Toshitaka Yoshii

The Effects of rhBMP-2 Released from Biodegradable Polyurethane/Microsphere Composite Scaffolds on New Bone Formation in Rat Femora

Scaffolds prepared from biodegradable polyurethanes (PUR) have been investigated as a supportive matrix and delivery system for skin, cardiovascular, and bone tissue engineering. While previous studies have suggested that PUR scaffolds are biocompatible and moderately osteoconductive, the effects of encapsulated osteoinductive molecules, such as recombinant human bone morphogenetic protein (rhBMP-2), on new bone formation have not been investigated for this class of biomaterials. The objective of this study was to investigate the effects of different rhBMP-2 release strategies on new bone formation in PUR scaffolds implanted in rat femoral plug defects. In the simplest approach, rhBMP-2 was added as a dry powder prior to the foaming reaction, which resulted in a burst release of 35% followed by a sustained release for 21 days. Encapsulation of rhBMP-2 in either 1.3-micron or 114-micron PLGA microspheres prior to the foaming reaction reduced the burst release. At 4 weeks post-implantation, all rhBMP-2 treatment groups enhanced new bone formation relative to the scaffolds without rhBMP-2. Scaffolds incorporating rhBMP-2 powder promoted the most extensive new bone formation, while scaffolds incorporating rhBMP-2 encapsulated in 1.3-micron microspheres, which exhibited the lowest burst release, promoted the least extensive new bone formation. Thus our observations suggest that an initial burst release followed by sustained release is better for promoting new bone formation.

Repair of large osteochondral defects in rabbits using porous hydroxyapatite/collagen (HAp/Col) and fibroblast growth factor-2 (FGF-2)

Articular cartilage has a limited capacity for self‐renewal. This article reports the development of a porous hydroxyapatite/collagen (HAp/Col) scaffold as a bone void filler and a vehicle for drug administration. The scaffold consists of HAp nanocrystals and type I atelocollagen. The purpose of this study was to investigate the efficacy of porous HAp/Col impregnated with FGF‐2 to repair large osteochondral defects in a rabbit model. Ninety‐six cylindrical osteochondral defects 5 mm in diameter and 5 mm in depth were created in the femoral trochlear groove of the right knee. Animals were assigned to one of four treatment groups: porous HAp/Col impregnated with 50 µl of FGF‐2 at a concentration of 10 or 100 µg/ml (FGF10 or FGF100 group); porous HAp/Col with 50 µl of PBS (HAp/Col group); and no implantation (defect group). The defect areas were examined grossly and histologically. Subchondral bone regeneration was quantified 3, 6, 12, and 24 weeks after surgery. Abundant bone formation was observed in the HAp/Col implanted groups as compared to the defect group. The FGF10 group displayed not only the most abundant bone regeneration but also the most satisfactory cartilage regeneration, with cartilage presenting a hyaline‐like appearance. These findings suggest that porous HAp/Col with FGF‐2 augments the cartilage repair process.

Quantitative measures of femoral fracture repair in rats derived by micro-computed tomography

Although fracture healing is frequently studied in pre-clinical models of long bone fractures using rodents, there is a dearth of objective quantitative techniques to assess successful healing. Biomechanical testing is possibly the most quantitative and relevant to a successful clinical outcome, but it is a destructive technique providing little insight into the cellular mechanisms associated with healing. The advent of X-ray computed tomography (CT) has provided the opportunity to quantitatively and non-destructively assess bone structure and density, but it is unknown how measurements derived using this technology relate to successful healing. To examine possible relationships, we used a pre-clinical model to test for statistically significant correlations between quantitative characteristics of the callus by micro-CT (microCT) and the bending strength, stiffness, and energy-to-failure of the callus as assessed by three-point bending of excised bones. A closed, transverse fracture was generated in the mid-shaft of rat femurs by impact loading. Shortly thereafter, the rats received a one-time, local injection of either the vehicle or one of four doses of lovastatin. Following sacrifice after 4 weeks of healing, fractured femurs were extracted for microCT analysis and then three-point bending. Setting the region of interest to be 3.2 mm above and below the fracture line, we acquired standard and new microCT-derived measurements. The mineralized callus volume and the mineral density of the callus correlated positively with callus strength (rxy = -0.315, p = 0.016 and rxy = 0.444, p<0.0005, respectively) and stiffness (rxy = -0.271, p = 0.040 and rxy = 0.325, p = 0.013, respectively), but the fraction of the callus that mineralized and the moment of inertia of the callus did not. This fraction did correlate with energy-to-failure (rxy = -0.343, p = 0.0085). Of the microCT-derived measurements, quantifying defects within the outer bridging cortices of the callus produced the strongest correlation with both callus strength (rxy = 0.557, p<0.0001) and stiffness (rxy = 0.468, p = 0.0002). By both reducing structural defects and increasing mineralization, lovastatin appears to increase the callus strength.

