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Background: There are currently no disease-modifying treatments available for knee osteoarthritis (OA), although cultured adipose-derived stromal cells (ASCs) have shown promise in experimental models. However, given the regulatory limits on the use of cultured cells in humans, previous trials have focused primarily on the stromal vascular fraction (SVF) intra-articular injection. Therefore, the therapeutic value of ASCs for knee OA remains unknown. Purpose: To study ASC versus SVF intra-articular injection in patients with Kellgren-Lawrence (KL) knee OA grades 2 to 4 in parallel single-arm trials. Study Design: Cohort study; Level of evidence, 2. Methods: A total of 80 patients were enrolled, with 42 (72 knees) receiving ASC intra-articular injection and 38 (69 knees) receiving SVF. Patient-reported outcome measures were assessed at 1, 3, 6, 12, and 24 months using the Knee injury and Osteoarthritis Outcome Score 5 (KOOS5) and pain visual analog scale (VAS). The percentages of patients achieving the minimal clinically important difference (MCID) and Patient Acceptable Symptom State (PASS) were also calculated. Per protocol, a subset of the ASC group received an ASC booster injection after 6 months. A repeated-measures analysis of variance compared results between treatment arms and by KL grade over time. Results: Patient-reported outcome measures improved substantially after both treatments ( P < .05 at all time points), with the ASC group more likely to achieve the MCID (50% vs 24%; P = .01) and PASS (45% vs 24%; P = .04) for the pain VAS and the MCID (43% vs 16%; P = .02) for the KOOS5 at 12 months, although not at 24 months. Knees treated with ASC for KL grade 2/3 OA had significantly superior outcomes compared with those with KL grade 4 OA for the KOOS5 ( P = .01) and pain VAS ( P = .03), but no such difference was observed in knees treated with SVF. Three patients receiving ASCs (7%; all KL grade 3) sought additional nonoperative treatment by 24 months versus 9 patients receiving SVF (24%; all KL grade 3) ( P = .06). ASC booster injections conferred no additional benefit. Notably, patients in the ASC cohort reported more injection-site pain and swelling after the booster injection than after the initial injection ( P < .01). Conclusion: This represents the first head-to-head comparison of ASCs and SVF for the treatment of knee OA in humans. ASC and SVF injections both substantially improved knee pain and function at all follow-up time points, although ASC injections demonstrated significantly better improvements with regard to the MCID and PASS for the pain VAS and the MCID for the KOOS5 at 12 months. There appears to be no benefit to a booster ASC injection after initial treatment. Given less donor-site morbidity and equivalent superior outcomes at 2 years, the use of ASCs over SVF in the treatment of knee OA may be warranted.

Posterior ankle impingement syndrome is mainly seen in ballet dancers and frequently associated with specific movements in ballet such as pointe and demi pointe in which the whole-body weight is applied to the maximally plantar flexed ankle. We performed arthroscopic debridement for 2 dedicated ballet dancers on the intervening soft tissue causing posterior ankle impingement syndrome (PAIS). In both cases, T2-weighted magnetic resonance imaging (MRI) revealed low-signal intensity of meniscus-like soft tissue without abnormal osseous findings, connecting from the posterior side of the talus to Kager's fat pad. To examine the intervening soft tissue in detail, we performed histological evaluation by hematoxylin and eosin staining, Safranin O fast green staining, and immunohistochemistry for type I collagen and type II collagen. Hematoxylin and eosin staining showed that there was cartilage-like tissue including chondrocyte-like cells in contact with fibrous tissue. The extracellular matrix in the cartilage zone was consistently stained by Safranin O staining and type II collagen without any staining with type I collagen. These findings suggested that the meniscus-like soft tissue appearing as low-signal intensity on MRI at the posterior side of talus included hyaline-like cartilage. To the extent of our knowledge, these were rare cases of hyaline-like cartilage generation causing PAIS in ballet dancers, which might be associated with ballet specific movements resulting in chondrogenesis.

