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Recently, deep learning (DL) algorithms have been adapted for the diagnosis of medical images. The purpose of this study was to detect image features using DL without measuring median nerve cross-sectional area (CSA) in ultrasonography (US) images of carpal tunnel syndrome (CTS) and calculate the diagnostic accuracy from the confusion matrix obtained. US images of 50 hands without CTS and 50 hands diagnosed with CTS were used in this study. The short-axis image of the median nerve was visualized, and 5000 images of both groups were prepared. Forty hands in each group were used as training data for the DL algorithm, while the remainder were used as test data. Transfer learning was performed using three pre-trained models. The confusion matrix and receiver operating characteristic curves were used to evaluate diagnostic accuracy. Furthermore, regions where DL was determined to be important were visualized. The highest score had an accuracy of 0.96, precision of 0.99 and recall of 0.94. Visualization of the important features revealed that the DL models focused on the epineurium of the median nerve and the surrounding soft tissue. The proposed technique enables the accurate prediction of CTS without measurement of the CSA.

Background: Advanced glycation end products (AGEs) are end products of protein glycation that bind to the receptor for AGEs (RAGE) and activate nicotinamide adenine dinucleotide phosphate oxidase (NOX), resulting in increased oxidative stress and rotator cuff fragility. Stump classification using the signal intensity ratio of the tendon rupture site to the deltoid muscle in the coronal view of T2-weighted fat-suppressed magnetic resonance imaging (MRI) scans is an indicator of clinical outcomes after rotator cuff repair surgery. Comparing the signal intensities of the deltoid (D) and rotator cuff tears (C), Ishitani et al. classified C/D 1.3 as type 3. Hypothesis/Purpose: It was hypothesized that the oxidative stress and collagen degeneration that occur in the rotator cuff due to accumulation of AGEs can be assessed on MRI scans (stump classification). Therefore, this study aimed to compare AGE-related factors in the rotator cuff tear site tissues based on stump classification. Study Design: Descriptive laboratory study. Methods: The authors included 30 patients (11 with type 1, 9 with type 2, and 10 with type 3; mean age, 62.3 years) who underwent surgery for complete rotator cuff tears at our hospital. Tendon tissue was harvested from the torn rotator cuff site during surgery for tissue and cell evaluation. Results: There was no significant difference in the mean age according to stump classification. The number of patients with diabetes was significantly larger in type 3 than in the other types ( P < .05). Tissue evaluation showed significantly higher expression of AGE and RAGE staining in type 3 than in the other types (~6.7-fold; P < .01). Cell evaluation showed that the expression rates of reactive oxygen species and apoptosis were significantly higher in type 3 than in the other types (~4.3-fold; P < .01). Gene expression by real-time polymerase chain reaction showed significantly higher RAGE (~5.1-fold), NOX (~5.3-fold), and IL (~3.0-fold) in type 3 than in the other types ( P < .05). Conclusion: Stump classification type 3 exhibited the highest accumulation of AGEs and the highest oxidative stress and apoptosis, suggesting a high degree of degeneration and inflammation. Imaging based on stump classification reflects the degeneration and fragility of the torn rotator cuff site. Clinical Relevance: This study provides evidence of a relationship between stump classification, which reflects rotator cuff fragility on MRI, and pathologies related to advanced glycation end products.

This article aims to evaluate the usefulness of ultrasonography for the measurement of thenar muscles in carpal tunnel syndrome (CTS). A total of 85 patients with CTS who had a carpal tunnel release procedure were included in this study. The transducer was applied onto the palmar surface of the hand perpendicularly to the longitudinal axis of the first metacarpal bone. Thenar atrophy was evaluated visually and classified using the visual grading scale. A nerve conduction test was performed and classified according to the electrophysiological severity scale. This technique is more precise than visual evaluation because it is a quantitative assessment.

Background While the precise measurement of the range of motion (ROM) of the elbow joint is important for clinical assessment and rehabilitation, problems include low accuracy and reproducibility in goniometer measurements due to the influence of soft tissue. The purpose of this study was to validate elbow joint motion analysis using a three-dimensional electromagnetic sensor system (EMS). Methods The accuracy and reproducibility of the EMS system were evaluated at four angles (0°, 45°, 90°, and 135°) using a model bone of the humerus and forearm. In addition, the maximum extension and maximum flexion of six elbows of six healthy volunteers were assessed by radiographic and EMS measurements. Accuracy was assessed by calculating the mean value of the measurement angle, standard deviation, Pearson’s correlation coefficient, and the Bland–Altman method. Reproducibility was assessed by calculating the intra-rater and inter-rater reliabilities using intraclass correlation coefficients. Results In the model bone evaluation, the mean angles of the EMS measurement were 1.2° ± 2.0°, 45.4° ± 2.1°, 91.7° ± 2.4°, and 134.6° ± 2.7° at 0°, 45°, 90°, and 135°, respectively. In the in vivo evaluation, the elbow angles at the maximum extension with the EMS and radiographic angles were 4.7° ± 3.0° and 2.7° ± 2.0°, respectively, and the angles at maximum flexion were 131.8° ± 13.0° and 130.8° ± 4.5°, respectively. There were statistically significant correlations between the EMS and radiographic measurements; the Bland–Altman plots indicated that the two methods were almost in agreement for both extension and flexion. Conclusions This method of measuring ROM of the elbow joint using EMS showed high accuracy, reliability, and reproducibility. The current results demonstrated the possibility of using the electromagnetic system to provide an accurate evaluation of the elbow joint in clinical settings.

Previous studies have reported that glenohumeral internal rotation deficit (GIRD) may increase the risk of throwing-shoulder injuries. The purpose of this study was to analyze the conditions of the throwing shoulder in professional baseball pitchers with GIRD by comparing with those in pitchers without GIRD.In total, 26 male professional baseball pitchers participated in this study. We evaluated passive range of motion (ROM) and isometric muscle strength at internal rotation (IR) and external rotation (ER) at 90° of abduction, as well as the muscle thickness of the supraspinatus (SSP) and infraspinatus (ISP) by ultrasound. The pitchers were divided into 2 groups: those who exhibited a loss of IR of ≥20° in the throwing shoulder (GIRD group) and those who did not (non-GIRD group).In the GIRD group, the total ROM deficit (throwing side - non-throwing side) (P.001), the muscle thickness ratio (throwing to non-throwing) of the SSP and ISP (P = .017 and P = .014, respectively), and the muscle strength ratio (throwing to non-throwing) of ER (P = .028) were significantly lower than those in the non-GIRD group. In contrast, the muscle strength ratio (throwing to non-throwing) of IR was significantly higher in the GIRD group than in the non-GIRD group (P = .0064).We have shown that GIRD has significant correlations with several conditions, such as atrophy of the SSP and ISP, weakness of ER strength, enhancement of IR strength, limitation of total ROM, and throwing side, all of which could be important factors for throwing-shoulder injuries.

Background/aim This study aimed to evaluate the association of clinical characteristics with treatment outcomes to ascertain the appropriate treatment options for soft tissue sarcomas (STS) patients with brain metastasis (BM). Patients and methods Medical records of STS patients with BM who were treated in our institutions were retrospectively reviewed, and analyzed to identify the factors associated with post-BM survival. Results Among the 509 STS patients, BM occurred in five patients (0.98%). The median survival after BM was 1.5 months. Histological subtypes of the primary lesions in the five BM patients were: two synovial sarcomas, one myxoid liposarcoma, one alveolar soft part sarcoma, and one rhabdomyosarcoma. Among the five BM patients, the post-BM survival of two patients, who underwent surgery and postoperative radiotherapy, was longer than that of the other patients (p Conclusion Combined surgery and postoperative radiotherapy effectively managed symptoms and prolonged survival in STS patients with BM.