Synthesis, characterization, and remodeling of weight-bearing allograft bone/polyurethane composites in the rabbit

The process of bone healing requires the restoration of both anatomy and physiology, and there is a recognized need for innovative biomaterials that facilitate remodeling throughout this complex process. While porous scaffolds with a high degree of interconnectivity are known to accelerate cellular infiltration and new bone formation, the presence of pores significantly diminishes the initial mechanical properties of the materials, rendering them largely unsuitable for load-bearing applications. In this study, a family of non-porous composites has been fabricated by reactive compression molding of mineralized allograft bone particles (MBPs) with a biodegradable polyurethane (PUR) binder, which is synthesized from a polyester polyol and a lysine-derived polyisocyanate. At volume fractions exceeding the random close-packing limit, the particulated allograft component presented a nearly continuous osteoconductive pathway for cells into the interior of the implant. By varying the molecular weight of the polyol and manipulating the surface chemistry of the MBP via surface demineralization, compressive modulus and strength values of 3-6 GPa and 107-172 MPa were achieved, respectively. When implanted in bilateral femoral condyle plug defects in New Zealand White rabbits, MBP/PUR composites exhibited resorption of the allograft and polymer components, extensive cellular infiltration deep into the interior of the implant, and new bone formation at 6 weeks. While later in vivo timepoints are necessary to determine the ultimate fate of the MBP/PUR composites, these observations suggest that allograft bone/polymer composites have potential for future development as weight-bearing devices for orthopedic applications.

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.

Hybrid grafting using bone marrow aspirate combined with porous β-tricalcium phosphate and trephine bone for lumbar posterolateral spinal fusion: a prospective, comparative study versus local bone grafting.

Study Design. A prospective, comparative study. Objective. We developed a hybrid graft (HBG) of porous &bgr;-tricalcium phosphate ceramics/percutaneously harvested bone sticks/autologous bone marrow aspirate for lumbar posterolateral fusion (PLF). The aim of this study was to investigate the efficacy of the HBG as a substitute for conventional corticocancellous iliac autografts. Summary of Background Data. Iliac crest bone graft (ICBG) has been traditionally used as the golden standard for lumbar spinal fusion. The significant complication rate associated with harvesting corticocancellous ICBG, however, has encouraged development of alternative graft substitutes. Methods. From September 2005, 61 consecutive patients underwent decompressive laminotomy and 1-level instrumented PLF. Each patient in this study had the constructs of the HBG placed on 1 side of the intertransverse process gutter. An autologous local bone graft (LBG) harvested during decompressive laminotomy was placed on the other side as a control. Radiographic evaluation was performed at 6 months, 1 year after surgery, and subsequently on an annual basis. The fusion statuses on either side of vertebra were compared. Results. The flexion-extension motion in the dynamic x-rays at the target level decreased over time. Only 1 case exhibited over 5° of angular motion 2 years after surgery. In the evaluation of fusion status, the fusion rate for the HBG side (68.9% at 6 months, 83.6% at 1 year, 93.5% at 2 years) was higher than that for the LBG side (49.2% at 6 months, 75.4% at 1 year, 89.1% at 2 years) with a significant difference at 6 months after surgery. No significant complications at the donor site were found postoperatively. Conclusion. The HBG promoted posterolateral spinal fusion without significant donor site morbidity. Because of its efficacy and safety, this hybrid construct seems promising as an alternative to conventional iliac bone grafts for lumbar spinal fusion.