Anterior cruciate ligament reconstruction (ACLR) has become a popular surgery in orthopedic practice today, and the technique has evolved significantly over time. Surgical procedure, graft choice, and fixation systems have varied over the years. Nonetheless, several challenges like insufficient graft ligamentization, tunnel enlargement, and insufficient reestablishment of proprioception remain in ACLR. A vision of better graft healing and integration for improved outcomes after ACLR introduced the idea of the biological ACLR. Various techniques with growth factors, cellular therapies, or tissue augment have been researched with ACLR surgery for better integration and ligamentization. This review highlights the tissue wrappingmodalities currently being explored in biological ACLR.

Both mesenchymal stromal cells (MSC) and induced pluripotent stem cells (iPSC) offer the potential for repair of damaged connective tissues. The use of hybrid implants containing both human MSC and iPSC was investigated to assess their combined potential to yield enhanced repair of osteochondral defects. Human iPSC-CP wrapped with tissue engineered constructs (TEC) containing human MSC attained secure defect filling with good integration to adjacent tissue in a rat osteochondral injury model. The presence of living MSC in the hybrid implants was required for effective biphasic osteochondral repair. Thus, the TEC component of such hybrid implants serves several critical functions including, adhesion to the defect site via the matrix and facilitation of the repair via live MSC, as well as enhanced angiogenesis and neovascularization. Based on these encouraging studies, such hybrid implants may offer an effective future intervention for repair of complex osteochondral defects.

A 17-year-old male patient suffered a radial lateral meniscus tear and underwent an arthroscopic all-inside suture repair. After 7 months, the patient experienced catching. Magnetic resonance imaging and computed tomography revealed an intra-articular loose body without calcification, which was removed surgically. The excised specimen was histopathologically confirmed to be a necrotic meniscus fragment with a suture knot. In addition, cartilage damage because of suspected impingement by a residual suture knot was observed. After removing the loose body and knot, the patient's symptoms were relieved, and he returned to sports.Suture knot-related complications should be considered while performing meniscal repairs.

Oxaliplatin is a platinum complex antineoplastic agent widely used for chemotherapy of colorectal cancer. However, one of its side effects is hypersensitivity reactions, the incidence of which increases with a cumulative dose, thereby posing a difficulty to continue oxaliplatin use. Our hospital changed the premedication of oxaliplatin in August 2009 and September 2012. We retrospectively investigated the usefulness of these premedication changes. The results showed no significant difference in the incidence of hypersensitivity between the control group(12.1%)and the group receiving H1 and H2-blockers (12.3%); however, the incidence of hypersensitivity was significantly reduced in the group receiving increased dexamethasone based on the number of courses(2.7%). Therefore, our regimen was found to be effective in preventing hypersensitivity reactions to oxaliplatin.

Human synovium-derived stem cells (hSSCs) are an attractive source of cells for cartilage repair. At present, the quality of tissue and techniques used for cartilage regeneration have scope for improvement. A small compound, TD-198946, was reported to enhance chondrogenic induction from hSSCs; however, other applications of TD-198946, such as priming the cell potential of hSSCs, remain unknown. Our study aimed to examine the effect of TD-198946 pretreatment on hSSCs. HSSCs were cultured with or without TD-198946 for 7 days during expansion culture and then converted into a three-dimensional pellet culture supplemented with bone morphogenetic protein-2 (BMP2) and/or transforming growth factor beta-3 (TGFβ3). Chondrogenesis in cultures was assessed based on the GAG content, histology, and expression levels of chondrogenic marker genes. Cell pellets derived from TD-198946-pretreated hSSCs showed enhanced chondrogenic potential when chondrogenesis was induced by both BMP2 and TGFβ3. Moreover, cartilaginous tissue was efficiently generated from TD-198946-pretreated hSSCs using a combination of BMP2 and TGFβ3. Microarray analysis revealed that NOTCH pathway-related genes and their target genes were significantly upregulated in TD-198946-treated hSSCs, although TD-198946 alone did not upregulate chondrogenesis related markers. The administration of the NOTCH signal inhibitor diminished the effect of TD-198946. Thus, TD-198946 enhances the chondrogenic potential of hSSCs via the NOTCH3 signaling pathway. This study is the first to demonstrate the gradual activation of NOTCH3 signaling during chondrogenesis in hSSCs. The priming of NOTCH3 using TD-198946 provides a novel insight regarding the regulation of the differentiation of hSSCs into chondrocytes.

