Yusuke Inagaki

The regeneration and augmentation of bone with injectable osteogenic cell sheet in a rat critical fracture healing model.

Limitations in the current treatment strategies make cases with compromised bone healing challenging clinical problems. Osteogenic cell sheets (OCSs), fabricated from rat bone marrow stromal cells (BMSCs), contain enriched osteoblasts and extracellular matrix. Here, we evaluated whether the minimally invasive percutaneous injection of OCSs without a scaffold could be used as a treatment to increase bone regeneration in a critical fracture healing model. Critical fracture healing model was created in the femora of 60 male Fischer 344 inbred rats using marrow ablation and periosteal removal. The rats were then randomly divided into two groups. Six hours after fracture, one group received an injection of OCSs (OCS group), while the second group was injected with phosphate-buffered saline (PBS) (control group). Fracture healing was evaluated using radiological, histological, micro-computed tomography (CT) and biomechanical analyses. The radiological and histological evaluations demonstrated enhanced bone regeneration in the OCS group compared with that in the control group. By 12 weeks, the hard callus had been remodelled via recorticalization in the OCS group. By contrast, no fracture union was found in the rats in the control group. Biomechanical testing revealed a significantly higher maximum bending load in the OCS group compared with that in the control group. The results of the present study demonstrate that the injection of entire OCSs can enhance bone regeneration and lead to bony union in a critical fracture healing model. Therefore, this procedure offers a minimally invasive technique to promote hard tissue reconstruction and, in particular, bone repair strategies for cases with compromised bone healing.

Osteogenic Matrix Cell Sheet Transplantation Enhances Early Tendon Graft to Bone Tunnel Healing in Rabbits

The objective of this study was to determine whether osteogenic matrix cell sheets (OMCS) could induce bone formation around grafted tendons, thereby enhancing early stage tendon to bone tunnel healing in skeletally mature male Japanese white rabbits. First, the osteogenic potential of rabbit OMCS was evaluated. Then, the OMCS were transplanted into the interface between the grafted tendon and the bone tunnel created at the tibia. Histological assessments and biomechanical tensile testing were performed after 3 weeks. The rabbit OMCS showed high alkaline phosphatase (ALP) activity, positive staining of ALP, and osteogenic potential when transplanted subcutaneously with beta tricalcium phosphate disks. Newly formed bony walls and positive collagen type I staining were seen around the grafted tendon with OMCS transplantation, whereas such bony walls were thinner or less frequent without OMCS transplantation. Micro-computed tomography images showed significantly higher bone volume in the OMCS transplantation group. The pullout strength was significantly higher with OMCS (0.74 ± 0.23 N/mm2) than without OMCS (0.58 ± 0.15 N/mm2). These results show that OMCS enhance early tendon to bone tunnel healing. This method can be applied to cases requiring early tendon to bone tunnel healing after ligament reconstruction surgery.

Utility of tricalcium phosphate and osteogenic matrix cell sheet constructs for bone defect reconstruction

AIM To determine the effects of transplanting osteogenic matrix cell sheets and beta-tricalcium phosphate (TCP) constructs on bone formation in bone defects. METHODS Osteogenic matrix cell sheets were prepared from bone marrow stromal cells (BMSCs), and a porous TCP ceramic was used as a scaffold. Three experimental groups were prepared, comprised of TCP scaffolds (1) seeded with BMSCs; (2) wrapped with osteogenic matrix cell sheets; or (3) both. Constructs were implanted into a femoral defect model in rats and bone growth was evaluated by radiography, histology, biochemistry, and mechanical testing after 8 wk. RESULTS In bone defects, constructs implanted with cell sheets showed callus formation with segmental or continuous bone formation at 8 wk, in contrast to TCP seeded with BMSCs, which resulted in bone non-union. Wrapping TCP constructs with osteogenic matrix cell sheets increased their osteogenic potential and resulting bone formation, compared with conventional bone tissue engineering TCP scaffolds seeded with BMSCs. The compressive stiffness (mean ± SD) values were 225.0 ± 95.7, 30.0 ± 11.5, and 26.3 ± 10.6 MPa for BMSC/TCP/Sheet constructs with continuous bone formation, BMSC/TCP/Sheet constructs with segmental bone formation, and BMSC/TCP constructs, respectively. The compressive stiffness of BMSC/TCP/Sheet constructs with continuous bone formation was significantly higher than those with segmental bone formation and BMSC/TCP constructs. CONCLUSION This technique is an improvement over current methods, such as TCP substitution, and is useful for hard tissue reconstruction and inducing earlier bone union in defects.