Background Limitations of shoulder range of motion (ROM) have been reported to be at high risk for shoulder disorders in baseball players. In this study, we assessed posterior shoulder muscle stiffness using shear wave elastography in baseball players with and without posterior shoulder tightness. Materials and methods In total, 21 college baseball players volunteered to participate in this study. Passive ROMs for shoulder abduction and horizontal adduction were measured using a goniometer. Subsequently, we divided all participants into 2 groups based on the differences between bilateral shoulder ROMs: STIFF+ and STIFF− groups. Thickness and elasticity of the posterior and inferior shoulder muscles were assessed using ultrasound. Results Shoulder abduction ROM on the throwing side was 114.5° ± 5.3° and 131.3° ± 5.7° in the STIFF+ and STIFF− groups, respectively, which was significantly different between the 2 groups (P = .023). Horizontal adduction ROM on the throwing side was 96.6° ± 4.9° and 110.9° ± 4.8° in the STIFF+ and STIFF− groups, respectively, which was also significantly different between the 2 groups (P = .014). The elasticity of infraspinatus and lower trapezius in the STIFF+ group was significantly greater than that in the STIFF− group (P = .018 and .033, respectively). Discussion In this study, we found that the elasticity of infraspinatus and lower trapezius in stiff shoulders was significantly greater than that in nonstiff shoulders, which indicated that the infraspinatus and lower trapezius could be the key muscles in posterior shoulder tightness.

Background Most degenerative rotator cuff tears (RCTs) are associated with a limited range of motion (ROM) of the shoulder joint. Additionally, patients with diabetes mellitus (DM) show a higher frequency of limited ROM. Recently, advanced glycation end products (AGEs) of proteins have been observed to cause tissue fibrosis, primarily through abnormal collagen cross-linking and oxidative stress. In this study, we investigated the effect of AGEs on ROM limitation in the shoulder capsule and its relationship with DM in the patients with RCTs. Methods Sixteen patients (eight in the DM and non-DM groups) who underwent arthroscopic surgery for RCT with limited shoulder ROM were included in this study. AGE-related pathologies in both groups were compared, and the relationship between AGE accumulation and shoulder joint ROM was evaluated. Shoulder capsule tissue was harvested and subjected to histological and in vitro evaluation. Results The DM group displayed high levels of AGEs and reactive oxygen species (ROS), and reduced cell viability. There was a significant positive correlation between ROS expression, apoptosis, and preoperative hemoglobin A1c. ROS expression, apoptosis, and ROM of the shoulder joint showed a negative correlation. The NADPH oxidase (NOX) expression and collagen III/I ratio were significantly higher in the DM group than in the non-DM group. Conclusions The DM group showed significant AGEs deposition in the shoulder capsule. Additionally, there was a significant association between AGEs and ROM limitation. Collectively, the findings suggest that the oxidative stress induced by AGEs deposition, which leads to fibrosis and local inflammation, might contribute to the limited ROM of the shoulder joint in patients with RCTs accompanied by DM.

Osteolytic bone metastasis leads to skeletal‑related events, resulting in a decline in the patient activities and survival; therefore, it is important to understand the mechanism underlying bone metastasis. Recent studies have suggested that microRNAs (miRNAs or miRs) are involved in osteoclast differentiation and/or osteolytic bone metastasis; however, the roles of miRNAs have not been elucidated. In the present study, the roles of miRNAs in bone destruction caused by breast cancer metastasis were investigated in vitro and in vivo. miR‑16, miR‑133a and miR‑223 were transfected into a human breast cancer cell line, MDA‑MB‑231. The expression of osteolytic factors in conditioned medium (miR‑CM) collected from the culture of transfected cells was assessed. To evaluate the effects of miRNAs on osteoclast differentiation and activities, tartrate‑resistant acid phosphatase (TRAP) staining and bone resorptive assays were performed in osteoclasts following miR‑CM treatment. To create in vivo bone metastasis models for histological and morphometric evaluation, miRNA‑transfected MDA‑MB‑231 cells were transplanted into the proximal tibia of nude mice. Expression of osteolytic factors, including receptor activator for nuclear factor‑κB ligand (RANKL), interleukin (IL)‑1β, IL‑6, parathyroid hormone‑related protein (PTHrP), and tumor necrosis factor (TNF), was increased in miR‑16‑CM, whereas it was decreased in both miR‑133a‑CM and miR‑223‑CM. TRAP staining and bone resorptive assays revealed that osteoclast function and activities were promoted by miR‑16‑CM treatment, whereas they were suppressed by miR‑133a‑CM and miR‑223‑CM. Consistent with in vitro findings, in vivo experiments revealed that the overexpression of miR‑16 increased osteoclast activities and bone destruction in MDA‑MB‑231 cells, whereas the opposite results were observed in both miR‑133a‑ and miR‑223‑transfected MDA‑MB‑231 cells. Our results indicated that miR‑16 promoted osteoclast activities and bone destruction caused by breast cancer metastasis in the bone microenvironment, whereas miR‑133a and miR‑223 suppressed them. These miRNAs could be potential biomarkers and therapeutic targets for breast cancer bone metastasis.

Ewing sarcoma (ES) is an aggressive primary malignant bone tumor that predominantly affects children and young adults. Multimodal treatment approaches have markedly improved the survival of patients with localized ES. However, local recurrence and distant metastasis following curative therapies remain a main concern for patients with ES. Recent studies have suggested that slow‑cycling cells (SCCs) are associated with tumor progression, local recurrence and distant metastasis in various types of cancers. According to the results of these studies, it was hypothesized that SCCs may play a critical role in tumor progression, chemoresistance and local/distal recurrence in patients with ES. The present study applied a label‑retaining system using carboxyfluorescein diacetate succinimidyl ester (CFSE) to identify and isolate SCCs in ES cell lines. In addition, the properties of SCCs, including sphere formation ability, cell cycle distribution and chemoresistance, in comparison with non‑SCCs were investigated. RNA sequencing also revealed several upregulated genes in SCCs as compared with non‑SCCs; the identified genes not only inhibited cell cycle progression, but also promoted the malignant properties of SCCs. On the whole, the present study successfully identified SCCs in ES cells through a label‑retaining system using CFSE. Moreover, to the best of our knowledge, the present study is the first to describe the characteristic properties of SCCs in ES. The findings of this study, if confirmed, may prove to be useful in elucidating the underlying molecular mechanisms and identifying effective therapeutic targets for ES.

To calculate the diagnostic accuracy from the confusion matrix using deep learning (DL) on ultrasound (US) images of Palmer 1B triangular fibrocartilage complex (TFCC) injury.Twenty-nine wrists of 15 healthy volunteers (11 men; mean age, 34.9 years ± 9.7) (control group) and 20 wrists of 17 patients (11 men; mean age 41.0 years ± 12.2) with TFCC injury (Palmer type IB) (injury group) were included in the study. The diagnosis of Palmer 1B TFCC injury was made using magnetic resonance imaging, computed tomography arthrography, and intraoperative arthroscopic findings. In total, 2,000 images were provided to each group, 80% of which were randomly selected by AI and used as training data; the remaining data were used as test data. Transfer learning was conducted using a pretrained 3 separate models (GoogLeNet, ResNet50, ResNet101). Model evaluation was performed using a confusion matrix. The area under a receiver operating characteristic curve was also calculated. The occlusion sensitivity was used to visualize the important features.For the prediction of TFCC injury by the DL model, the best score of accuracy was 0.85 in GoogLeNet, a recall was 1.0 in ResNet50 and ResNet101, and a specificity was 0.78 in GoogLeNet. In predicting the TFCC injury for the test data, the best score of the AUC was 0.97 on ResNet101. Visualization of important features showed that AI predicted the presence of injury by focusing on the morphology of the articular disc.US images using the DL model predicted Palmer 1B TFCC injury with high accuracy, with the best scores of 0.85 for accuracy on GoogLeNet, 1.00 for sensitivity on ResNet50 and ResNet101, and 0.78 for specificity on GoogLeNet. The use of DL for US imaging of Palmer 1B TFCC injury predicted the injury as well as magnetic resonance imaging and computed tomography arthrography LEVEL OF EVIDENCE: IV; retrospective case series study.

