Masayuki Umeda

Treating Achilles Tendon Rupture in Rats with Bone-Marrow-Cell Transplantation Therapy

BACKGROUND Bone marrow cells possess multipotentiality and have been used for several treatments. We hypothesized that bone marrow cells might differentiate into regenerated tendon and that several cytokines within bone marrow cells might accelerate tendon healing. Therefore, we treated Achilles tendon ruptures in a rat model with transplantation of whole bone marrow cells. METHODS Nine F344/Nslc (Fisher) rats were the source of bone marrow cells and mesenchymal stem cells as well as normal Achilles tendons. Eighty-seven Fisher rats were used for the experiments. The rats were divided into three groups: the BMC group (bone marrow cells injected around the tendon), the MSC group (mesenchymal stem cells injected around the tendon), and the non-treated control group (incision only). Outcome measures included mechanical testing, collagen immunohistochemistry, histological analysis, and reverse transcription-polymerase chain reaction to detect expression of transforming growth factor-β (TGF-β) and vascular endothelial growth factor (VEGF). RESULTS The ultimate failure load in the BMC group was significantly greater than that in the non-treated or the MSC group at seven days after incision (3.8 N vs. 0.9 N or 2.1 N, p < 0.016) and at fourteen days after incision (10.2 N vs. 6.1 N or 8.2 N, p < 0.016). The ultimate failure load in the BMC group at twenty-eight days after incision (33.8 N) was the same as that of normal tendon (34.8 N). The BMC group demonstrated stronger staining for type-III collagen at seven days after incision and stronger staining for type-I collagen at twenty-eight days than did the MSC group. Expression of TGF-β and VEGF in the BMC group was significantly increased compared with that in the other groups at four days after incision (TGF-β: 1.6 vs. 1.3 or 0.6, p < 0.01; VEGF: 1.7 vs. 1.1 or 0.9, p < 0.01). CONCLUSIONS Transplantation of whole bone marrow cells may be a better and more readily available treatment for Achilles tendon rupture than cultured mesenchymal stem cells.

Microsurgical bilateral decompression via a unilateral approach for lumbar spinal canal stenosis including degenerative spondylolisthesis

OBJECT Surgical outcome and radiographic changes after microsurgical bilateral decompression via a unilateral approach (MBDU) for lumbar spinal canal stenosis during midterm follow-up periods (> 2 years) have not been reported. The authors retrospectively investigated surgical outcomes after MBDU in patients with lumbar degenerative spondylolisthesis with stenosis in comparison with patients with degenerative stenosis during a minimum follow-up period of 2 years. Radiographic changes at the affected intervertebral level were analyzed during that follow-up period. METHODS Forty-eight patients (23 in the spondylolisthesis group, 25 in the degenerative stenosis group) were included in the study. The average follow-up period was 46 months (range 24-71 months). Surgical outcome was evaluated using the Neurogenic Claudication Outcome Score (NCOS) and the Oswestry Disability Index (ODI). Additionally, the back pain score within the NCOS was also compared. There were no statistically significant differences between the spondylolisthesis group and the degenerative stenosis group with regard to sex, age, follow-up period, operating time, blood loss, surgical sites, approach side, preoperative NCOS, preoperative back pain score, and preoperative ODI. Comparisons were also made between groups using 2 satisfaction measurements at the last follow-up visit. Radiographically, intervertebral angles of 80 sites and slip percentages of 24 sites were measured preoperatively and at the last follow-up. RESULTS No patient in either group had additional surgery in the lumbar spine, including fusion procedures. The NCOS, back pain score, and ODI had significantly improved at the last follow-up in both groups. There were no significant differences between the 2 groups in these 3 parameters and the 2 satisfaction measurements at the last follow-up, although those for the spondylolisthesis group indicated a somewhat worse outcome. Intervertebral angles, dynamic intervertebral angles, and dynamic slip percentage did not significantly change after surgery, whereas only slip percentage significantly increased postoperatively (p = 0.0319). CONCLUSIONS A satisfactory outcome of MBDU persisted for a period longer than 2 years for patients with degenerative spondylolisthesis with stenosis as well as for those with degenerative stenosis. Radiographically in both groups this less invasive procedure was not likely to result in postoperative dynamic instability at the affected level, although the slippage progressed in the spondylolisthesis group.