Meniscal Bearing Dislocation of Unicompartmental Knee Arthroplasty with Faint Symptom.

We experienced two cases of atypical lateral dislocations of meniscal bearing in UKA (unicompartmental knee arthroplasty) without manifest symptoms. The dislocated bearing, which jumped onto the wall of tibial components, was found on radiographs in periodic medical examination although they could walk. Two thicker size bearing exchanges were promptly performed before metallosis and loosening of components. Continual examination is important to mobile bearing type of UKA because slight or less symptoms may disclose such unique dislocation. One case showed malrotation of the femoral component on 3D image. Anteroposterior view hardly disclosed the malrotation of the femoral component. Epicondylar view is an indispensable view of importance, and it can demonstrate the rotation of the femoral component. The the femoral distal end is wedge shaped and is wider posteriorly. If the femoral component is set according to the shape of medial condyle, the femoral component shifts to medial site compared with tibial component in flexion. It can account for such rare dislocation as follows. If excessive force applies on most medial side of the bearing during flexion, the lateral part of the bearing pops and the force squeezes it laterally simultaneously. Finally, the bearing jumps onto the lateral wall of the tibial component.

Nontraumatic tibial polyethylene insert cone fracture in mobile-bearing posterior-stabilized total knee arthroplasty

A 72-year-old male patient underwent mobile-bearing posterior-stabilized total knee arthroplasty for osteoarthritis. He experienced a nontraumatic polyethylene tibial insert cone fracture 27 months after surgery. Scanning electron microscopy of the fracture surface of the tibial insert cone suggested progress of ductile breaking from the posterior toward the anterior of the cone due to repeated longitudinal bending stress, leading to fatigue breaking at the anterior side of the cone, followed by the tibial insert cone fracture at the anterior side of the cone, resulting in fracture at the base of the cone. This analysis shows the risk of tibial insert cone fracture due to longitudinal stress in mobile-bearing posterior-stabilized total knee arthroplasty in which an insert is designed to highly conform to the femoral component.

Implant failure in bilateral metal-on-metal hip resurfacing arthroplasties: a clinical and pathological study.

AbstractMetal-on-metal hip resurfacing arthroplasties (MoMHRAs) have a high failure rate due to pseudotumour formation. It is not certain whether pseudotumours in bilateral MoMHRAs form on the basis of an adverse reaction to metal debris (ARMD) that is entirely due to a local innate and adaptive immune response to Cobalt-Chrome (Co-Cr) wear particles. To determine if there is a systemic component to ARMD in bilateral MoMHRAs, we examined the histology of ARMD in unilateral and bilateral MoMHRAs revised for pseudotumour and determined whether implant survival differed between these two groups. Periprosthetic tissue specimens from 119 hips revised for pseudotumour were examined. These were derived from 109 patients including 10 patients with bilateral MoMHRAs and 99 with sunilateral MoMHRAs including a cohort of 20 patients with bilateral MoMHRAs that had undergone only one MoMHRA revision for pseudotumour. The mean time to revision for pseudotumour of unilateral and bilateral MoMHRAs was determined. The histology of periprosthetic tissue was examined for evidence of the innate and adaptive immune response and scored semi-quantitatively. There was no significant difference in histological features of the innate / adaptive response between Group 1 bilateral pseudotumours and Group 2 and Group 3 unilateral pseudotumours. Histological features, including ALVAL scores, were similar in the periprosthetic tissues of right and left hips in Group 1 bilateral MoMHRAs. The mean time to first revision for pseudotumour of bilateral MoMHRAs (6.59 years) was not decreased compared with unilateral MoMHRAs (5.66 years) or bilateral MoMHRAs that had only one revision (7.05 years). Right and left hip pseudotumours in bilateral MoMHRAs exhibit similar histological features of the innate and adaptive immune response. Mean implant survival is not decreased in bilateral compared with unilateral MoMHRA cases. The findings suggest that pseudotumour formation is due more to a local than a systemic innate /adaptive immune response to components of metal wear.