Background Most rotator cuff tears are the result of age-related degenerative changes, but the mechanisms underlying these changes have not been reported. Recently, advanced glycation end products (AGEs) have been regarded as an important factor in senescence. Therefore, we hypothesized that AGEs would have detrimental effects on rotator cuff–derived cells. In this study, we investigated the influence of AGEs on rotator cuff–derived cells in vitro and ex vivo. Methods Rotator cuff–derived cells were obtained from human supraspinatus tendons. The cells were cultured in the following media: (1) regular medium with 500 μg/mL AGEs (High-AGEs), (2) regular medium with 100 μg/mL AGEs (Low-AGEs), and (3) regular medium alone (Control). Cell viability, secretion of vascular endothelial growth factor, and the expressions of hypoxia-inducible factor-1α, reactive oxygen species, and apoptosis were assessed after cultivation. An ex vivo tissue culture with AGEs was also performed to measure the tensile strength. Results Cell viability in the High-AGEs group was significantly suppressed relative to that in the Controls. The amount of vascular endothelial growth factor secretion was significantly greater in the High- and Low-AGEs groups than in the Controls. Immunofluorescence stain demonstrated enhancement of hypoxia-inducible factor-1α and reactive oxygen species expressions and cell apoptosis in the High- and Low-AGEs groups relative to that in the Controls. In ex vivo mechanical testing, tensile strength was significantly higher in the Control group than in the AGEs groups. Discussion These results indicated that AGEs caused age-related degenerative rotator cuff changes. The reduction of AGEs might prevent rotator cuff senescence-related degeneration.

Background Quercetin, a flavonoid abundantly in vegetables and fruits, exerts antioxidant and anti-inflammatory effects. We investigated the protective effects of quercetin against oxidative stress in the Achilles tendons of diabetic rats. Methods Cells were collected from the Achilles tendons of Sprague–Dawley rats and cultured under four conditions: regular glucose (RG) without quercetin (Quer-), RG with quercetin (Quer +), high-glucose (HG) Quer-, and HG Quer + . The expression of genes related to NADPH oxidase (NOX) and inflammation, reactive oxygen species accumulation, and apoptosis rates was analyzed. Additionally, diabetic rats were divided into two groups and subjected to quercetin (group Q) or no quercetin (group C) treatment. Histological evaluation and expression analysis of relevant genes in the Achilles tendon were performed. Results In rat tendon-derived cells, the expression of Nox1, Nox4, and Il6; reactive oxygen species accumulation; and apoptosis rates were significantly decreased by quercetin treatment in the HG group. The collagen fiber arrangement was significantly disorganized in the diabetic rat Achilles tendons in group C compared with that in group Q. The mRNA and protein expression levels of NOX1 and NOX4 were significantly decreased upon quercetin treatment. Furthermore, the expression of Il6, type III collagen, Mmp2, and Timp2 was significantly decreased, whereas that of type I collagen was significantly increased in group Q compared with that in group C. Conclusions Quercetin treatment decreases NOX expression and thus exerts antioxidant and anti-inflammatory effects in the Achilles tendons of diabetic rats. Quercetin treatment may be effective against diabetic tendinopathy.

We report the case of a 37-year-old male with autism spectrum disorder who was hospitalized for chronic elbow dislocation. He had suffered a posterior elbow dislocation 5 months ago. His elbow dislocation was easily reduced, but the reduction position could not be maintained. Severe varus and valgus instabilities were observed in his right elbow. He was diagnosed with chronic elbow dislocation due to bilateral collateral ligament dysfunction and was surgically treated. Bilateral ligament reconstruction using the bilateral palmaris longus (PL) tendon combined with a temporary ulnohumeral joint fixation was performed. Cast immobilization was applied for 6 weeks. One year after surgery, his range of motion was −15° in extension, 140° in flexion, 80° in pronation, and 90° in supination. He did not face any problems in daily activities.

BACKGROUND: Common peroneal nerve (CPN) injury following a knee dislocation is a serious problem, and an optimal treatment is yet to be established. We report a case of complete CPN palsy following a knee dislocation treated with sural nerve grafting. CASE: A 19-year-old man suffered a knee injury during a hurdle race. Diagnosis in a previous hospital revealed a complex ligament injury with CPN palsy. Ten weeks following injury, he was admitted to our institution because of a lack of neurological improvement. Considering the grade 0 results obtained in the manual muscle test (MMT) of tibialis anterior (TA) and extensor hallucis longus (EHL), the patient was diagnosed with complete neurotmesis of CPN, and surgery was performed. Operative findings revealed CPN discontinuity and an extended nerve defect length of 15 cm; therefore, sural nerve grafting was performed to repair the CPN injury. One year postoperatively, a grade 1 result from MMT of TA and EHL indicated a gradual neurological recovery. Three years postoperatively, MMT of TA and EHL showed significant improvement to grade 4+ and grade 4, respectively, and he could walk and jog without a knee brace. DISCUSSION: Nerve graft length of >6 cm has shown limited success, and their efficacy for the treatment of CPN palsy following knee dislocations is controversial. However, young patients with complete CPN lesion are more likely to recover regardless of the length of nerve injury. Therefore, in such cases, nerve grafting can be considered as one of the treatments for complete CPN lesion following knee dislocations.

The triangular fibrocartilage complex (TFCC) is a significant stabilizer of the distal radioulnar joint. Diagnosing TFCC injury is currently difficult, but ultrasonography (US) has emerged as a low-cost, minimally invasive diagnostic tool. We aimed to quantitatively analyze TFCC by performing motion analysis by using US. Twelve healthy volunteers, comprising 24 wrists (control group), and 15 patients with TFCC Palmer type 1B injuries (injury group) participated. The US transducer was positioned between the ulnar styloid process and triquetrum and was tilted ulnarly 30° from the vertical line. The wrist was then actively moved from 10° of radial deviation to 20° of ulnar deviation in a 60-rounds-per-minute rhythm that was paced by a metronome. The articular disc displacement velocity magnitude was analyzed by using particle image velocimetry fluid measurement software. The mean area of the articular discs was larger on ulnar deviation in the control group. The mean articular disc area on radial deviation was larger in the injury group. The average articular disc velocity magnitude for the injury group was significantly higher than that for the control group. The results suggest that patients with TFCC injury lose articular disc cushioning and static stability, and subsequent abnormal motion can be analyzed by using US.

Background: Strong graft-bone integration is a prerequisite for successful graft remodeling after reconstruction of the anterior cruciate ligament (ACL) using soft tissue grafts. Novel strategies to accelerate soft tissue graft-bone integration are needed to reduce the need for bone-tendon-bone graft harvest, reduce patient convalescence, facilitate rehabilitation, and reduce total recovery time after ACL reconstruction. Hypothesis: The application of ACL-derived stem cells with enhanced expression of bone morphogenetic protein 2 (BMP2) onto soft tissue grafts in the form of cell sheets will both accelerate and improve the quality of graft-bone integration after ACL reconstruction in a rat model. Study Design: Controlled laboratory study. Methods: ACL-derived CD34+ cells were isolated from remnant human ACL tissues, virally transduced to express BMP2, and embedded within cell sheets. In a rat model of ACL injury, bilateral single-bundle ACL reconstructions were performed, in which cell sheets were wrapped around tendon autografts before reconstruction. Four groups containing a total of 48 rats (96 knees) were established (n = 12 rats; 24 knees per group): CD34+BMP2 (100%), CD34+BMP2 (25%), CD34+ (untransduced), and a control group containing no cells. Six rats from each group were euthanized 2 and 4 weeks after surgery, and each graft was harvested for immunohistochemical and histological analyses. The remaining 6 rats in each group were euthanized at 4 and 8 weeks to evaluate in situ tensile load to failure in each femur-graft-tibia complex. Results: In vitro, BMP2 transduction promoted the osteogenic differentiation of ACL-derived CD34+ cells while retaining their intrinsic multipotent capabilities. Osteoblast densities were greatest in the BMP2 (100%) and BMP2 (25%) groups. Bone tunnels in the CD34+BMP2 (100%) and CD34+BMP2 (25%) groups had the smallest cross-sectional areas according to micro–computed tomography analyses. Graft-bone integration occurred most rapidly in the CD34+BMP2 (25%) group. Tensile load to failure was significantly greater in the groups containing stem cells at 4 and 8 weeks after surgery. Tensile strength was greatest in the CD34+BMP2 (100%) group at 4 weeks, and in the CD34+BMP2 (25%) group at 8 weeks. Conclusion: ACL-derived CD34+ cells transduced with BMP2 accelerated graft-bone integration after ACL reconstruction using soft tissue autografts in a rat model, as evidenced by improved histological appearance and graft-bone interface biology along with tensile load to failure at each time point up to 8 weeks after surgery. Clinical Relevance: A primary disadvantage of using soft tissue grafts for ACL reconstruction is the prolonged time required for bony ingrowth, which delays the initiation of midsubstance graft remodeling. The lack of consistent correlation between the appearance of a “healed” ACL on postoperative magnetic resonance imaging and readiness to return to sport results in athletes being released to sport before the graft is ready to handle high-intensity loading. Therefore, it is desirable to identify strategies that accelerate graft-bone integration, which would reduce the time to biologic fixation, improve the reliability of biologic fixation, allow for accelerated rehabilitation, and potentially reduce the incidence of early graft pullout and late midsubstance failure.

