Differentiation and proliferation potencies of human bone tissue-derived mesenchymal stromal cells (hBT-MSCs) after long-term cryopreservation －Comparison among cells stored for 1, 5, 10, 15, and 20 years.

INTRODUCTION: We investigated bone differentiation and proliferation potencies of human bone tissue-derived mesenchymal stromal cells (hBT-MSCs) after long-term cryopreservation. We determined the presence of any morphological and characteristic changes due to freezing to identify issues that need to be solved for future clinical applications. SUBJECTS AND METHODS: A total of 15 samples of hBT-MSCs that had been cryopreserved for different lengths of time, ranging from one year to 20 years (n = 3 each), were thawed and recultivated after being collected from excess iliac cancellous bone specimens of patients who underwent secondary alveolar bone grafting for cleft lip and palate in our department. We determined viability by observing calcein/EthD-stained cells under a confocal microscope, and the cell proliferation experiment was performed for one week using the Water Soluble Tetrazolium salts (WST) assay method. A confocal microscope was also used to identify any excessively accumulated senescence-associated growth factor SA-ßgal. Differentiation potency was assessed in the following three groups: bone differentiation, adipocyte differentiation, and nondifferentiation induction. We examined bone/adipocyte differentiation potencies using Alizarin Red staining, Ca quantitation, and Oil Red staining after continuously culturing cells for four weeks. RESULTS: Viability test results indicated that the proportion of viable cells decreased as the number of years of cryopreservation increased. The cell proliferation experiment showed that cells cryopreserved for a shorter duration multiplied exponentially. In the aging test, cells cryopreserved for ≥5 years showed similar positive reactions independent of the number of years of cryopreservation. In the cell proliferation test, there was no statistically significant difference between the years of cryopreserving. We compared bone differentiation and adipocyte differentiation ability with the non-induction group, and the induction group was confirmed to have a statistical advantage. However, there was no significant difference in the induction group pertaining to different ages. CONCLUSIONS: Samples cryopreserved for 20 years remained competent in bone and adipocyte differentiation. However, their differentiation direction tended to skew to either bone or adipocyte differentiation. Our results suggest that freezing does not accelerate aging, and samples cryopreserved for a long time are useful in future clinical applications.

Diagnosis of skin and soft tissue infections using near-infrared spectroscopy.

AIM: Skin and soft tissue infections are classified into cellulitis and necrotizing fasciitis, which are difficult to distinguish. Necrotizing fasciitis has a poor prognosis and requires immediate intensive care. The diagnostic gold standard is to incise the lesion to determine whether necrosis has reached the fascia. We aimed to show that these infections can be differentiated using near-infrared spectroscopy. METHODS: We describe two cases in an observational study about the utility of near-infrared spectroscopy. Case 1 involved a 77-year-old man with a chief complaint of pain, redness, and swelling in the right lower leg for 1 week. Computed tomography of his legs showed no gas formation. Case 2 involved an 82-year-old man. He visited another hospital because of pain, redness, and swelling in the right thigh. Based on the X-ray examination, necrotizing fasciitis was suspected, and he was transferred to our hospital. RESULTS: In Case 1, the regional oxygen saturation value was lower on the lesion side (41%) than on the healthy side (55%). We confirmed the depth of invasion by incision, leading to a diagnosis of necrotizing fasciitis. In Case 2, the thigh's regional oxygen saturation was higher on the affected side (76%) than on the healthy side (61%). An incision was made for diagnosis, but the fascia was not necrotized. Thus, we diagnosed cellulitis and provided conservative treatment using antibiotics. CONCLUSION: Near-infrared spectroscopy can be utilized to measure tissue blood flow, and it could be useful as a non-invasive diagnostic tool for skin and soft tissue infections.

Clinical features of primary vein grafts in free tissue transfers.