Modifying oxygen tension affects bone marrow stromal cell osteogenesis for regenerative medicine

AIM To establish a hypoxic environment for promoting osteogenesis in rat marrow stromal cells (MSCs) using osteogenic matrix cell sheets (OMCSs). METHODS Rat MSCs were cultured in osteogenic media under one of four varying oxygen conditions: Normoxia (21% O2) for 14 d (NN), normoxia for 7 d followed by hypoxia (5% O2) for 7 d (NH), hypoxia for 7 d followed by normoxia for 7 d (HN), or hypoxia for 14 d (HH). Osteogenesis was evaluated by observing changes in cell morphology and calcium deposition, and by measuring osteocalcin secretion (ELISA) and calcium content. In vivo syngeneic transplantation using OMCSs and β-tricalcium phosphate discs, preconditioned under NN or HN conditions, was also evaluated by histology, calcium content measurements, and real-time quantitative PCR. RESULTS In the NN and HN groups, differentiated, cuboidal-shaped cells were readily observed, along with calcium deposits. In the HN group, the levels of secreted osteocalcin increased rapidly from day 10 as compared with the other groups, and plateaued at day 12 (P < 0.05). At day 14, the HN group showed the highest amount of calcium deposition. In vivo, the HN group showed histologically prominent new bone formation, increased calcium deposition, and higher collagen type I messenger RNA expression as compared with the NN group. CONCLUSION The results of this study indicate that modifying oxygen tension is an effective method to enhance the osteogenic ability of MSCs used for OMCSs.

Calcium Concentration in Culture Medium as a Nondestructive and Rapid Marker of Osteogenesis.

Artificial bones made of β-tricalcium phosphate (β-TCP) combined with bone marrow-derived mesenchymal stromal cells (BM-MSCs) are used for effective reconstruction of bone defects caused by genetic defects, traumatic injury, or surgical resection of bone tumors. However, the selection of constructs with high osteogenic potential before implantation is challenging. The purpose of this study was to determine whether the calcium concentration in BM-MSC culture medium can be used as a nondestructive and simple osteogenic marker for selecting tissue-engineered grafts constructed using β-TCP and BM-MSCs. We prepared three cell passages of BM-MSCs derived from three 7-week-old, male Fischer 344 rats; the cells were cultured in osteoinductive medium in the presence of β-TCP for 15 days. The medium was replaced with fresh medium on day 1 in culture and subsequently changed every 48 h; it was collected for measurement of osteocalcin secretion and calcium concentration by enzyme-linked immunosorbent assay and X-ray fluorescence spectrometry, respectively. After cultivation, the constructs were implanted subcutaneously into the backs of recipient rats. Four weeks after implantation, the alkaline phosphatase (ALP) activity and osteocalcin content of the constructs were measured. A strong inverse correlation was observed between the calcium concentration in the medium and the ALP activity and osteocalcin content of the constructs, with Pearsons correlation coefficients of 0.92 and 0.90, respectively. These results indicate that tissue-engineered bone with high osteogenic ability can be selected before implantation based on low calcium content of the culture medium, resulting in successful bone formation after implantation. This nondestructive, simple method shows great promise for assessing the osteogenic ability of tissue-engineered bone.

Quantification of neutrophil polymorphs in infected and noninfected second-stage revision hip arthroplasties

Aims In the diagnosis of periprosthetic joint infection (PJI), a heavy neutrophil polymorph (NP) infiltrate (>5 per high-power field [HPF] by MSIS criteria) is characteristically seen in periprosthetic tissues. PJI is commonly treated by a two-stage procedure with surgical clearance of infected tissues followed by intensive antibiotic treatment before re-implantation; tissues are sampled at the time of second stage but whether MSIS histological criteria can be used to diagnose the presence or absence of infection in second stage samples has not been established. Methods Periprosthetic tissues from 31 cases of second-stage revision hip arthroplasty (including 3 cases fulfilling the microbiological MSIS criteria for PJI), were analysed histologically after haematoxylin-eosin and chloroacetate esterase (CAE) staining. The extent of the NP infiltrate was determined semiquantitatively and correlated with the microbiological diagnosis. Results CAE staining facilitated identification of NPs in arthroplasty tissues and showed that in those cases where an organism was cultured in 2 or more samples, meeting the MSIS microbiological criteria for definite diagnosis of PJI, there was a heavy polymorph infiltrate (>5 NP per HPF on average). It was noted that isolated or scattered NPs were seen in 42.8% of periprosthetic tissues from noninfected second-stage revisions. Conclusions The MSIS histological criteria which support a diagnosis of PJI in specimens from a primary revision hip arthroplasty (i.e. >5 NPs per HPF on average) are also valid for the assessment of a second-stage specimens. NPs can be seen in samples of periprosthetic tissue from uninfected second-stage revisions, indicating that strict histological criteria should be used in evaluating their significance in this context.