New Strategies for Anterior Cruciate Ligament Partial Rupture Using Bone Marrow Transplantation in Rats

The purpose of this study was to compare anterior cruciate ligament (ACL) regeneration between animal groups subjected to intra-articular injection of fresh whole bone marrow cells (BMCs), cultured mesenchymal stem cells (MSCs), or saline. Partially transected ACLs in Fischer 344/Nslc rats were prepared, followed by injection of BMCs, MSCs, or saline into the articular cavity at 1 week after transection. Donor cells expressing green fluorescent protein were detected in the recipients transected ACLs at 4 weeks in the BMC and MSC groups, and their ACLs appeared almost normal histologically. Further, there were significantly more mature spindle cells in the BMC group than in the saline group at 4 weeks. Biomechanically, the tensile strength in the BMC group reached near normal levels at 4 weeks after injection. The levels of transforming growth factor-β1 in the ACL tissue and knee joint fluid in the BMC group were increased significantly compared with that of the saline group at 4 weeks as detected by immunohistochemical analysis. In conclusion, intra-articular bone marrow transplantation using fresh whole BMCs is an effective treatment for ACL partial rupture. This therapy is easy to apply in a clinical setting because no culture system is required for collecting MSCs.

Prevention of corticosteroid-induced osteonecrosis in rabbits by intra-bone marrow injection of autologous bone marrow cells.

OBJECTIVES Femoral head osteonecrosis (ON) is a serious complication of steroid administration. We evaluated bone marrow transplantation (BMT) for preventing corticosteroid-induced ON. METHODS Rabbits, injected with methylprednisolone (MPSL; 20 mg/kg), were divided into four groups: (i) MPSL alone; MPSL injection only, (ii) MPSL+needling; 2 days after MPSL injection, a hole (1.2 mm diameter) was drilled from the outer cortex 2.5 cm distal to the proximal end of the greater trochanter, (iii) MPSL+saline; 2 days after MPSL injection, 2 ml saline was injected directly into the bone marrow cavity, and (iv) MPSL+BMT; 2 days after MPSL injection, 1 x 10(7)/2 ml bone marrow cells (BMCs) were injected directly into the bone marrow cavity. Platelets, fibrinogen, prothrombin time and total cholesterol in peripheral blood were measured before and after treatment. Tissues were stained with haematoxylin and eosion and terminal deoxynucleotidyl-mediated deoxyuridine triphosphate nick-end labelling stain and immunostained for VEGF, while cell proliferation and viability of whole BMCs in the femur were analysed by cell cycle analysis and [(3)H]-thymidine uptake. RESULTS The ON incidence in rabbits treated with MPSL alone, MPSL+needling and MPSL+saline was 72.7, 70.0 and 66.7%, respectively, while in the MPSL+BMT group, the incidence was 0%. Serological findings in the MPSL+BMT group were almost normalized. VEGF and TUNEL staining were reduced in the MPSL+BMT group compared with all other groups. There were significantly fewer BMCs in G1 phase from the MPSL+BMT group than the other groups, while uptake of [(3)H]-thymidine was significantly increased. CONCLUSION Direct injection of autologous BMCs into femurs prevents corticosteroid-induced ON following treatment with high-dose, short-term steroids.

Allogeneic intra-bone marrow transplantation prevents rheumatoidarthritis in SKG/Jcl mice

The treatment of autoimmune diseases by allogeneic bone marrow transplantation remains a promising therapeutic avenue. However, more intensive studies on murine models are essential before application to a large number of human patients. In particular, the use of bone marrow transplantation to treat rheumatoid arthritis has been problematic. We have taken advantage of the SKG/Jcl mouse that develops a chronic T cell-mediated autoimmune disease that mimics rheumatoid arthritis which attempted to prevent the development of immunopathology in these mice by allogeneic bone marrow transplantation (BMT). In particular, we utilized our unique technology in which bone marrow cells (BMCs) of control C57BL/6J mice are directly injected into the bone marrow cavity in the tibia of SKG mice (intra-bone marrow [IBM]-BMT). As controls, SKG/Jcl mice were transplanted with whole BMCs from syngeneic SKG mice. Importantly, 12 months after IBM-BMT [B6-->SKG] demonstrated no evidence of arthritis, whereas the control [SKG-->SKG] mice demonstrated, the expected immunopathology of a rheumatoid arthritis-like condition. Further, hematolymphoid cells in [B6-->SKG] mice were reconstituted by donor-derived cells and the percentages of Treg (Foxp3+/CD4+) cells, the percentages of receptor activator of nuclear factor-kappaB ligand (RANKL)+ cells on the CD4+ T cells and the serum levels of tumor necrosis factor-alpha, interleukin-1 and interleukin-6 were normalized in the [B6-->SKG] mice. These data suggest that IBM-BMT is a viable method of immunological manipulation that suppresses the severe joint destruction and bone absorption in SKG/Jcl mice and lends further credence to the use of this methodology in humans with intractable rheumatoid arthritis.

