized that BMSCs and ß-tricalcium phosphate (ß-TCP) composites would promote the remodeling of large-sized autologous devitalized bone grafts; therefore, the aim of this study was to evaluate the effects of the composites on the remodeling of autologous devitalized bone grafts. Autologous BMSCs cultured in culture medium containing dexamethasone (10(-7) M) were loaded into porous ß-TCP granules under low-pressure. Theses BMSC/TCP composites were put into the bone marrow cavity of autologous heat-treated bone (femoral diaphysis, 65-mm long, 100°C, 30 min) and put back to the harvest site. In the contralateral side, ß-TCP without BMSC were used in the same manner as the opposite side as the control. Treatment with the BMSC/TCP composites resulted in a significant increase in thickness, bone mineral density, and matured bone volume of the cortical bone at the center of the graft compared to the control. Histological analysis showed matured regenerated bone in the BMSC loaded group. These results indicate that BMSC/TCP composites facilitated bone regeneration and maturation at the graft site of large-sized devitalized bone. This method could potentially be applied for clinical use in the reconstruction of large bone defects such as those associated with bone tumors.

Porous/dense composite hydroxyapatite for anterior cervical discectomy and fusion.

STUDY DESIGN: A prospective analysis OBJECTIVE: Our aim was to investigate the efficacy of new synthetic porous/dense composite hydroxyapatite (HA) for use in anterior cervical discectomy and fusion (ACDF). SUMMARY OF BACKGROUND DATA: Iliac crest bone graft (ICBG) has been traditionally used as the "gold standard" for ACDF. The significant complication rate associated with harvesting tricortical ICBG, however, has encouraged development of alternative graft substitutes. METHODS: The morphology of the porous/dense HA was observed by scanning electron microscopy (SEM), and the in vitro compressive strength of the composite HA was measured. From April 2005, 51 consecutive patients underwent 81 levels of ACDF using the composite HA with percutaneously harvested trephine bone chips. Clinical and radiological evaluation was performed during the postoperative hospital stay and at follow-up. Furthermore, the outcomes in ACDF using the composite HA were compared with those using tricortical ICBG. RESULTS: The SEM images demonstrated 100- to 300-µm pores (approximately 40% of porosity) in the porous layers of the HA. The compressive strength of the composite HA was 203.1 ± 4.1 MPa. In the clinical study, the demographic data of the patients were similar in HA and ICBG groups. The fusion rates in HA group were comparable with those in ICBG group. The cervical lordosis was enhanced postoperatively in both groups and well preserved at 2-year follow-up without significant differences between the groups. The intraoperative blood loss in HA group was significantly lesser than that in ICBG group. Donor site complications were found in 29.2% of the patients in ICBG group, whereas no donor site morbidity was found in HA group. No major collapse or fragmentation of the composite HA was found. CONCLUSION: The hybrid graft of composite HA and percutaneously harvested trephine chips seemed promising as a graft substitute for ACDF. LEVEL OF EVIDENCE: 4.

Intrathecal shRNA-AAV9 inhibits target protein expression in the spinal cord and dorsal root ganglia of adult mice.

Gene therapy for neurological diseases requires efficient gene delivery to target tissues in the central and peripheral nervous systems. Although adeno-associated virus is one of the most promising vectors for clinical use against neurological diseases, it is difficult to get it across the blood-brain barrier. A clinically practical approach to using a vector based on adeno-associated virus to decrease the expression of a specific gene in both the central and the peripheral nervous system has yet to be established. Here, we analyzed whether upper lumbar intrathecal administration of a therapeutic vector incorporating adeno-associated virus and short-hairpin RNA against superoxide dismutase-1 bypassed the blood-brain barrier to target the spinal cord and dorsal root ganglia. The therapeutic vector effectively suppressed mRNA and protein expression of endogenous superoxide dismutase-1 in the lumbar spinal cord and dorsal root ganglia. Moreover, neither neurological side effects nor toxicity due to the incorporated short-hairpin RNA occurred after the injection. We propose that this approach could be developed into novel therapies for motor neuron diseases and chronic pain conditions, such as complex regional pain syndrome, through silencing of the genes responsible for pathologies in the spinal cord and dorsal root ganglia.

Subcutaneous injections of platelet-rich plasma into skin flaps modulate proangiogenic gene expression and improve survival rates.

BACKGROUND: Flap necrosis remains a major complication of reconstructive surgery. To improve skin flap survival, various treatments with vasodilators, antiplatelet drugs, or the local administration of growth factors have been performed. However, the sufficient prevention of skin necrosis is not well established. Platelet-rich plasma has been used as an autologous factor and includes various growth factors. The authors evaluated whether or not platelet-rich plasma can improve skin flap survival in an experimental rat model. METHODS: Cranially based dorsal cutaneous flaps were elevated in 48 rats. The animals received subcutaneous injections of either platelet-rich plasma (100 µl) or platelet-poor plasma (100 µl). The rats were divided into three groups: the platelet-rich plasma group (n = 16), the platelet-poor plasma group (n = 16), and the nontreatment group (n = 16). Flap survival was measured and histologic specimens were collected on day 7. Real-time polymerase chain reaction specimens were collected after 8 hours, 24 hours, 3 days, and 7 days. RESULTS: Platelet-rich plasma significantly improved flap survival rates (61.2 percent) compared with the platelet-poor plasma treatment (35.8 percent) and nontreatment groups (28.0 percent). A histologic analysis showed that significantly fewer inflammatory cells and an increased blood vessel density were observed in the platelet-rich plasma rats versus the platelet-poor plasma or nontreatment rats. In addition, platelet-rich plasma treatment significantly increased the mRNA levels of vascular endothelial growth factor and platelet-derived growth factor. CONCLUSION: Platelet-rich plasma modulates the genes involved in angiogenesis and improves skin flap survival.