Tsutomu Kira

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.

Dynamic analysis of the ulnar nerve in the cubital tunnel using ultrasonography

BACKGROUND We investigated the dynamics of the ulnar nerve during elbow flexion and the relationships between these dynamics and the morphology of the ulnar nerve groove in healthy individuals. MATERIALS AND METHODS Twenty healthy volunteers (40 elbows) underwent ultrasonographic examination of the ulnar nerve at the elbow. We measured the breadth and depth of the ulnar nerve groove at 90° of elbow flexion and calculated the depth-to-breadth ratio. We recorded the distance from the trochlea of the humerus to the nerve and the short-axis diameter of the nerve at 30°, 60°, 90°, and 120° of elbow flexion. We calculated the medial shift and flattening of the ulnar nerve at each angle relative to 30° of flexion, compared the values among the different angles, and compared the depth-to-breadth ratio with the location, medial shift, and flattening ratio of the ulnar nerve. RESULTS The medial shift was significantly greater at 120° than at other angles (P < .001). Flattening increased with increasing elbow flexion and was significantly different at 60°, 90°, and 120° (all P < .001). The flattening ratios were significantly correlated with the depth-to-breadth ratio at 120° (r = -0.43, P = .005). CONCLUSIONS The ulnar nerve moves medially and is flattened with the elbow flexed between 90° and 120°. When the ulnar nerve groove is shallow, high degrees of elbow flexion result in flattening of the ulnar nerve in the groove.

Ultrasound-guided Selective Sensory Nerve Block for Wide-awake Forearm Tendon Reconstruction

Background: Wide-awake hand surgery is useful for tendon reconstruction because surgeons can observe the actual movement of the reconstructed tendons during the surgery. We hypothesized that accurate ultrasound-guided injection of local anesthetics into the sensory nerves contributes to reliable analgesia with a relatively small amount of anesthetic. Methods: We enrolled 8 patients who underwent forearm tendon transfer. Three patients underwent reconstruction of flexor tendon ruptures in zones 4 and 5, 3 underwent opponensplasty, and 2 underwent multiple tendon transfers according to Brand’s procedure. All patients underwent ultrasound-guided injection of ropivacaine to each sensory nerve branch of the upper arm and forearm and into the subfascial layer of the forearm. The mean amount of total ropivacaine was 193 mg. Results: In 7 of the 8 patients, we confirmed adequate active contraction of the flexor or extensor muscles during surgery. The expected active motion of the flexor pollicis longus was not found in 1 patient during surgery because the effect of the anesthetic had spread too widely, involving the motor branch of the median nerve. Two patients required additional infiltration of 2–3 mL of local anesthetic because of local wound pain. All patients gained satisfactory function of the transferred tendons after the surgery, and no remarkable perioperative complications related to local anesthetic systemic toxicity occurred. Conclusions: Selective administration of an anesthetic to the sensory nerve branches and subfascial layer enables the performance of wide-awake forearm tendon surgery. The ultrasound-guided injection technique provides safe and effective regional anesthesia for wide-awake surgery.

Biomechanical study of distal radioulnar joint ballottement test

We investigated the reliability and accuracy of the distal radioulnar joint (DRUJ) ballottement test using five fresh‐frozen cadaver specimens in triangular fibrocartilage complex (TFCC)‐intact, and TFCC‐sectioned wrists. The humerus and proximal ulna were fixed. The ulna was allowed to translate in dorsopalmar directions without rotation, and the radius was allowed to move freely. Four sensors of a magnetic tracking system were attached to the radius and ulna, and the nails of each examiners thumbs. Five examiners conducted the DRUJ ballottement test before and after TFCC sectioning. We used two techniques: With holding and without holding the carpal bones to the radius (holding and non‐holding tests, respectively). We compared the magnitudes of bone‐to‐bone (absolute DRUJ) movement with that of the examiners nail‐to‐nail (relative DRUJ) movement. The intrarater intraclass correlation coefficients (ICCs) were 0.92 (holding) and 0.94 (non‐holding). The interrater ICCs were 0.84 (holding) and 0.75 (non‐holding). Magnitudes of absolute and relative movements averaged 11.5 and 11.8 mm, respectively (p < 0.05). Before TFCC sectioning, the DRUJ movement during the holding and non‐holding techniques averaged 9.8 and 10.8 mm, respectively (p < 0.05). The increase in DRUJ movement after TFCC sectioning was greater with the holding technique (average 2.3 mm) than with the non‐holding technique (average 1.6 mm). The DRUJ ballottement test with magnetic markers is relatively accurate and reliable for detecting unstable joints. We recommend the holding technique for assessing DRUJ instability in clinical practice.

culturing bone marrow cells with dexamethasone and ascorbic acid improves osteogenic cell sheet structure