Inadequate blood supply frequently impedes the viability of tissue-engineered constructs in the initial phase after implantation, and can lead to improper cell integration or cell death. Vascularization using stem cells has continued to evolve as a potential solution to this problem. In this review, we summarize studies that utilize endothelial progenitor cells (EPCs) for musculoskeletal regeneration. This review will also highlight recent concepts for EPC identification in conjunction with the development of EPC biology research. EPCs promote bone regeneration in animal models through a variety of mechanisms. By differentiating toward endothelial cell lineages and osteoblasts, EPCs stimulate vasculogenesis, angiogenesis and osteogenesis. Moreover, EPCs influence supporting cells through the secretion of growth factors and cytokines. Phase I/II clinical trials have applied circulating CD34+ cells/EPCs to nonunion bone fractures and have exhibited promising results including accelerated bone healing. Similar mechanisms of angiogenesis and osteogenesis are proposed for anterior cruciate ligament (ACL) ruptured tissue derived CD34+ cells, and thus EPCs have implied a critical role at the site of tendon-bone integration. EPCs are an emerging strategy among other cell-based therapies in the field of orthopaedics for the promotion of musculoskeletal regeneration.

No studies have reported the use of ultrasound for the evaluation of trigger finger after steroid injection. We evaluated the clinical features and ultrasound appearance of trigger finger before and after steroid injection under ultrasound guidance. Thirty-eight digits with triggering were included. A single steroid injection into the tendon sheath was administered. Ultrasound findings and clinical symptoms, including pain, triggering, and the Quinnell score, were analyzed before injection and at 1 and 3 weeks after injection. Ultrasound indicated that the thickness of the flexor tendons and the thickness of A1 pulleys were significantly greater in the trigger fingers than in controls before steroid injection. Three weeks after injection, these two parameters decreased, and there was no significant difference in the two parameters between the trigger finger and the controls. The visual analogue scales at 1 and 3 weeks after the injection were significantly lower than those before the injection. The Quinnell grading system scores significantly improved at 1 and 3 weeks after injection compared with the scores before injection. Moreover, a tendency was seen for the more clinically severe cases to show more swelling of the tendon and pulley. In conclusion, ultrasound assessments could reveal that the thickened A1 pulley and flexor tendon significantly improved after steroid injection, which enables us to confirm the therapeutic effects of the steroid injection. Therefore, ultrasound assessments can be a useful adjunct to understand the response to treatment with the steroid injection.

Ultrasound measurement of the cross-sectional area (CSA) of the median nerve can give information about regeneration of the nerve after carpal tunnel release (CTR), but the changes at selected follow-up points up to 1 year compared to electrodiagnostic findings are not known. We postoperatively measured the CSA of the median nerve with ultrasound and compared the measurements with electrophysiological findings over 12 months after open CTR. In 21 hands that underwent open CTR, the CSA of the median nerve was measured at the inlet (proximal CSA) and outlet (distal CSA) of the carpal tunnel at 3- 6- and 12-month follow-up. The respective ratios [(CSA postoperatively/CSA preoperatively) × 100] were calculated and correlated with distal motor latency (DML) and sensory nerve conduction velocity (SCV). The proximal CSA ratio was 88.9, 84.5, and 78.4 % at 3-, 6-, and 12-month follow-up, respectively. Each value was significantly lower than that before surgery. The distal CSA ratio was 104.3, 99.1, and 91.8 % at 3-, 6-, and 12-month follow-up, respectively. The values were not significantly different compared to preoperative values. The proximal CSA of the median nerve decreased continuously over the time after CTR while the distal CSA increased up to 3 months before it decreased continuously, too.

Background Advanced glycation end products are associated with aging, hyperglycemia, and oxidative stress. Accumulation of advanced glycation end products can cause various pathological conditions; however, the association of Dupuytren’s disease with advanced glycation end products has not been demonstrated yet. The aim of this study is to investigate the association of Dupuytren’s disease with advanced glycation end products. Methods Normal palmar fascia from five patients with carpal tunnel syndrome (control group) and Dupuytren’s cords from five patients (Dupuytren’s disease group) were harvested. The tissues were stained using an anti-advanced glycation end products antibody, anti-receptor for advanced glycation end products antibody, and an anti-reactive oxygen species modulator 1 antibody. The expression of nicotinamide adenine dinucleotide phosphate oxidase-1 and nicotinamide adenine dinucleotide phosphate oxidase-4 genes was also assessed using real-time PCR. For in vitro analysis, the cells harvested from the control and Dupuytren’s disease groups were used. After 3 days of exposure to four types of media (control group, control + advanced glycation end products group, Dupuytren’s disease group, Dupuytren’s disease + advanced glycation end products group), superoxide detection reagent was detected using a total reactive oxygen species/superoxide detection kit. Results Immunostaining of the palmar fasciae of the Dupuytren’s disease group showed higher expressions of advanced glycation end products and receptor for advanced glycation end products than that in the control group. The expression of nicotinamide adenine dinucleotide phosphate oxidase oxidase-1 and nicotinamide adenine dinucleotide phosphate oxidase-4 as well as reactive oxygen species modulator 1, an oxidatively damaged protein, was also higher in the Dupuytren’s disease group than in the control group. In an in vitro cell culture, the addition of advanced glycation end products to the Dupuytren’s disease-derived cells produced more superoxide free radicals. Conclusions These data suggest that the advanced glycation end products receptor for advanced glycation end products interaction produced free radicals via nicotinamide adenine dinucleotide phosphate oxidase activation in Dupuytren’s disease patients. Further studies are required to confirm these results.

Ruptured human anterior cruciate ligaments (ACL) contain vascular stem cells capable of enhancing the healing of tendon grafts. In the current study we explored the role that neo-angiogenesis plays in ACL healing. ACL-derived CD34+ cells were isolated via Fluorescence Activated Cell Sorting (FACS) from the rupture sites of human ACLs. The cells were then virally transduced to express either vascular endothelial growth factor (VEGF) or soluble FLT-1 (sFLT-1), which is an antagonist of VEGF. We established five groups: CD34+VEGF(100%), where 100% of the cells were transduced with VEGF, CD34+VEGF(25%), where only 25% of the cells were transduced with VEGF, CD34+, CD34+sFLT-1, and a No cells group. The CD34+sFLT1 group had a significant reduction in biomechanical strength compared to the CD34+ group at 4 and 8 weeks; whereas the biomechanical strength of the CD34+VEGF(25%) group was significantly greater than the CD34+ group at week 4; however, no difference was observed by week 8. Immunohistochemical staining demonstrated a significantly lower number of isolectin B4 and hCD31 positive cells, markers associated with angiogenesis, in the CD34+sFLT1 group, and a higher number of isolectin B4 and hCD31 positive cells in the CD34+VEGF(100%) and CD34+VEGF(25%) groups compared to the CD34+ group. Graft maturation was significantly delayed in the CD34+sFLT1 group and accelerated in the CD34+VEGF(25%) group compared to the CD34+ group. In conclusion, blocking VEGF reduced angiogenesis, graft maturation and biomechanical strength following ACL reconstruction. Native expression of VEGF by the CD34+ cells improved tendon graft maturation and biomechanical strength; however, over-expression of VEGF impeded improvements in biomechanical strength.

