Shinichi Sotome

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 &mgr;l) or platelet-poor plasma (100 &mgr;l). 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.

Transplanted neural progenitor cells expressing mutant NT3 promote myelination and partial hindlimb recovery in the chronic phase after spinal cord injury

Neutrotrophin-3 (NT3) plays a protective role in injured central nervous system tissues through interaction with trk receptors. To enhance the regeneration of damaged tissue, a combination therapy with cell transplantation and neurotrophins has been under development. We examined whether the transplantation of neural progenitor cells (NPCs) secreting NT3/D15A, a multi-neurotrophin with the capacity to bind both trkB and trkC, would enhance the repair of damaged tissues and the functional recovery in a chronic phase of spinal cord injury. The cultured NPCs with lentiviral vector containing either GFP or NT3/D15A were transplanted into the contused spinal cord at 6 weeks after the initial thoracic injury. Eight weeks after the transplantation, the NT3/D15A transplants displayed better survival than the GFP transplants, and they exhibited enhanced myelin formation and partial improvement of hindlimb function. Our study revealed that NT3/D15A produced positive effects in injured spinal cords even in the chronic phase. These effects suggest an enhanced neurotrophin-trk signaling by NT3/D15A.

Fresh bone marrow introduction into porous scaffolds using a simple low-pressure loading method for effective osteogenesis in a rabbit model.

Recent advances in tissue engineering techniques have allowed porous biomaterials to be combined with osteogenic cells for effective bone regeneration. We developed a simple low‐pressure cell‐loading method using only syringes and stopcocks, and examined the effect of this method on osteogenesis when applied to the combination of highly porous β‐tricalcium phosphate (β‐TCP) and fresh autologous bone marrow. Both block and granule β‐TCP scaffolds were used to prepare implants in three different ways: without bone marrow as a control, with bone marrow that was allowed to penetrate spontaneously under atmospheric pressure (AP group), and with bone marrow that was seeded under low pressure (ULP group). These implants were transplanted into rabbit intramuscular sites, and the samples were examined biologically and histologically. The penetration efficiency of the block implants after marrow introduction was significantly higher in the ULP group than in the AP group. In the transplanted block samples, alkaline phosphatase activity was significantly higher in the ULP group at 2 weeks after implantation, and significantly more newly formed bone was observed in the ULP group at both 5 and 10 weeks compared with the AP group. Similar results were observed even in the experiment using β‐TCP granules, which are smaller than the blocks and frequently used clinically. Because of its convenience and safety, this low‐pressure method might be a novel, effective treatment to promote osteogenesis with bone marrow in clinical bone reconstruction surgeries.

Bone Regeneration with Autologous Plasma, Bone Marrow Stromal Cells, and Porous β-Tricalcium Phosphate in Nonhuman Primates

To potentiate the bone formation capability of bone marrow stromal cell (BMSC)/beta-tricalcium phosphate (beta-TCP) constructs, we devised an autologous plasma-based construct. We tested its effectiveness and investigated the effects of its components on a monkey ectopic bone formation model. The autologous plasma (platelet-rich plasma, PRP, or platelet-poor plasma, PPP)/BMSC/beta-TCP construct (R group or P group) showed significantly more bone formation at 3 and 6 weeks after implantation than a conventional BMSC/beta-TCP construct using a culture medium (M group). There was no significant difference between the P and R groups. Moreover, the P group constructs with a 10-fold lower cell concentration yielded equivalent bone formation to the M group at 5 weeks after implantation. To elucidate the effect of fibrin and serum contained in the plasma, five constructs were prepared using the following cell vehicles: autologous serum + fibrinogen (0, 1, 4, or 16 mg/mL) or phosphate-buffered saline + fibrinogen (4 mg/mL). The serum + fibrinogen (4 mg/mL, physiological concentration of monkeys) construct showed the most abundant bone formation at 3 weeks after implantation, though at 5 weeks no statistical difference existed among the groups. Autologous plasma efficiently promoted osteogenesis of BMSCs/porous beta-TCP constructs, and both fibrin and serum proved to play significant roles in the mechanism.

Novel cell seeding system into a porous scaffold using a modified low-pressure method to enhance cell seeding efficiency and bone formation.

The efficient seeding of cells into porous scaffolds is important in bone tissue engineering techniques. To enhance efficiency, we modified the previously reported cell seeding techniques using low-pressure conditions. In this study, the effects of low pressure on bone marrow-derived stromal cells (BMSCs) of rats and the usefulness of the modified technique were assessed. There was no significant difference found in the proliferative and osteogenic capabilities among various low-pressure (50–760 mmHg, 1–10 min) conditions. To analyze the efficacies of the cell seeding techniques, BMSCs suspended in the plasma of rats were seeded into porous β-tricalcium phosphate (β-TCP) blocks by the following three procedures: 1) spontaneous penetration of cell suspension under atmospheric pressure (SP); 2) spontaneous penetration and subsequent low pressure treatment (SPSL), the conventional technique; and 3) spontaneous penetration under low pressure conditions (SPUL), the modified technique. Subsequently, these BMSCs/β-TCP composites were used for the analysis of cell seeding efficiency or in vivo bone formation capability. Both the number of BMSCs seeded into β-TCP blocks and the amount of bone formation of the SPUL group were significantly higher than those of the other groups. The SPUL method with a simple technique permits high cell seeding efficiency and is useful for bone tissue engineering using BMSCs and porous scaffolds.

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.

Dexamethasone Enhances Osteogenic Differentiation of Bone Marrow- and Muscle-Derived Stromal Cells and Augments Ectopic Bone Formation Induced by Bone Morphogenetic Protein-2

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.

Three-dimensional porous hydroxyapatite/collagen composite with rubber-like elasticity

A three-dimensional porous hydroxyapatite/collagen (HAp/Col) composite with a random pore structure was fabricated using freeze-drying processes; the self-organized HAp/Col nanocomposite with a weight ratio of 80.5:19.5, freeze-dried, was kneaded in 100 mM sodium phosphate buffer, frozen at −20°C and freeze-dried. The cross-linkage of Col molecules was introduced dehydrothermally at 140°C in vacuo. The porous composite had a porosity of 94.7% with pore sizes between 200 and 500 μm. The compressive stress for the wet porous composite in phosphate buffer saline (PBS) was gradually decreased during 20 days incubation with a small amount of weight loss. The cyclic and time-course compression tests showed good repeatability of stress and well-recovery of its height, and caused no collapse of the porous composite. The implantation of the porous composite in rat bone holes showed the biodegradable property and new bone formation occurred in the pores without inflammatory response. The porous composite fabricated has good flexibility and rubber-like elasticity, and is a promising bone regenerative material.

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 Paulis 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. Mankins 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.

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.