A less-invasive cervical laminoplasty for spondylotic myelopathy that preserves the semispinalis cervicis muscles and nuchal ligament

OBJECT Modified cervical laminoplasty techniques have been developed to reduce postoperative axial neck pain and preserve function in patients with cervical spondylotic myelopathy (CSM). However, the previous studies demonstrating satisfactory surgical outcomes had a retrospective design. Here, the authors aimed to prospectively evaluate the 2-year outcomes of a modified cervical laminoplasty technique for CSM that preserves the paravertebral muscles. METHODS Outcomes were analyzed for 40 patients (22 men and 18 women; mean age, 66.6 years; age range 44-92 years) with CSM who underwent C4-6 laminoplasty with C-3 and C-7 partial laminectomies or C-3 total and C-7 partial laminectomies and received hydroxyapatite spacers. Neurological, pain severity, and spinal radiographic evaluations were performed preoperatively and at 3, 6, 12, 18, and 24 months postoperatively. Plain radiography and MRI of the cervical spine were performed to evaluate the range of motion (ROM), sagittal alignment, and cross-sectional areas of the deep extensor muscles. The extent of bone-spacer bonding and bony union at the gutter was assessed by CT. RESULTS The mean preoperative Japanese Orthopaedic Association CSM score was 10.2, but it increased to 14.4 by 24 months after surgery. Eleven patients had axial neck pain preoperatively, but only 3 reported mild pain at 24 months, and in all 3 cases the pain was mild. The mean angle of lordosis was 11.7° preoperatively and 12.0° 2 years postoperatively. Although the ROM at the C2-7 levels was significantly reduced 3 months postoperatively, an increasing trend was observed up to 12 months, and 86% of the preoperative ROM was achieved by 2 years postoperatively. The mean paravertebral muscle cross-sectional areas were 833 ± 215 mm(2) preoperatively and 763 ± 197 mm(2) 24 months postoperatively, but the difference was not statistically significant. The rates of bone-spacer bonding and bony union at the gutter were low during the early stages but increased to 90% and 93%, respectively, by 2 years after surgery. CONCLUSIONS The modified laminoplasty technique used in this study ensured very good neurological status and ROM after 2 years and was associated with low incidences of axial neck pain and serious complications. This simple and easy operative method could benefit future laminoplasty protocols.

Activation of rat nucleus pulposus cells by coculture with whole bone marrow cells collected by the perfusion method.

Cell proliferation and matrix synthesis were compared for rat nucleus pulposus cells cocultured with mesenchymal stem cells (MSCs) or fresh whole bone marrow cells (BMCs), harvested by the perfusion or aspiration methods. Nucleus pulposus cells were isolated from tail intervertebral discs of F344/slc rats, and BMCs were obtained from femora. Proteoglycan synthesis, DNA synthesis, and aggrecan mRNA expression were measured. The level of transforming growth factor‐β in supernatants from the culture system was also measured. Cell number, aggrecan mRNA expression, and uptake of [35S]‐sulfate and [3H]‐thymidine by nucleus pulposus cells cocultured with fresh whole BMCs all increased significantly compared with nucleus pulposus cells cocultured with MSCs. TGF‐β secreted by nucleus pulposus cells cocultured with fresh whole BMCs also significantly increased when compared with cocultures with MSCs. The perfusion method was superior to the aspiration method for preventing contamination of BMCs with peripheral red blood cells and lymphocytes, which may cause an autoimmune response in the disc. In conclusion, we suggest that fresh whole BMCs harvested by the perfusion method are more effective for increasing the proliferative and matrix synthesis capacity of nucleus pulposus cells.