Failures in fracture healing after conventional autologous and allogenic bone grafting are mainly due to poor vascularization. To meet the clinical demand, recent attentions in the regeneration and repair of bone have been focused on the use of stem cells such as bone marrow mesenchymal stem cells and circulating skeletal stem cells. Circulating stem cells are currently paid a lot of attention due to their ease of clinical setting and high potential for osteogenesis and angiogenesis. In this report, we focus on the first proof-of-principle experiments demonstrating the collaborative characteristics of circulating CD34(+) cells, known as endothelial and hematopoietic progenitor cell-rich population, which are capable to differentiate into both endothelial cells and osteoblasts. Transplantation of circulating CD34(+) cells provides a favorable environment for fracture healing via angiogenesis/vasculogenesis and osteogenesis, finally leading to functional recovery from fracture. Based on a series of basic studies, we performed a phase 1/2 clinical trial of autologous CD34(+) cell transplantation in patients with tibial or femoral nonunions and reported the safety and efficacy of this novel therapy. In this review, the current concepts and strategies in circulating CD34(+) cell-based therapy and its potential applications for bone repair will be highlighted.

Atrophy with fatty degeneration is often seen in rotator cuff muscles with torn tendons. PRP has been reported to enhance tissue repair processes after tendon ruptures. However, the effect of PRP on atrophy and fatty degeneration of the muscle is not yet known. The aim of this study is to examine the effect of PRP on degeneration change of rotator cuff muscles in vitro and in vivo. A murine myogenic cell line and a rat rotator cuff tear model were used in this study and PRP was administrated into subacromial space which is widely used in clinical practice. In in vitro study, administration of PRP to C2C12 cells stimulated cell proliferation while inhibited both myogenic and adipogenic differentiation. In in vivo study, administration of PRP suppressed Oil Red-O positive lipid droplet formation. The expression of adipogenic genes was also decreased by PRP administration. In conclusion, PRP promoted proliferation of myoblast cells, while inhibiting adipogenic differentiation of myoblast cells and suppressing fatty degeneration change in rat torn rotator cuff muscles. Further investigations are needed to determine the clinical applicability of the PRP. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1806-1815, 2017.

Purpose To investigate the biomechanics of the intermediate (IM), anteromedial (AM), and posterolateral (PL) bundles in the human anterior cruciate ligament (ACL). Methods Eighteen human cadaveric knees were tested with a robotic/universal force-moment sensor testing system. Anterior tibial translation (ATT) was determined under an 89-N anterior tibial load. Coupled ATT was determined under a combined rotatory load of 7-Nm valgus and 5-Nm internal rotation torque (pivot moment). Each bundle's in situ forces were measured under identical external loading conditions. Results Under anterior load, the PL bundle's in situ force was highest at 0° and decreased during flexion. Under the anterior load, the AM bundle's in situ force was significantly higher than the IM and PL bundles' force at 15°, 30°, and 60°. Under the pivot moment, the AM bundle's in situ force was significantly higher than the PL and IM bundles' force at 0° and 15°, and the IM bundle had the lowest in situ force at 0° but higher in situ force than the AM and PL bundles at 30° and 45°. IM and AM bundle removal increased ATT under the anterior load at all angles. Cutting the PL bundle after IM and AM bundle removal (whole ACL removal) significantly increased ATT under the anterior load at 0°, 15°, and 30° of knee flexion and increased coupled ATT under the pivot moment at 0° and 15°. Conclusions The biomechanical role of each of the 3 ACL bundles (AM, IM, and PL) was measured with a robotic/universal force-moment sensor testing system. The AM bundle stabilized the knee against both the anterior and rotatory loads. The PL bundle stabilized the knee especially near full extension. The IM bundle supported the AM and PL bundles through all flexion angles, especially from 30° to 45°, against the rotatory load. Clinical Relevance Knowledge of functions of the different ACL bundles will help improve ACL reconstruction techniques to enable restoration of normal knee function.

Background Autologous chondrocyte implantation (ACI) is considered a promising choice for the treatment of cartilage defects. However, the application of ACI to osteoarthritic patients is, in general, contraindicated. The purpose of this study is to evaluate the efficiency of three-dimensionallystructured ACI (3D-ACI; CaReS) in a rat model of knee osteoarthritis (OA). Methods OA-like degenerative changes in the articular cartilage were created by transecting the anterior cruciate ligament (ACLT) in athymic nude rats. Two weeks later, CaReS was transplanted at the cartilage injury sites created by micro-drilling in the patella groove (Chondrocyte-implanted (CI) group: CaReS collagen with human chondrocytes; Collagen group: CaReS collagen without cells; and Sham group: sham operation; n = 15/group). Results Reverse Transcription Polymerase Chain Reaction (RT-PCR) analysis demonstrated the expression of human-specific type 2 collagen and Sry-type high-mobility-group box 9 (SOX9) in the CI group—not in the other groups—throughout the study period. Double immunohistochemistry for human-specific type 2 collagen and human leukocyte antigen-abacavir (HLA-ABC) at week 4 showed positive staining in the CI group only. Macroscopic assessment showed better repair at the cartilage defect sites in the CI group, compared to the other groups. Histological assessment with toluidine blue staining showed that the thickness of the articular cartilage and semi-quantitative histological scores were higher in the CI group than in the other groups up to week 20. Conclusions We demonstrate, for the first time, that 3D-ACI is effective in repairing cartilage defects in a rat model of ACLT-induced OA.

Adipose tissue is one of the promising sources of multipotent stem cells in human. Human multipotent adipose-derived stem (hMADS) cells have recently been isolated and showed differentiation potential into multiple mesenchymal lineages in vitro and in vivo. On the basis of these evidences, we examined the therapeutic efficacy of hMADS cells for fracture healing in an immunodeficient rat femur non-union fracture model. Local transplantation of hMADS cells radiographically and histologically promoted fracture healing with significant improvement of biomechanical function at the fracture sites compared with local transplantation of human fibroblasts (hFB) or PBS administration. Histological capillary density and physiological blood flow by laser Doppler perfusion imaging were significantly greater in hMADS group than hFB and PBS groups. Expressions of intrinsic (rat) bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF) and angiopoietin-1 in peri-fracture tissue were upregulated in hMADS group than other groups. In addition, presence of BMP-2 or VEGF activated the proliferation and migration of hMADS cells in vitro. These results indicate that hMADS cells stimulate the interaction between the transplanted cells and the resident cells stronger than other cells, and they promote fracture healing more effectively. Furthermore, immunohistochemistry for human-specific antibodies revealed direct differentiation of hMADS cells into osteoblasts or endothelial cells in newly formed callus or vasculature, respectively. RT-PCR for human-specific primers for osteogenic/endothelial markers also disclosed osteogenic and vasculogenic plasticity of the transplanted hMADS cells at the early stage of fracture healing. The present results suggest that transplantation of hMADS cells may become a useful strategy for cell-based bone regeneration in the future clinical setting.

Only a few cases of double-layered meniscus have been described in the English literature. We report two cases of bilateral double-layered lateral meniscus, where an additional semicircular meniscus was observed over the normal lateral meniscus. One of the patients exhibited a bucket-handle tear with a double-layered meniscus. To our knowledge, this abnormality is extremely rare and the incidence of double layered meniscus with bucket-handle tear has not been previously reported.

An emerging strategy in the regeneration and repair of bone is to use stem cells, including bone marrow mesenchymal stem cells, which are the most investigated and reliable source for tissue engineering, as well as circulating skeletal stem/progenitor cells, which are receiving abundant attention in regenerative medicine due to their ease of isolation and high osteogenic potential. Because failures in fracture healing are largely due to poor vascularization among many environmental factors, we highlight the first proof-of-principle experiments that elucidated the collaborative multi-lineage differentiation of circulating CD34 positive cells - a cell-enriched population of endothelial/hematopoietic progenitor cells - into not only endothelial cells but also osteoblasts. These cells develop a favorable environment for fracture healing via vasculogenesis/angiogenesis and osteogenesis, ultimately leading to functional recovery from fracture. This review will also highlight current concepts of circulating stem/progenitor cell-based therapy and their potential application for bone repair.