The rapidly growing and emerging nature of biologics have made indications for regenerative and reparative hip therapies ever changing, with at times only early-stage evidence for their use. The purpose of this study was to review and summarize the currently available data on the management of hip cartilage injuries and osteoarthritis. A scoping review of the available scientific literature for hip biologics was performed, with available evidence for hyaluronic acid (HA), platelet rich plasma (PRP), stem/stromal cells, microfracture, mosaicplasty, osteochondral allograft, and cell-based therapies investigated. To date, there exist better guidelines and further consensus concerning knee joint biologic treatments than the hip due to a greater number of studies as well as the more recent emergence of hip preservation approaches. However, increasing evidence is available for the selective implementation of biologics on an individualized basis with attention to lesion size and location. Orthopedic surgeons are at an exciting crossroads in medicine, where hip biologic therapies are evolving and increasingly available. Timetested interventions such as arthroplasty have shown good results and still have a major role to play but newer, regenerative approaches have the potential to effectively delay or reduce the requirement for such invasive procedures.

With the advancement of cell-based technologies for cartilage regeneration, many tissue engineering approaches have taken advantage of scaffolds made of either synthetic polymer or natural materials, providing an appropriate three-dimensional environment for cells to optimize their proliferation and chondrogenic differentiation and effectively delivering cells into cartilage defects. As an alternative, a scaffold-free cell delivery system could be an excellent choice due to its simplicity in development and ease of subsequent implantation, as well as lower cost, eliminating expensive biomaterials. In this chapter, we discuss the recent developments regarding scaffold-free approaches for cartilage regeneration, including using technique for a unique mesenchymal stem cell-based scaffold-free approach, and taking a bench to bedside perspective for clinical application.

Osteoarthritis (OA) is the most common joint disorder due to poor regenerative properties of cartilage. Existing approaches leave a treatment gap for patients with moderate OA. The intra-articular administration of a biological agent derived from adipose tissue is one of the novel therapies currently in development to address the limitations of standard treatments.

There is no therapy currently available for fully repairing articular cartilage lesions. Our laboratory has recently developed a visible light-activatable methacrylated gelatin (mGL) hydrogel, with the potential for cartilage regeneration. In this study, we further optimized mGL scaffolds by supplementing methacrylated hyaluronic acid (mHA), which has been shown to stimulate chondrogenesis via activation of critical cellular signalling pathways. We hypothesized that the introduction of an optimal ratio of mHA would enhance the biological properties of mGL scaffolds and augment chondrogenesis of human bone marrow-derived mesenchymal stem cells (hBMSCs). To test this hypothesis, hybrid scaffolds consisting of mGL and mHA at different weight ratios were fabricated with hBMSCs encapsulated at 20 × 106 cells/ml and maintained in a chondrogenesis-promoting medium. The chondrogenenic differentiation of hBMSCs, within different scaffolds, was estimated after 8 weeks of culture. Our results showed that mGL/mHA at a 9:1 (%, w/v) ratio resulted in the lowest hBMSC hypertrophy and highest glycosaminoglycan production, with a slightly increased volume of the entire construct. The applicability of this optimally designed mGL/mHA hybrid scaffold for cartilage repair was then examined in vivo. A full-thickness cylindrical osteochondral defect was surgically created in the rabbit femoral condyle, and a three-dimensional cell-biomaterial construct was fabricated by in situ photocrosslinking to fully fill the lesion site. The results showed that implantation of the mGL/mHA (9:1) construct resulted in both cartilage and subchondral bone regeneration after 12 weeks, supporting its use as a promising scaffold for repair and resurfacing of articular cartilage defects, in the clinical setting.

