Masato Yuasa

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.

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.

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.

Fibrinolysis is essential for fracture repair and prevention of heterotopic ossification.

Bone formation during fracture repair inevitably initiates within or around extravascular deposits of a fibrin-rich matrix. In addition to a central role in hemostasis, fibrin is thought to enhance bone repair by supporting inflammatory and mesenchymal progenitor egress into the zone of injury. However, given that a failure of efficient fibrin clearance can impede normal wound repair, the precise contribution of fibrin to bone fracture repair, whether supportive or detrimental, is unknown. Here, we employed mice with genetically and pharmacologically imposed deficits in the fibrin precursor fibrinogen and fibrin-degrading plasminogen to explore the hypothesis that fibrin is vital to the initiation of fracture repair, but impaired fibrin clearance results in derangements in bone fracture repair. In contrast to our hypothesis, fibrin was entirely dispensable for long-bone fracture repair, as healing fractures in fibrinogen-deficient mice were indistinguishable from those in control animals. However, failure to clear fibrin from the fracture site in plasminogen-deficient mice severely impaired fracture vascularization, precluded bone union, and resulted in robust heterotopic ossification. Pharmacological fibrinogen depletion in plasminogen-deficient animals restored a normal pattern of fracture repair and substantially limited heterotopic ossification. Fibrin is therefore not essential for fracture repair, but inefficient fibrinolysis decreases endochondral angiogenesis and ossification, thereby inhibiting fracture repair.

Jagged1 is essential for osteoblast development during maxillary ossification

Maxillary hypoplasia occurs due to insufficient maxillary intramembranous ossification, leading to poor dental occlusion, respiratory obstruction and cosmetic deformities. Conditional deletion of Jagged1 (Jag1) in cranial neural crest (CNC) cells using Wnt1-cre; Jagged1(f/f) (Jag1CKO) led to maxillary hypoplasia characterized by intrinsic differences in bone morphology and density using μCT evaluation. Jag1CKO maxillas revealed altered collagen deposition, delayed ossification, and reduced expression of early and late determinants of osteoblast development during maxillary ossification. In vitro bone cultures on Jag1CKO mouse embryonic maxillary mesenchymal (MEMM) cells demonstrated decreased mineralization that was also associated with diminished induction of osteoblast determinants. BMP receptor expression was dysregulated in the Jag1CKO MEMM cells suggesting that these cells were unable to respond to BMP-induced differentiation. JAG1-Fc rescued in vitro mineralization and osteoblast gene expression changes. These data suggest that JAG1 signaling in CNC-derived MEMM cells is required for osteoblast development and differentiation during maxillary ossification.

Modified K-line in magnetic resonance imaging predicts insufficient decompression of cervical laminoplasty.

Study Design. A retrospective single-center study. Objective. To clarify preoperative factors predicting unsatisfactory indirect decompression after laminoplasty in patients with cervical spondylotic myelopathy. Summary of Background Data. Many authors have shown that inadequate indirect decompression after laminoplasty can inhibit neural recovery and should be considered a complication. We previously demonstrated that residual anterior compression of the spinal cord (ACS) impaired recovery of upper extremity motor function. Although the K-line has been established as a predictive index indicating that laminoplasty is required in patients with ossification of the posterior longitudinal ligament, it remains unclear what preoperative factors can predict insufficient posterior cord decompression in patients with cervical spondylotic myelopathy. Methods. Forty-six consecutive patients who underwent laminoplasty for the treatment of cervical spondylotic myelopathy at our hospital were reviewed. A modified K-line was defined as the line connecting the midpoints of the spinal cord at C2 and C7 on a T1-weighted sagittal magnetic resonance image. We also determined the minimum interval between the tip of local kyphosis and a line connecting the midpoint of the cord at the level of the inferior endplates of C2 and C7 (INTmin) on the midsagittal image. Data analysis involved logistic regression and receiver operating characteristic curve analysis to select the most valuable index for predicting postoperative ACS. Results. Ten patients had ACS immediately after laminoplasty. Logistic regression analysis showed that INTmin was a significant predictive factor for the occurrence of postoperative ACS (odds ratio = 0.485; 95% confidence interval = 0.29–0.81; P = 0.02). Receiver operating characteristic curve analysis showed an area under the curve of 0.871. A cutoff of 4.0 mm had a sensitivity of 80% and a specificity of 80.6% for prediction of postoperative ACS. Conclusion. The parameter INTmin correlated with the occurrence of postoperative ACS. A cutoff point of 4.0 mm is most appropriate for alerting spine surgeons to a high likelihood of postoperative ACS.