Background— Therapeutic effect of stem cell transplantation (SCTx) for myocardial neovascularization has been evaluated by histological capillary density in small animals. However, it has been technically difficult to obtain imaging evidence of collateral formation by conventional angiography. Methods and Results— Peripheral blood CD34+ and CD34− cells were isolated from patients with critical limb ischemia. PBS, CD34− cells, or CD34+ cells were intramyocardially transplanted after ligating LAD of nude rats. Coronary angiography of ex vivo beating hearts 5 and 28 days after the treatment was performed using the third generation synchrotron radiation microangiography (SRM), which has potential to visualize vessels as small as 20 μm in diameter. The SRM was performed pre and post sodium nitroprusside (SNP) to examine vascular physiology at each time point. Diameter of most collateral vessels was 20 to 120 μm, apparently invisible size in conventional angiography. Rentrop scores at day 28 pre and post SNP were significantly greater in CD34+ cell group than other groups ( P P R =0.82, P Conclusions— The SRM, capable of visualizing microvessels, may be useful for morphometric and physiological evaluation of coronary collateral formation by SCTx. The novel imaging system may be an essential tool in future preclinical/translational research of stem cell biology.

Avascular necrosis of the humeral capitellum (Panner's disease), a relatively rare disorder in the juvenile. We diagnosed three patients using radiographs and magnetic resonance imaging, who were previously misdiagnosed with osteochondritis dissecans of the humeral capitellum. All patients were successfully treated by restriction of sports activities using the upper extremities. It is often difficult to distinguish Panner's disease from osteochondritis dissecans of the humeral capitellum. Panner's disease has been reported to occur in boys between 7 and 10 years old, which is younger than susceptible age of osteochondritis dissecans. In MRI, low intensity area in the ossification center of the humeral capitellum in T1-weighted image is useful findings for diagnosis. For the treatment, the conservative treatment has been recommended in the early stage of Panner's disease, while surgical intervention is required in some cases of osteochondritis dissecans.

To achieve biological regeneration of tendon-bone junctions, cell sheets of human rotator-cuff derived cells were used in a rat rotator cuff injury model. Human rotator-cuff derived cells were isolated, and cell sheets were made using temperature-responsive culture plates. Infraspinatus tendons in immunodeficient rats were resected bilaterally at the enthesis. In right shoulders, infraspinatus tendons were repaired by the transosseous method and covered with the cell sheet (sheet group), whereas the left infraspinatus tendons were repaired in the same way without the cell sheet (control group). Histological examinations (safranin-O and fast green staining, isolectin B4, type II collagen, and human-specific CD31) and mRNA expression (vascular endothelial growth factor; VEGF, type II collagen; Col2, and tenomodulin; TeM) were analyzed 4 weeks after surgery. Biomechanical tests were performed at 8 weeks. In the sheet group, proteoglycan at the enthesis with more type II collagen and isolectin B4 positive cells were seen compared with in the control group. Human specific CD31-positive cells were detected only in the sheet group. VEGF and Col2 gene expressions were higher and TeM gene expression was lower in the sheet group than in the control group. In mechanical testing, the sheet group showed a significantly higher ultimate failure load than the control group at 8 weeks. Our results indicated that the rotator-cuff derived cell sheet could promote cartilage regeneration and angiogenesis at the enthesis, with superior mechanical strength compared with the control. Treatment for rotator cuff injury using cell sheets could be a promising strategy for enthesis of tendon tissue engineering. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:289-296, 2017.

We recently demonstrated that the local transplantation of human peripheral blood (PB) CD34+ cells, an endothelial/hematopoietic progenitor cell-rich population, contributes to fracture repair via vasculogenesis/angiogenesis and osteogenesis. Human PB mononuclear cells (MNCs) are also considered a potential cell fraction for neovascularization. We have previously shown the feasibility of human PB MNCs to enhance fracture healing. However, there is no report directly comparing the efficacy for fracture repair between CD34+ cells and MNCs. In addition, an unhealing fracture model, which does not accurately resemble a clinical setting, was used in our previous studies. To overcome these issues, we compared the capacity of human granulocyte colony-stimulating factor-mobilized PB (GM-PB) CD34+ cells and human GM-PB MNCs in a nonunion model, which more closely resembles a clinical setting. First, the effect of local transplantation of 1 × 105 GM-PB CD34+ cells (CD34+ group), 1 × 107 GM-PB MNCs (containing approximately 1 × 105 GM-PB CD34+ cells) (MNC group), and phosphate-buffered saline (PBS) (PBS group) on nonunion healing was compared. Similar augmentation of blood flow recovery at perinonunion sites was observed in the CD34+ and MNC groups. Meanwhile, a superior effect on nonunion repair was revealed by radiological, histological, and functional assessment in the CD34+ group compared with the other groups. Moreover, through in vivo and in vitro experiments, excessive inflammation induced by GM-PB MNCs was confirmed and believed to be one of the mechanisms underlying this potency difference. These results strongly suggest that local transplantation of GM-PB CD34+ cells is a practical and effective strategy for treatment of nonunion after fracture.

DNA damage is a cause of age related pathologies, including osteoarthritis (OA). Excision repair cross complementation group 1 (ERCC1) is an endonuclease required for DNA damage repair. In this study we investigated the function of ERCC1 in chondrocytes and its association with the pathophysiology of OA. ERCC1 expression in normal and osteoarthritic cartilage was assessed, as were changes in ERCC1 expression in chondrocytes under catabolic stress. Inhibiting ERCC1 in chondrocytes under interleukin-1β stimulation using small interfering RNA (siRNA) was also evaluated. Finally, cellular senescence and apoptosis were examined in relation to ERCC1 function. ERCC1 expression was decreased in OA cartilage and increased within 4 h of exposure to interleukin (IL)-1β, but decreased after 12 h. The inhibition of ERCC1 by siRNA increased the expression of matrix metallopeptidase 13 and decreased collagen type II. ERCC1 inhibition also increased the number of apoptotic and senescent cells. The inhibition of ERCC1 in chondrocytes increased their expression of OA related proteins, apoptosis, cellular senescence, and hypertrophic-like changes which suggest that ERCC1 is critical for protecting human chondrocytes (HCs) from catabolic stresses and provides insights into the pathophysiology of OA and a potential target for its treatment. (191)

Introduction. Some patients showed unusual responses to the immobilization without any objective findings with casts in upper extremities. We hypothesized their that intolerance with excessive anxiety to casts is due to claustrophobia triggered by cast immobilization. The aim of this study is to analyze the relevance of cast immobilization to the feeling of claustrophobia and discover how to handle them.Methods. There were nine patients who showed the caustrophobic symptoms with their casts. They were assesed whether they were aware of their claustrophobis themselves. Further we investigated the alternative immobilization to casts.Results. Seven out of nine cases that were aware of their claustrophobic tendencies either were given removable splints initially or had the casts converted to removable splints when they exhibited symptoms. The two patients who were unaware of their latent claustrophobic tendencies were identified when they showed similar claustrophobic symptoms to the previous patients soon after short arm cast application. We replaced the casts with removable splints. This resolved the issue in all cases.Conclusions. We should be aware of the claustrophobia if patients showed unusual responses to the immobilization without any objective findings with casts in upper extremities, where removal splint is practical alternative to cast to continue the treatment successfully.

Idiopathic necrosis of the second metatarsal head is quite unusual in adults. We present a rare case of idiopathic osteonecrosis of the second metatarsal head in an adult via magnetic resonance imaging, which enabled an early diagnosis of the necrosis before osteochondral deformity could develop. This case is noteworthy due to its rarity and its successful nonoperative treatment.

Most bone fractures typically heal, although a significant proportion (5%–10%) of fractures fail to heal, resulting in delayed union or persistent nonunion. Some preclinical evidence shows the therapeutic potential of peripheral blood CD34+ cells, a hematopoietic/endothelial progenitor cell-enriched population, for bone fracture healing; however, clinical outcome following transplantation of CD34+ cells in patients with fracture has never been reported. We report a phase I/IIa clinical trial regarding transplantation of autologous, granulocyte colony stimulating factor-mobilized CD34+ cells with atelocollagen scaffold for patients with femoral or tibial fracture nonunion (n = 7). The primary endpoint of this study is radiological fracture healing (union) by evaluating anteroposterior and lateral views at week 12 following cell therapy. For the safety evaluation, incidence, severity, and outcome of all adverse events were recorded. Radiological fracture healing at week 12 was achieved in five of seven cases (71.4%), which was greater than the threshold (18.1%) predefined by the historical outcome of the standard of care. The interval between cell transplantation and union, the secondary endpoint, was 12.6 ± 5.4 weeks (range, 8–24 weeks) for clinical healing and 16.1 ± 10.2 weeks (range, 8–36 weeks) for radiological healing. Neither deaths nor life-threatening adverse events were observed during the 1-year follow-up after the cell therapy. These results suggest feasibility, safety, and potential effectiveness of CD34+ cell therapy in patients with nonunion.