The outcomes of free tissue transfers combined with vein grafts have been inconsistent, and discussions continue regarding their appropriate use. Of the 142 free tissue transfers that we performed from January 2004 to December 2011, we retrospectively analyzed 15 consecutive patients who underwent free tissue transfers in combination with vein grafts. Etiologies included trauma (8 patients), infection (4), and tumor (3). Types of free tissue transfers were fibula (4), anterolateral thigh (3), groin (3), jejunum (3), latissimus dorsi (1), and dorsal pedis (1). Vein grafts were used for the artery (6), vein (2), or both (7). The donor veins were the saphenous vein (12) and the external jugular vein (3). The mean length of the grafted veins was 10.8 cm (range: 4-18 cm). Even though complications of congestion occurred in 2 patients, these flaps survived by reexploration. The flap success rate was 15 of 15 (100%) of vein grafted free flaps versus 124 of 127 (97.6%) of free flaps not requiring vein grafts. To improve the success rate of free tissue transfers combined with vein grafts, securing healthy recipient vessels, meticulous surgical handling, a reliable vascular anastomosis technique, and strict postoperative monitoring are crucial.

Elective use of surgical cricothyroidotomy for maxillofacial fracture fixation with contraindication of nasotracheal intubation: a case report.

We report three cases of airway management with elective surgical cricothyroidotomy (SCT) for anesthetic management during surgical repair of maxillofacial injury involving basal skull fracture or nasal-bone fracture. In all patients, general anesthesia was induced, a supraglottic airway (SGA) device inserted, and SCT performed. Tracheal intubation was performed through SCT site, and the SGA device was removed. After surgery of maxillofacial fixation, the SGA device was re-inserted and the tracheal tube was removed. No major complications, such as subglottic stenosis or voice change, occurred. SCT holds potential as an alternative to tracheostomy because of ease of performance, fewer complications, and better cosmetic outcomes.

Osteogenic potential of human umbilical cord-derived mesenchymal stromal cells cultured with umbilical cord blood-derived fibrin: a preliminary study.

This study examined the potential for osteogenesis via regenerative medicine using autologous tissues (umbilical cord (UC) and umbilical cord blood (UCB)) in nude mice. The study was designed to provide the three elements required for regenerative medicine (cell, scaffold, and growth factor) and autoserum for culture by means of autologous tissues. Mesenchymal stromal cells were obtained from UC (UC-MSCs). Fibrin, platelet-rich-plasma, and autoserum were obtained from UCB as scaffold, growth factor and serum for culture respectively. UC-MSCs were obtained from Wharton jelly and cultured with UCB-derived fibrin (UCB-fibrin) for 3-4 weeks to induce their differentiation into osteoblasts. They were implanted subcutaneously into the dorsum of male nude mice for 6 weeks prior to undergoing assessment. The assessments performed were haematoxylin and eosin, and alizarin red staining, immunohistochemical staining of human mitochondria, scanning electron microscopy, scanning electron microscopy with energy dispersive X-ray spectrometry and real-time reverse transcriptase-polymerase chain reaction to assess the expressions of osteoblast markers. Consequently, the differentiation of UC-MSCs into osteoblasts and the production of hydroxyapatite were verified. This study suggested the possible formation of bone tissue using biomedical materials obtained from UC and UCB.

Transradial approach for transcatheter arterial chemoembolization in patients with hepatocellular carcinoma: comparison with conventional transfemoral approach.

We evaluated the clinical usefulness and safety of transradial approach for transcatheter arterial chemoembolization (TACE) in patients with hepatocellular carcinoma (HCC) compared with that of conventional transfemoral approach. The two groups (radial group, n = 177; femoral group, n = 150) of cases were retrospectively compared with regard to the successful rate of angiography or TACE, time required for catheterizaiton and complications. Hepatic angiography and TACE were completed in 174 (98.3%) of 177 cases in the radial group. There was no intergroup difference of time required for catheterization. Minor complications (dull pain, numbness) occurred in 8 (4.6%) patients in the radial group, and there were lower complications in the radial group compared to the femoral group. TACE by our new transradial approach was found to have therapeutic efficacy with lower complications comparable to that of the conventional transfemoral approach.