Cervical Sagittal Imbalance is a Predictor of Kyphotic Deformity After Laminoplasty in Cervical Spondylotic Myelopathy Patients Without Preoperative Kyphotic Alignment.

Study Design. A retrospective cohort study. Objective. The aim of this study is to investigate the preoperative factors for postlaminoplasty kyphotic deformity in cervical spondylotic myelopathy (CSM) patients without preoperative kyphotic alignment focused on the cervical sagittal balance. Summary of Background Data. After laminoplasty (LAMP), appropriate decompression may be obtained when cervical lordosis is maintained to allow the posterior shift of the spinal cord. Therefore, LAMP is not suitable for patients with preoperative cervical kyphosis. However, we sometimes encounter patients who developed postoperative kyphosis despite normal preoperative alignment. The risk factors of postlaminoplasty kyphotic deformity for the patients without preoperative kyphotic alignment are not well known. Methods. A total of 174 consecutive patients who received a double-door LAMP for CSM without preoperative kyphotic alignment and completed a 1-year follow-up were enrolled. Cervical lateral X-ray images obtained in the standing position were measured at the preoperative stage and during a 1-year follow-up visit. The radiographic measurements included the following: (1) C2-7 lordotic angle (C2-7 angle), (2) C2-7 range of motion (C2-7 ROM), (3) CGH (center of gravity of the head)-C7 SVA, and (4) C7 slope. The clinical results were evaluated using the Japanese Orthopedic Association score system for cervical myelopathy (C-JOA score). Results. Postoperative kyphotic deformity was observed in 9 patients (5.2%). The recovery rates of the C-JOA scores at the 1-year follow-up period in the kyphotic deformity (+) group were inferior to those of the kyphotic deformity (−) group. The CGH-C7 SVA and advanced age were detected as preoperative risk factors using multivariate analysis. Cutoff values for predicting postlaminoplasty kyphotic deformity were a CGH-C7 SVA = 42 mm and an age of 75 years. Conclusion. Cervical sagittal imbalance and advanced age were the preoperative risk factors for kyphotic deformity after LAMP for CSM in patients without preoperative cervical kyphotic alignment. Level of Evidence: 4

Synthesis, characterization of calcium phosphates/polyurethane composites for weight-bearing implants†

Calcium phosphate (CaP)/polymer composites have been studied as an alternative graft material for the treatment of bone defects. In this study, lysine-triisocyanate-based polyurethane (PUR) composites were synthesized from both hydroxyapatite (HA) and β-tricalcium phosphate (TCP) to reduce the brittleness of CaP and increase the bioactivity of the polymer. The mechanical properties and in vitro cellular response were investigated for both HA/PUR and TCP/PUR composites. The composites were implanted in femoral defects in rats, and in vivo bioactivity was evaluated by X-rays, micro-computed tomography (μCT), and histological sections. In biomechanical testing, PUR improved the mechanical properties of the CaP, thus rendering it potentially suitable for weight-bearing applications. In vitro cell culture studies showed that CaP/PUR composites are biocompatible, with β-TCP enhancing the cell viability and proliferation relative to HA. CaP/PUR composites also supported the differentiation of osteoblastic cells on the materials. When implanted in rat femoral defects, the CaP/PUR composites were biocompatible and osteoconductive with no adverse inflammatory response, as evidenced by X-rays, μCT images, and histological sections. Additionally, a histological examination showed evidence of cellular infiltration and appositional remodeling. These results suggest that CaP/PUR composites could be potentially useful biomaterials for weight-bearing orthopaedic implants.

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.