Differential development of the distal and proximal femoral epiphysis and physis in mice

Many pathologic conditions of hip development result from aberrant vascularity with subsequent effects on physeal resorption and epiphyseal fusion. To elucidate the mechanisms of these developmental disorders, researchers have mainly focused on larger mammals as they have been well characterized and are known to provide a model similar to humans in which a secondary ossification center is formed through an independent blood supply followed by physeal resorption. Murine models of hip development, however, have never been characterized as it was previously assumed that all physes in rodents never resorb and therefore not suitable as a model of the human condition. The purpose of this study was to determine if murine hip development was comparable to humans as laboratory mice provide an ideal model in which genetic knockouts are readily available with a short developmental time span. Here we show for the first time the unique developmental patterns of the murine hip in which a secondary ossification center never develops. Instead, the epiphysis undergoes a prolonged phase of mineralization through chondrocyte, not osteoblast, dependent mechanisms. After skeletal growth, transphyseal vessels develop from the metaphysis resulting in resorption of the physis, ossification of the mineralized cartilage of the epiphysis, and epiphyseal fusion. Although the development of the murine hip is markedly different from that in humans, we believe that these findings have direct implications for studying the vascularity of developing bone, particularly during physeal resorption, chondrocyte-mediated mineralization and more importantly, developmental diseases of the human physis and epiphysis.

Effects of the systemic administration of alendronate on bone formation in a porous hydroxyapatite/collagen composite and resorption by osteoclasts in a bone defect model in rabbits

Several bisphosphonates are now available for the treatment of osteoporosis. Porous hydroxyapatite/collagen (HA/Col) composite is an osteoconductive bone substitute which is resorbed by osteoclasts. The effects of the bisphosphonate alendronate on the formation of bone in porous HA/Col and its resorption by osteoclasts were evaluated using a rabbit model. Porous HA/Col cylinders measuring 6 mm in diameter and 8 mm in length, with a pore size of 100 μm to 500 μm and 95% porosity, were inserted into a defect produced in the lateral femoral condyles of 72 rabbits. The rabbits were divided into four groups based on the protocol of alendronate administration: the control group did not receive any alendronate, the pre group had alendronate treatment for three weeks prior to the implantation of the HA/Col, the post group had alendronate treatment following implantation until euthanasia, and the pre+post group had continuous alendronate treatment from three weeks prior to surgery until euthanasia. All rabbits were injected intravenously with either saline or alendronate (7.5 μg/kg) once a week. Each group had 18 rabbits, six in each group being killed at three, six and 12 weeks post-operatively. Alendronate administration suppressed the resorption of the implants. Additionally, the mineral densities of newly formed bone in the alendronate-treated groups were lower than those in the control group at 12 weeks post-operatively. Interestingly, the number of osteoclasts attached to the implant correlated with the extent of bone formation at three weeks. In conclusion, the systemic administration of alendronate in our rabbit model at a dose-for-weight equivalent to the clinical dose used in the treatment of osteoporosis in Japan affected the mineral density and remodelling of bone tissue in implanted porous HA/Col composites.

The temporal and spatial development of vascularity in a healing displaced fracture

Underlying vascular disease is an important pathophysiologic factor shared among many co-morbid conditions associated with poor fracture healing, such as diabetes, obesity, and age. Determining the temporal and spatial patterns of revascularization following a fracture is essential for devising therapeutic strategies to augment this critical reparative process. Seminal studies conducted in the last century have investigated the pattern of vascularity in bone following a fracture. The consensus model culminating from these classical studies depicts a combination of angiogenesis emanating from both the intact intramedullary and periosteal vasculature. Subsequent to the plethora of experimental fracture angiography in the early to mid-20th century there has been a paucity of reports describing the pattern of revascularization of a healing fracture. Consequently the classical model of revascularization of a displaced fracture has remained largely unchanged. Here, we have overcome the limitations of animal fracture models performed in the above described classical studies by combining novel techniques of bone angiography and a reproducible murine femur fracture model to demonstrate for the first time the complete temporal and spatial pattern of revascularization in a displaced/stabilized fracture. These studies were designed specifically to i) validate the classical model of fracture revascularization of a displaced/stabilized fracture, ii) assess the association between intramedullary and periosteal angiogenesis and iii) elucidate the expression of VEGF/VEGF-R in relation to the classical model. From the studies, in conjunction with classic studies of angiogenesis during fracture repair, we propose a novel model (see abstract graphic) that defines the process of bone revascularization subsequent to injury to guide future approaches to enhance fracture healing. This new model validates and advances the classical model by providing evidence that during the process of revascularization of a displaced fracture 1) periosteal angiogenesis occurs in direct communication with the remaining intact intramedullary vasculature as a result of a vascular shunt and 2) vascular union occurs through an intricate interplay between intramembranous and endochondral VEGF/VEGF-R mediated angiogenesis.

Bone Defect Regeneration by a Combination of a β-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 β-tricalcium phosphate (β-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 β-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 β-TCP block under low-pressure conditions. The BMSC/β-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 β-TCP/BMSC composites, and bone union was evaluated radiographically. Results: After the primary surgery and treatment with β-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 β-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 β-TCP/BMSC composite for large bone defect regeneration and confirmed the bone union capability of the regenerated bone.