Recent reports indicated that human isolated CD271+ bone marrow mesenchymal stromal cells (BM-MSCs) have a greater expansion and potential for multipotent differentiation including chondrogenesis than classical plastic adherent (PA) BM-MSCs in vitro. Therefore, we set up a hypothesis that CD271+ MSCs may have a greater chondrogenic potential than PA-MSCs in vitro and in vivo. We investigated the superiority of CD271+ MSCs on chondrogenesis using in vitro expansion and pellet culture system and in vivo rat model of cartilage defect when compared to PA-MSCs. In the in vitro study, CD271+ MSCs showed higher expansion potential and produced larger pellets with higher expressions of chondrogenic genes when compared to the control groups. During the culture, CD271 expression decreased, which resulted in decreased chondrogenesis. In the in vivo study, immunohistochemical staining demonstrated differentiated human chondrocytes identified as double-stained cells with human-specific collagen type 2 and human leukocyte antigen-ABC in CD271+ and PA groups. The number of double-stained cells was significantly higher in the CD271+ group than PA group. Real-time RT-PCR analysis of tissue RNA isolated from the chondral defect site for human-specific chondrogenic markers demonstrated a significantly higher expression in CD271+ group than PA group. Macroscopic examination of chondral defect sites at week 8 revealed glossy white and well-integrated repaired tissues in the CD271+ and PA groups, but not in the PBS group. The average histological score in the CD271+ group was significantly greater than in the other groups. Apoptosis analysis at the cell transplanted site with TUNEL staining showed that the CD271+ group had significantly fewer apoptotic chondrocytes compared with the PA group. These results indicate that CD271+ MSCs have a greater chondrogenic potential than PA-MSCs in both in vitro and in vivo conditions.

A case presented dysfunctional flexion of the thumb and index fingers after volar plate fixation for of distal radius fractures was diagnosed anterior interosseous nerve palsy and confirmed by 3D-CT. 3D-CT was useful to confirm the continuities of tendons, which excluded the most common complication of tendon ruptures after volar plate fixation. Also, it was superior to needle electromyography which is unreliable due to the accompanying damage of the pronator quadratus following volar plate fixation.

As a new strategy for enhancing recovery after anterior cruciate ligament (ACL) reconstruction, we developed a cell sheet wrapped graft using ACL-derived CD34+ cells. Our results from an ex vivo cell migration assay demonstrated gradual cell migration into the graft from the cell sheet, and an enzyme-linked immunosorbent assay indicated that the cell sheet cultures secreted a significantly greater amount of VEGF than typical monolayer cultures. In the in vivo study, the rats received one of three possible ACL reconstruction treatments; 1) a tendon graft wrapped in an ACL-derived CD34+ cell sheet, 2) a tendon graft and ACL-derived CD34+ cell injection, or 3) the control group that received just the tendon graft. The in vivo studies revealed that the ACL-derived CD34+ cell sheet wrapped grafts demonstrated a greater number of the cells derived from the cell sheets incorporated within the bone tunnel site and grafted tendon. We believe the incorporated CD34+ cells enhanced the healing of both the bone-tendon junction and the grafted tendon in the reconstructed rat ACLs by increasing proprioceptive recovery, graft maturation, and biomechanical strength. ACL-derived CD34+ cell sheet wrapped grafts could become a promising strategy to revitalize tendon autografts and recovery after ACL reconstruction resulting in superior and quicker recovery in patients requiring ACL reconstruction.

AB Purpose: Surgical repairs of tears in the vascular region of the meniscus usually heal better than repairs performed in the avascular region; thus, we hypothesized that this region might possess a richer supply of vascular-derived stem cells than the avascular region. Methods: In this study, we analyzed 6 menisci extracted from aborted human fetuses and 12 human lateral menisci extracted from adult human subjects undergoing total knee arthroplasty. Menisci were immunostained for CD34 (a stem cell marker) and CD146 (a pericyte marker) in situ, whereas other menisci were dissected into two regions (peripheral and inner) and used to isolate meniscus-derived cells by flow cytometry. Cell populations expressing CD34 and CD146 were tested for their multilineage differentiation potentials, including chondrogenic, osteogenic, and adipogenic lineages. Fetal peripheral meniscus cells were transplanted by intracapsular injection into the knee joints of an athymic rat meniscal tear model. Rat menisci were extracted and histologically evaluated after 4 wk posttransplantation. Results: Immunohistochemistry and flow cytometric analyses demonstrated that a higher number of CD34- and CD146-positive cells were found in the peripheral region compared with the inner region. The CD34- and CD146-positive cells isolated from the vascular region of both fetal and adult menisci demonstrated multilineage differentiation capacities and were more potent than cells isolated from the inner (avascular) region. Fetal CD34- and CD146-positive cells transplanted into the athymic rat knee joint were recruited into the meniscal tear sites and contributed to meniscus repair. Conclusions: The vascularized region of the meniscus contains more stem cells than the avascular region. These meniscal-derived stem cells were multipotent and contributed to meniscal regeneration.

Transplantation of bone marrow (BM) CD34+ cells, an endothelial/hematopoietic progenitor-enriched cell population, has shown therapeutic efficiency in the treatment of ischemic diseases enhancing neovascularization. However, the number of CD34+ cells obtained from bone marrow is not sufficient for routine clinical application. To overcome this issue, we developed a more efficient and clinically applicable CD34+ cell expansion method. Seven-day ex vivo expansion culture of BM CD34+ cells with a cocktail of five growth factors containing VEGF, SCF, IL-6, Flt-3 ligand, and TPO resulted in reproducible more than 20-fold increase in cell number. The favorable effect of the local transplantation of culture expanded (cEx)-BM CD34+ cells on rat unhealing fractures was equivalent or higher than that of nonexpanded (fresh) BM CD34+ cells exhibiting sufficient therapeutic outcome with frequent vasculogenic/osteogenic differentiation of transplanted cEx-BM CD34+ cells and fresh BM CD34+ cells as well as intrinsic enhancement of angiogenesis/osteogenesis at the treated fracture sites. Specifically, cEx-BM CD34+ cell treatment demonstrated the best blood flow recovery at fracture sites compared with the nonexpanded BM CD34+ cells. In vitro, cEx-BM CD34+ cells showed higher colony/tube-forming capacity than nonexpanded BM CD34+ cells. Both cells demonstrated differentiation potential into osteoblasts. Since fresh BM CD34+ cells can be easily collected from fracture sites at the time of primary operation and stored for future use, autologous cEx-BM CD34+ cell transplantation would be not only a simple but also a promising therapeutic strategy for unhealing fractures in the field of orthopedic trauma surgery.

We report a case of flexor pollicis longus and flexor digitorum profundus(II) laceration as a result of the severe tenosynovitis after volar locking plate fixation of distal radius fracture.

Triamcinolone acetonide (TA) injections are widely used to treat enthesopathy, but they may induce adverse effects such as tendon impairment and rupture. Platelet-rich plasma (PRP) is a blood fraction containing high platelet concentrations and various growth factors that play a role in tissue repair processes. The purpose of this study is to investigate whether TA has deleterious effects on human rotator cuff-derived cells, and if PRP can protect these cells from the effects of TA. Human rotator cuff-derived cells were cultured with and without TA and PRP, and the culture without any additive served as the control. Cell morphology was assessed at days 7 and 21. Cell viability was evaluated at days 1, 7, 14, and 21 by a water-soluble tetrazolium salt assay. Induction of apoptosis was measured by immunofluorescence staining and flow cytometry at day 7. Induction of cleaved caspase-3 was measured by immunofluorescence staining at day 7. The cells cultured with TA had a flattened and polygonal shape at day 7. The cells cultured with both TA and PRP were similar in appearance to control cells. Exposure to TA also significantly decreased cell viability, but cell viability did not decrease when PRP was added along with TA. The number of apoptotic cells increased with TA exposure, while addition of PRP prevented cell apoptosis. In conclusion, the deleterious effect of TA was prevented by PRP, which can be used as a protective agent for patients receiving local TA injections.