Objectives To assess the structure and extracellular matrix molecule expression of osteogenic cell sheets created via culture in medium with both dexamethasone (Dex) and ascorbic acid phosphate (AscP) compared either Dex or AscP alone. Methods Osteogenic cell sheets were prepared by culturing rat bone marrow stromal cells in a minimal essential medium (MEM), MEM with AscP, MEM with Dex, and MEM with Dex and AscP (Dex/AscP). The cell number and messenger (m)RNA expression were assessed in vitro, and the appearance of the cell sheets was observed after mechanical retrieval using a scraper. β-tricalcium phosphate (β-TCP) was then wrapped with the cell sheets from the four different groups and subcutaneously implanted into rats. Results After mechanical retrieval, the osteogenic cell sheets from the MEM, MEM with AscP, and MEM with Dex groups appeared to be fragmented or incomplete structures. The cell sheets cultured with Dex/AscP remained intact after mechanical retrieval, without any identifiable tears. Culture with Dex/AscP increased the mRNA and protein expression of extracellular matrix proteins and cell number compared with those of the other three groups. More bridging bone formation was observed after transplantation of the β-TCP scaffold wrapped with cell sheets cultured with Dex/AscP, than in the other groups. Conclusions These results suggest that culture with Dex/AscP improves the mechanical integrity of the osteogenic cell sheets, allowing retrieval of the confluent cells in a single cell sheet structure. This method may be beneficial when applied in cases of difficult tissue reconstruction, such as nonunion, bone defects, and osteonecrosis. Cite this article: M. Akahane, T. Shimizu, T. Kira, T. Onishi, Y. Uchihara, T. Imamura, Y. Tanaka. Culturing bone marrow cells with dexamethasone and ascorbic acid improves osteogenic cell sheet structure. Bone Joint Res 2016;5:569–576. DOI: 10.1302/2046-3758.511.BJR-2016-0013.R1.

Bone regeneration with osteogenic matrix cell sheet and tricalcium phosphate: An experimental study in sheep

AIM To determine the effects of a cell sheet created from sheep bone marrow and tricalcium phosphate (TCP) on osteogenesis. METHODS Bone marrow cells were harvested from a sheep and cultured in a minimal essential medium (MEM) containing ascorbic acid phosphate (AscP) and dexamethasone (Dex). After 2 wk, the formed osteogenic matrix cell sheet was lifted from the culture dish using a scraper. Additionally, harvested bone marrow cells were cultured in MEM only as a negative control group, and in MEM with AscP, Dex, and β-glycerophosphate as a positive control group. For in vitro evaluation, we measured the alkaline phosphatase (ALP) activity and osteocalcin (OC) content in the media of the cultured cells from each group. For in vivo analysis, a porous TCP ceramic was used as a scaffold. We prepared an experimental group comprising TCP scaffolds wrapped with the osteogenic matrix cell sheets and a control group consisting of the TCP scaffold only. The constructs were implanted subcutaneously into athymic rats and the cell donor sheep, and bone formation was confirmed by histology after 4 wk. RESULTS In the in vitro part, the mean ALP activity was 0.39 ± 0.03 mg/well in the negative control group, 0.67 ± 0.04 mg/well in the sheet group, and 0.65 ± 0.07 mg/well in the positive control group. The mean OC levels were 1.46 ± 0.33 ng/well in the negative control group, 3.92 ± 0.16 ng/well in the sheet group, and 4.4 ± 0.47 ng/well in the positive control group, respectively. The ALP activity and OC levels were significantly higher in the cell sheet and positive control groups than in the negative control group (P < 0.05). There was no significant difference in ALP activity or OC levels between the cell sheet group and the positive control group (P > 0.05). TCP constructs wrapped with cell sheets prior to implantation showed bone formation, in contrast to TCP scaffolds alone, which exhibited poor bone formation when implanted, in the subcutaneous layer both in athymic rats and in the sheep. CONCLUSION This technique for preparing highly osteoinductive TCP may promote regeneration in large bone defects.

A Biomechanical Perspective on Distal Radioulnar Joint Instability

Background The purpose of this article was to review the anatomy, kinematics of the distal radioulnar joint (DRUJ), and to discuss definition, classification, and diagnosis of DRUJ instability. Methods A biomechanical perspective on physical examination of DRUJ ballottement test was documented. Physiological dynamic DRUJ translation and differences of the translation following sequential ligament sectioning and changes in different forearm and wrist positions were demonstrated. The clinical significance of each ligaments contribution to joint stability in specific wrist positions was addressed. Conclusion Each ligament stabilizing the DRUJ contributed to joint stability depending on the direction (palmer or dorsal) and different positions of the wrist and forearm. DRUJ ballottement test in each wrist and forearm position may detect tears of specific ligament stabilizing the DRUJ.

