Tomokazu Yoshioka

Rotating three-dimensional dynamic culture of adult human bone marrow-derived cells for tissue engineering of hyaline cartilage

The method of constructing cartilage tissue from bone marrow‐derived cells in vitro is considered a valuable technique for hyaline cartilage regenerative medicine. Using a rotating wall vessel (RWV) bioreactor developed in a NASA space experiment, we attempted to efficiently construct hyaline cartilage tissue from human bone marrow‐derived cells without using a scaffold. Bone marrow aspirates were obtained from the iliac crest of nine patients during orthopedic operation. After their proliferation in monolayer culture, the adherent cells were cultured in the RWV bioreactor with chondrogenic medium for 2 weeks. Cells from the same source were cultured in pellet culture as controls. Histological and immunohistological evaluations (collagen type I and II) and quantification of glycosaminoglycan were performed on formed tissues and compared. The engineered constructs obtained using the RWV bioreactor showed strong features of hyaline cartilage in terms of their morphology as determined by histological and immunohistological evaluations. The glycosaminoglycan contents per µg DNA of the tissues were 10.01 ± 3.49 µg/µg DNA in the case of the RWV bioreactor and 6.27 ± 3.41 µg/µg DNA in the case of the pellet culture, and their difference was significant. The RWV bioreactor could provide an excellent environment for three‐dimensional cartilage tissue architecture that can promote the chondrogenic differentiation of adult human bone marrow‐derived cells.

Stable and nondisruptive in vitro/in vivo labeling of mesenchymal stem cells by internalizing quantum dots.

Progress in stem cell research has prioritized the refinement of cell-labeling techniques for in vitro and in vivo basic and therapeutic studies. Although quantum dots, because of their optical properties, are emerging as favorable nanoparticles for bioimaging, substantial refinements or modifications that would improve their biocompatibility are still required. We report here that internalizing quantum dots (i-QDs) generated by their conjugation with an internalizing antibody against a heat shock protein-70 family stress chaperone, mortalin, offered an efficient, genetically noninvasive, nontoxic, and functionally inert way to label mesenchymal stem cells (MSCs). The i-QD-labeled MSCs underwent normal adipocyte, osteocyte, and chondrocyte differentiation in vitro and in vivo, suggesting the potential application of i-QDs in in vivo diagnostics, regenerative and therapeutic medicine.

Concentration of bone marrow aspirate for osteogenic repair using simple centrifugal methods

Bone marrow transplantation can lead to osteogenic repair of intractable bone conditions. To achieve optimal clinical results, it is necessary to transplant as many cells with osteogenetic potential as possible. However, approaches involving special equipment and reagents for the extraction and purification of cells are expensive, and the complicated procedures involved are a hindrance to widespread acceptance of bone marrow transplantation for osteogenic repair. To standardize bone marrow transplantation for bone regeneration, a simple, safe, clean, and low‐cost system is required. We describe an easy‐to‐use method using a conventional manual blood bag centrifugation technique traditionally used for extracting buffy coats, for concentration of cells from bone marrow aspirates (BMAs) to obtain osteogenic progenitors.

Ultrasound-guided platelet-rich plasma injections for the treatment of common peroneal nerve palsy associated with multiple ligament injuries of the knee

PurposePeroneal nerve palsy in traumatic knee dislocations associated with multiple ligament injuries is common. Several surgical approaches are described for this lesion with less-than-optimal outcomes. The present case represents the application of plasma rich in growth factors (PRGF) technology for the treatment of peroneal nerve palsy with drop foot. This technology has already been proven its therapeutic potential for various musculoskeletal disorders. Based on these results, we hypothesized that PRGF could stimulate the healing process of traumatic peroneal nerve palsy with drop foot.MethodsThe patient was a healthy 28-year-old man. He suffered peroneal nerve palsy with drop foot after multiple ligament injuries of the knee. PRGF was prepared according to the manufactured instruction. Eleven months after the trauma with severe axonotmesis, serial intraneural infiltrations of PRGF were started using ultrasound guidance. The therapeutic effect was assessed by electromyography (EMG), echogenicity of the peroneal nerve under ultrasound (US) and manual muscle testing.ResultsTwenty-one months after the first injection, not complete but partial useful recovery is obtained. He is satisfied with walking and running without orthosis. Sensitivity demonstrates almost full recovery in the peroneal nerve distribution area. EMG controls show complete reinnervation for the peroneus longus and a better reinnervation for the tibialis anterior muscle, compared with previous examinations.ConclusionPlasma rich in growth factors (PRGF) infiltrations could enhance healing process of peroneal nerve palsy with drop foot. This case report demonstrates the therapeutic potential of this technology for traumatic peripheral nerve palsy and the usefulness of US-guided PRGF.Level of evidenceV.