The anterior cruciate ligament (ACL) usually fails to heal after rupture mainly due to the inability of the cells within the ACL tissue to establish an adequate healing process, making graft reconstruction surgery a necessity. However, some reports have shown that there is a healing potential of ACL with primary suture repair. Although some reports showed the existence of mesenchymal stem cell-like cells in human ACL tissues, their origin still remains unclear. Recently, blood vessels have been reported to represent a rich supply of stem/progenitor cells with a characteristic expression of CD34 and CD146. In this study, we attempted to validate the hypothesis that CD34- and CD146-expressing vascular cells exist in hACL tissues, have a potential for multi-lineage differentiation, and are recruited to the rupture site to participate in the intrinsic healing of injured ACL. Immunohistochemistry and flow cytometry analysis of hACL tissues demonstrated that it contains significantly more CD34 and CD146-positive cells in the ACL ruptured site compared with the noninjured midsubstance. CD34+CD45- cells isolated from ACL ruptured site showed higher expansionary potentials than CD146+CD45- and CD34-CD146-CD45- cells, and displayed higher differentiation potentials into osteogenic, adipogenic, and angiogenic lineages than the other cell populations. Immunohistochemistry of fetal and adult hACL tissues demonstrated a higher number of CD34 and CD146-positive cells in the ACL septum region compared with the midsubstance. In conclusion, our findings suggest that the ACL septum region contains a population of vascular-derived stem cells that may contribute to ligament regeneration and repair at the site of rupture.

Although implantation of crude bone marrow cells has been applied in a small number of patients for fracture healing, transplantation of peripheral blood CD34+ cells, the hematopoietic/endothelial progenitor cell-enriched population, in patients with fracture has never been reported. Here, we report the first case of tibial nonunion receiving autologous, granulocyte colony stimulating factor mobilized CD34+ cells accompanied with autologous bone grafting. No serious adverse event occurred, and the novel therapy performed 9 months after the primary operation resulted in bone union 3 months later without any symptoms including pain and gait disturbance.

Human multipotent adipose-derived stem cells (hMADSCs) have recently been isolated featuring extensive expansion capacity ex vivo. We tested the hypothesis that hMADSC transplantation might contribute to cardiac functional recovery by its direct or indirect effect on myocardial infarction (MI). Nude rats were either transplanted with hMADSCs or PBS (control) in ischemic myocardium immediately following MI. Echocardiographical assessment of cardiac function after MI with hMADSCs showed significant improvement of each parameter compared to that with PBS. Histological analysis also showed significantly reduced infarct size and increased capillary density in peri-infarct myocardium by hMADSC treatment. However, remarkable transdifferentiation of hMADSCs into cardiac or vascular lineage cells was not observed. Despite the less transdifferentiation capacity, hMADSCs produced robust multiple pro-angiogenic growth factors and chemokines, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and stromal cell-derived factor-1α (SDF-1α). Specifically, hMADSC-derived SDF-1α had a crucial role for cooperative angiogenesis, with the paracrine effect of hMADSCs and Tie2-positive bone marrow (BM) progenitor recruitment in ischemic myocardium. hMADSCs exhibit a therapeutic effect on cardiac preservation following MI, with the production of VEGF, bFGF, and SDF-1α showing paracrine effects and endogenous BM stem/progenitor recruitment to ischemic myocardium rather than its direct contribution to tissue regeneration.

We recently reported that i.v. transplantation of adult human circulating CD34+ cells, an endothelial/hematopoietic progenitor-enriched cell population, contributes to fracture healing through the enhancement of vasculogenesis and osteogenesis. However, the scarcity of CD34+ cells in the adult human is a critical issue for the future clinical application of this method. To overcome this issue, we assessed in vitro and in vivo capacity of granulocyte colony-stimulating factor-mobilized peripheral blood (GM-PB) human CD34+ cells for vasculogenesis and osteogenesis. First, we confirmed the differentiation capability of GM-PB CD34+ cells into osteoblasts in vitro. Second, local transplantation of GM-PB CD34+ cells on atelocollagen scaffold was performed in nude rats in a model of unhealing fractures. Immunostaining for human leukocyte antigen-ABC of tissue samples 1 week after fracture and cell therapy showed the superior incorporation after local transplantation compared with systemic infusion. Third, the effects of local transplantation of 105 (Hi), 104 (Mid), or 103 (Lo) doses of GM-PB CD34+ cells or phosphate-buffered saline (PBS) on fracture healing were compared. Extrinsic vasculogenic and osteogenic differentiation of GM-PB CD34+ cells, enhancement of the intrinsic angio-osteogenesis by recipient cells, augmentation of blood flow recovery at the fracture sites, and radiological and histological confirmation of fracture healing were observed only in the Hi and Mid groups but not in the Lo and PBS groups. These results strongly suggest that local transplantation of GM-PB CD34+ cells with atelocollagen scaffold is a feasible strategy for therapeutic vasculogenesis and osteogenesis needed for fracture healing. Disclosure of potential conflicts of interest is found at the end of this article.

Neoangiogenesis is a key process in the initial phase of ligament healing. Adult human circulating CD34+ cells, an endothelial/hematopoietic progenitor-enriched cell population, have been reported to contribute to neoangiogenesis; however, the therapeutic potential of CD34+ cells for ligament healing is still unclear. Therefore, we performed a series of experiments to test our hypothesis that ligament healing is supported by CD34+ cells via vasculogenesis. Granulocyte colony-stimulating factor-mobilized peripheral blood (GM-PB) CD34+ cells with atelocollagen (CD34+ group), GM-PB mononuclear cells (MNCs) with atelocollagen (MNC group), or atelocollagen alone (control group) was locally transplanted after the creation of medial collateral ligament injury in immunodeficient rats. Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemical staining at the injury site demonstrated that molecular and histological expression of human-specific markers for endothelial cells was higher in the CD34+ group compared with the other groups at week 1. Endogenous effect, assessed by capillary density and mRNA expression of vascular endothelial growth factor, was significantly higher in CD34+ cell group than the other groups. In addition to the observation that, as assessed by real-time RT-PCR, gene expression of ligament-specific marker was significantly higher in the CD34+ group than in the other groups, ligament healing assessed by macroscopic, histological, and biomechanical examination was significantly enhanced by CD34+ cell transplantation compared with the other groups. Our data strongly suggest that local transplantation of circulating human CD34+ cells may augment the ligament healing process by promoting a favorable environment through neovascularization. Disclosure of potential conflicts of interest is found at the end of this article.

Failures in fracture healing are mainly caused by a lack of vascularization. Adult human circulating CD34+ cells, an endothelial/hematopoietic progenitor-enriched cell population, have been reported to differentiate into osteoblasts in vitro; however, the therapeutic potential of CD34+ cells for fracture healing is still unclear. Therefore, we performed a series of experiments to test our hypothesis that functional fracture healing is supported by vasculogenesis and osteogenesis via regenerative plasticity of CD34+ cells. Peripheral blood CD34+ cells, isolated from total mononuclear cells of adult human volunteers, showed gene expression of osteocalcin in 4 of 20 freshly isolated cells by single cell reverse transcriptase-polymerase chain reaction analysis. Phosphate-buffered saline, mononuclear cells, or CD34+ cells were intravenously transplanted after producing nonhealing femoral fractures in nude rats. Reverse transcriptase-polymerase chain reaction and immunohistochemical staining at the peri-fracture site demonstrated molecular and histological expression of human-specific markers for endothelial cells and osteoblasts at week 2. Functional bone healing assessed by biomechanical as well as radiological and histological examinations was significantly enhanced by CD34+ cell transplantation compared with the other groups. Our data suggest circulating human CD34+ cells have therapeutic potential to promote an environment conducive to neovascularization and osteogenesis in damaged skeletal tissue, allowing the complete healing of fractures.