Vascularized Bone Grafts from the Dorsal Wrist for the Treatment of Kienböck Disease

Purpose The objective of this article is to evaluate functional and radiological outcomes of vascularized bone grafts for stage 2 and 3 Kienböck disease. The outcomes of three different donor sites via dorsal approach of the wrist were compared. Pearls and pitfalls in surgical technique were discussed. Methods There were 28 patients who underwent vascularized bone grafts, including the extensor fourth and fifth compartmental artery graft of distal radius in 8 patients, the first and second supraretinacular intercompartmental artery graft of distal radius in 12 patients, and the second dorsal metacarpal neck graft in 8 patients. Average age was 32 years, and radiological grading according to Lichtman classification was stage 2 in 8 patients, stage 3A in 10 patients, and stage 3B in 10 patients. Temporary pinning fixing the midcarpal joint was conducted for 10 weeks postoperatively. Results Follow-up periods averaged 70 months. Pain reduced in 27 patients, and visual analog scale for pain of pre- and postoperative level averaged 59 and 18. Range of wrist flexion and extension motion improved from 87 to 117 degrees, and average grip strength improved from 21 kg preoperatively to 33 kg postoperatively. Carpal height ratio had almost no change from 0.52 to 0.53. Fragmentation of necrotic bone healed in 7 of the 14 cases. Comparative analyses of functional and radiological outcomes between three donor sites found no significant difference. Conclusion Three different vascularized bone grafts from the dorsal wrist and hand area demonstrated favorable and comparable functional outcomes. It was technically important to elevate vascular bundle with surrounding retinaculum or fascia, to include sufficient periosteum, and to insert the vascularized bone as the cortex aligned longitudinally.

Biceps Femoris Musculocutaneous Flap for Reconstruction of Refractory Ulceration at the Popliteal Fossa

Background: There is a lack of information about the possibility of transfer of the long head of the biceps femoris (LHBF) musculocutaneous flap to the knee area. We discuss the use of the LHBF musculocutaneous flap to treat refractory ulceration at the popliteal fossa and the results of a preliminary study investigating the anatomical possibility of transferring this flap to the popliteal region. Methods: Five lower extremities of 5 fresh cadaveric specimens were dissected following injection of a silicone compound into the deep femoral artery. We investigated the number, location, and diameter of nutrient branches to the LHBF originating from the deep femoral artery. Based on these results, we treated a 76-year-old woman with a refractory postradiation ulcer at the popliteal fossa associated with popliteal artery obstruction using a 25 × 7 cm LHBF musculocutaneous flap. Results: The mean number of nutrient branches to the LHBF muscle was 3.6, with a mean diameter of 1.9 mm. One to two branches consistently arose from the distal aspect of the posterior thigh. Most branches followed an intramuscular route, giving rise to fine cutaneous branches. The distal border reached by the musculocutaneous flap was located 6.7 cm distal to the bicondylar line. The flap survived completely without complications, and the patient was able to walk with a walking frame postoperatively. Conclusions: The LHBF musculocutaneous flap may offer a reliable treatment option for soft-tissue defects of the popliteal fossa, especially in patients with significant damage to the popliteal artery from trauma or radiation therapy.

Osteogenesis of osteogenic matrix cell sheets preserved in culture medium in a rat model

Osteogenic matrix cell sheets (OMCSs) are ideal for bone regeneration. Transportation of OMCSs may be necessary, during which their osteogenic ability must be maintained. Here, we evaluated different media and temperatures for OMCS preservation. Bone marrow stromal/stem cells (BMSCs) were obtained from Fischer rats and analyzed for stem cell markers by flow cytometry. OMCSs were prepared from BMSCs by treatment with dexamethasone and ascorbic acid phosphate. After OMCS collection, they were stored in minimum essential medium (MEM) or Hank’s balanced salt solution (HBSS) at 37, 22, or 4°C for 24 hours. Cell viability and cytotoxic effects in the preservation conditions were determined by adenosine triphosphate (ATP) contents and lactate dehydrogenase (LDH) release, respectively. Osteogenesis was assessed by subcutaneously implanting preserved OMCSs around β-tricalcium phosphate ceramic disks into syngeneic rats. Implants were evaluated by alkaline phosphatase (ALP) activities, osteocalcin contents, and histology. Mesenchymal stem cells comprised 51% of primary cultured BMSCs. ATP contents were significantly different in OMCSs stored in MEM or HBSS at 22°C and 4°C. LDH release was significantly different in OMCSs stored in HBSS at 22°C and 4°C. The highest LDH release was observed in OMCSs stored in HBSS at 37°C. ALP activities and osteocalcin contents were the lowest in implanted OMCSs stored in HBSS at 37°C at four weeks after subcutaneous implantation. There was a significant difference in the osteocalcin levels of implanted OMCSs stored in MEM at 37°C and HBSS at 4°C. Abundant bone tissue around and inside disks was found in histological sections of OMCSs stored in all preservation conditions except for MEM and HBSS at 37°C. Maintaining the osteogenic ability of OMCSs during transport is important, and preservation of OMCSs in MEM or HBSS at 4°C or 22°C is a simple and inexpensive method.