[Usefulness of gemcitabine in the treatment of advanced pancreatic cancer in terms of its clinical benefits].

Since pancreatic cancers are frequently discovered in an advanced stage, the expectations for chemotherapy are high. However, the response rate to chemotherapy of advanced cancer remains low. In Western countries, gemcitabine (GEM), a newly approved drug in Japan, was reported to be useful in improving symptoms and prolonging survival. In the present study, we compared 13 patients with unresectable advanced pancreatic cancer who were treated with GEM alone and 13 patients with pancreatic cancer treated with 5-fluorouracil/cisplatin (FP) as historical controls, with reference to therapeutic results, adverse reactions, and clinical benefit response. Although the response rate to GEM was low, at 7.7% (1/13), prolonged NC was seen in 76.9% (10/13) of cases, suggesting a contribution to prolonged survival (p = 0.0025). A clinical benefit response was seen in 38.5% (5/13) of the GEM group, as compared with the lower rate of 7.7% (1/13) in the FP group (p = 0.063). Since GEM is convenient to-administer, and does not diminish a patient's QOL, it is considered to be useful as a drug of first choice in the treatment of advanced pancreatic cancer in terms of clinical benefits.

Pylorus-preserving pancreaticoduodenectomy without homologous blood transfusion in a Jehovah's witness with pancreatic cancer: report of a case.

A case of successfully treated pancreatic cancer without homologous blood transfusion in a Jehovah's witness is reported. The patient was a 60-year-old Japanese man and he was diagnosed with pancreatic cancer. Based on the patient's informed consent, human recombinant erythropoietin (12000 IU/day), saccharated ferric oxide (60 mg/day), and 25% human serum albumin (100 mL/day) were administrated for 7 consecutive days prior to the operation. As a result, the hemoglobin level increased by 1.6 g/dL during the week, pylorus-preserving pancreaticoduodenectomy was performed on June 5, 1998. We used a technique of transfusing diluted autologous blood, hypervolemic hemodilution, and a cell-saver device for autologous transfusion during the operation. The operation took 10 hours and 35 minutes, blood loss was 600 mL, and the hemoglobin level at the end of surgery was 12.0 g/dL. He had an uneventful postoperative course, and was discharged on the 31st postoperative day. Although there have been numerous reports on surgery without homologous blood transfusion in Jehovah's witnesses, many of the cases of major surgery have involved cardiovascular procedures. However, major digestive surgery may be feasible in more Jehovah's witnesses if adequate alternative treatments and strict nutritional management are carefully implemented, as in our case.

[Transradial approach for transcatheter arterial chemoembolization in patients with hepatocellular carcinoma].

We devised a catheter designed for a transradial artery approach and carried out selective hepatic arteriography and transcatheter arterial chemoembolization (TACE) in 1999. To evaluate the clinical usefulness and safety of our new catheterization, we compared the results of transradial approach with those of the conventional transfemoral approach. In 164 (98.9%) of 166 patients in the radial group, hepatic arteries and the SMA were successfully visualized by an approach via the left radial artery. TACE via the radial artery was attempted in all 164 patients in whom the sheath was able to be inserted. When the amount of time required from puncture to sheath removal was compared, the two groups of patients showed no intergroup difference. Complications occurred in 4 patients (2.6%) in the radial group: there were 2 cases of temporary dull pain and 2 cases of numbness at the site of puncture, all of which improved after slight release of the tourniquet. In conclusion, the present study reported that TACE by our new transradial approach was found to have therapeutic efficacy comparable to that of the conventional transfemoral approach. This technique is only minimally invasive and is associated with few complications. We consider that the transradial artery catheterization may become a technique of first choice of TACE.