Fate of bone marrow mesenchymal stem cells following the allogeneic transplantation of cartilaginous aggregates into osteochondral defects of rabbits.

The purpose of this study was to track mesenchymal stem cells (MSCs) labelled with internalizing quantum dots (i‐QDs) in the reparative tissues, following the allogeneic transplantation of three‐dimensional (3D) cartilaginous aggregates into the osteochondral defects of rabbits. QDs were conjugated with a unique internalizing antibody against a heat shock protein‐70 (hsp70) family stress chaperone, mortalin, which is upregulated and expressed on the surface of dividing cells. The i‐QDs were added to the culture medium for 24 h. Scaffold‐free cartilaginous aggregates formed from i‐QD‐labelled MSCs (i‐MSCs), using a 3D culture system with chondrogenic supplements for 1 week, were transplanted into osteochondral defects of rabbits. At 4, 8 and 26 weeks after the transplantation, the reparative tissues were evaluated macroscopically, histologically and fluoroscopically. At as early as 4 weeks, the defects were covered with a white tissue resembling articular cartilage. In histological appearance, the reparative tissues resembled hyaline cartilage on safranin‐O staining throughout the 26 weeks. In the deeper portion, subchondral bone and bone marrow were well remodelled. On fluoroscopic evaluation, QDs were tracked mainly in bone marrow stromata, with some signals detected in cartilage and the subchondral bone layer. We showed that the labelling of rabbit MSCs with anti‐mortalin antibody‐conjugated i‐QDs is a tolerable procedure and provides a stable fluorescence signal during the cartilage repair process for up to 26 weeks after transplantation. The results suggest that i‐MSCs did not inhibit, and indeed contributed to, the regeneration of osteochondral defects. Copyright

Ultrasound-guided plasma rich in growth factors injections and scaffolds hasten motor nerve functional recovery in an ovine model of nerve crush injury.

In the present study we evaluated the motor recovery process of peripheral nerve injury (PNI), based on electrophysiological and histomorphometric criteria, after treatment with plasma rich in growth factors (PRGF) injections and scaffolds in an ovine model. Three groups of sheep underwent a nerve crush lesion: the first group (n = 3) was left to recover spontaneously (SR); the second group was administered saline injections (SI; n = 5) and a third group (n = 6) received PRGF injections and scaffolds immediately after the crush injury. At post‐intervention week 8, 70% of sheep in the PRGF group were CMAP‐positive, with no electrophysiological response in the rest of the groups. Histomorphometric analysis 12 weeks after the surgical intervention revealed that the average axonal density of the SR (1184 ± 864 axons/µm2) and SI (3109 ± 2450 axons/µm2) groups was significantly inferior to the control (8427 ± 2433 axons/µm2) and also inferior to the PRGF group (5276 ± 4148 axons/µm2), showing no significant differences between the control and PRGF groups. The axonal size of the SR and SI groups was significantly smaller compared with the control group (18 ± 4 µm2), whereas the axonal size of the PRGF group (6 ± 5 µm2) did not show statistical differences from the control. Morphometry of the target muscles indicated that the PRGF group had the lowest percentage volume reduction 12 weeks after the crush injury. The PRGF group had larger muscle fibre areas than the SI and SR groups, although the differences did not reach statistical significance. Overall, these data suggest that the PRGF injections and scaffolds hastened functional axon recovery and dampened atrophy of the target muscles in an ovine model. Copyright

Knee-Extension Training with a Single-Joint Hybrid Assistive Limb during the Early Postoperative Period after Total Knee Arthroplasty in a Patient with Osteoarthritis

The knee range of motion is an important outcome of total knee arthroplasty (TKA). According to previous studies, the knee range of motion temporarily decreases for approximately 1 month after TKA due to postoperative pain and quadriceps dysfunction following surgical invasion into the knee extensor mechanism. We describe our experience with a knee-extension training program based on a single-joint hybrid assistive limb (HAL-SJ, Cyberdyne Inc., Tsukuba, Japan) during the acute recovery phase after TKA. HAL-SJ is a wearable robot suit that facilitates the voluntary control of knee joint motion. A 76-year-old man underwent HAL-SJ-based knee-extension training, which enabled him to perform knee function training during the acute phase after TKA without causing increased pain. Thus, he regained the ability to fully extend his knee postoperatively. HAL-SJ-based knee-extension training can be used as a novel post-TKA rehabilitation modality.

Circadian variation of growth factor levels in platelet-rich plasma.

Objective:The influence of circadian rhythms has recently been considered in advanced studies of chronopharmacology and chronotherapeutics. Although emerging studies have reported the usefulness of platelet-rich plasma (PRP), no reports have described the diurnal variations in PRP growth factor levels. In the present study, we hypothesized that growth factor levels in PRP show a significant diurnal variation. Design:Controlled laboratory study. Setting:Institutional. Participants:Ten adult male volunteers (mean age, 30.0 ± 2.6 years; range, 26-34 years). Interventions:Each subject donated a 20-mL sample of fasting blood, 4 times per day (7 AM, 11 AM, 3 PM, and 7 PM). Main Outcome Measures:Each blood sample was processed to produce PRP. Complete blood counts of peripheral blood (PB) and PRP, as well as quantitative determination of PRP growth factor levels (platelet-derived growth factor and transforming growth factor) were performed. Statistical analyses were conducted using a one-way repeated measures analysis of variance. Results:No significant differences were observed in platelet, white blood cell, and red blood cell counts in the PB or in the PRP samples throughout the course of the day. In addition, there were no significant differences in PRP growth factor levels at the various time points. Conclusions:Significant diurnal variations in platelet counts and growth factor levels were not observed in PRP. Therefore, adjustments for diurnal variation in chronotherapy may not be important when PRP is used in clinical practice.

Autologous Concentrated Bone Marrow Grafting for the Treatment of Osteonecrosis of the Humeral Head: A Report of Five Shoulders in Four Cases

Five shoulders in four patients affected by advanced osteonecrosis of the humeral head were treated with autologous concentrated bone marrow grafting. Bone marrow sample was aspirated from the iliac crests, concentrated by a centrifugation technique, and injected into the necrotic site. The shoulders were evaluated radiologically with X-ray scoring and clinically with measurement of range of motion and pain score (visual analogue scale, VAS). The mean follow-up period was 49.4 (range, 24–73) months. The concentration ratio of nucleated cells was calculated and the number of transplanted mesenchymal stem cells (MSC) was estimated by a colony-forming assay. All four shoulders with stage 3 disease achieved joint sparing. One shoulder with stage 4 disease required replacement surgery. Clinical evaluation of the spared joints showed improvement in range of motion in two cases and deterioration in two cases. VAS scores were 0 after surgery in three cases. The mean concentration ratio was 2.73, and the mean number of transplanted MSC was 1125. The outcomes of autologous concentrated bone marrow grafting for advanced osteonecrosis of the humeral head were varied. Further research is needed to determine the effectiveness and the indications of the present surgery.

Fate of bone marrow mesenchymal stromal cells following autologous transplantation in a rabbit model of osteonecrosis

BACKGROUND AIMS Internalizing quantum dots (i-QDs) are a useful tool for tracking cells in vivo in models of tissue regeneration. We previously synthesized i-QDs by conjugating QDs with a unique internalizing antibody against a heat shock protein 70 family stress chaperone. In the present study, i-QDs were used to label rabbit mesenchymal stromal cells (MSCs) that were then transplanted into rabbits to assess differentiation potential in an osteonecrosis model. METHODS The i-QDs were taken up by bone marrow-derived MSCs collected from the iliac of 12-week-old Japanese white rabbits that were positive for cluster of differentiation (CD)81 and negative for CD34 and human leukocyte antigen DR. The average rate of i-QD internalization was 93.3%. At 4, 8, 12, and 24 weeks after transplantation, tissue repair was evaluated histologically and by epifluorescence and electron microscopy. RESULTS The i-QDs were detected at the margins of the drill holes and in the necrotized bone trabecular. There was significant colocalization of the i-QD signal in transplanted cells and markers of osteoblast and mineralization at 4, 8, and 12 weeks post-transplantation, while i-QDs were detected in areas of mineralization at 12 and 24 weeks post-transplantation. Moreover, i-QDs were observed in osteoblasts in regenerated tissue by electron microscopy, demonstrating that the tissue was derived from transplanted cells. CONCLUSION These results indicate that transplanted MSCs can differentiate into osteoblasts and induce tissue repair in an osteonecrosis model and can be tracked over the long term by i-QD labeling.