Objective The aim of this study was to elucidate the efficacy of T2-mapping MRI and correlation with histology for the evaluation of tissue repair quality following the first-in-human implantation of an autologous tissue engineered construct. Design We directly compared the results of T2-mapping MRI of cartilage repair tissue with the histology of a biopsy specimen from the corresponding area at 48 weeks postoperatively in 5 patients who underwent the implantation of a scaffold-free tissue-engineered construct generated from autologous synovial mesenchymal stem cells to repair an isolated cartilage lesion. T2 values and histological scores were compared at each of 2 layers of equally divided halves of the repair tissue (upper and lower zones). Results Histology showed that the repair tissue in the upper zone was dominated by fibrous tissue and the ratio of hyaline-like matrix increased with the depth of the repair tissue. There were significant differences between upper and lower zones in histological scores. Conversely, there were no detectable statistically significant differences in T2 value detected among zones of the repair tissue, but zonal differences were detected in corresponding healthy cartilage. Accordingly, there were no correlations detected between histological scores and T2 values for each repair cartilage zone. Conclusion Discrepancies in the findings between T2 mapping and histology suggest that T2 mapping was limited in ability to detect details in the architecture and composition of the repair cartilage.

Purpose To compare the suture slippage on a hamstring tendon graft prepared with a modified finger-trap device (SPEEDTRAP) with one prepared with Krackow stitch during graft passage through the tibial tunnel in ACL reconstruction. Methods Thirty-eight patients underwent anatomic triple-bundle anterior cruciate ligament reconstruction with 2 femoral and 3 tibial tunnels. After semitendinosus tendon was cut in half to make 2 grafts, the free ends of the proximal membranous portion (posterolateral [PL] graft) were prepared together with 2 sutures: (1) one SPEEDTRAP and one Krackow stitch for 20 cases (group A) and (2) double Krackow stitches on both sides for 18 cases (group B). Then, the PL graft was dye-marked at the proximal suture of SPEEDTRAP in group A and Krackow suture in group B and was inserted into the joint via tibial tunnel ahead of the loop side. The distance between the mark on the graft and the proximal suture of SPEEDTRAP or Krackow stitch was measured under arthroscopy after graft fixation at femur. Slippage was defined as 1 mm and more of distance between the mark and the proximal suture. Results Slippage was observed in 16 cases for SPEEDTRAP and in 2 for Krackow suture in group A, whereas one case showed slippage in group B. The slippage distance was 4.0 ± 2.9 mm for SPEEDTRAP and 0.2 ± 0.5 mm for Krackow stitch in group A (P < .001), whereas it was 0.1 ± 0.2 mm for double Krackow stitch in group B, showing a significant difference from SPEEDTRAP suture (P

Chondral regeneration and repair is a field of orthopaedics that has had exponential growth in the last decade. The use of various cells, stem cells and repair techniques have shown encouraging results for addressing chondral joint pathologies. The biomechanics of cartilage has been further researched to understand the requirements of the regenerate tissue and the possible materials that may be utilised in combination to improve results. A variety of materials have been used each having their own benefits and drawbacks. The function and structure of chondral tissue is complex and so in order for a biomaterial to resemble cartilage it must be carefully selected and modified accordingly. In this chapter, we discuss the biomechanical roles of cartilage and the current available biomaterials that have been developed to aid in chondral repair techniques.

The high mortality rate of cancer is strongly correlated with the development of distant metastases at secondary sites. Although Rho GTPases, such as RhoA, RhoB, RhoC, and RhoE, promote tumor metastasis, the main roles of Rho GTPases remain unidentified. It is also unclear whether rhosin, a Rho inhibitor, acts by suppressing metastasis by a downstream inhibition of Rho. In this study, we investigated this mechanism of metastasis in highly metastatic melanoma and breast cancer cells, and the mechanism of inhibition of metastasis by rhosin. We found that rhosin suppressed the RhoA and RhoC activation, the nuclear localization of YAP, but did not affect ERK1/2, Akt, or NF-&kappa, B activation in the highly metastatic cell lines B16BL6 and 4T1. High expression of YAP was associated with poor overall and recurrence-free survival in patients with breast cancer or melanoma. Treatment with rhosin inhibited lung metastasis in vivo. Moreover, rhosin inhibited tumor cell adhesion to the extracellular matrix via suppression of RHAMM expression, and inhibited SDF-1-induced cell migration and invasion by decreasing CXCR4 expression in B16BL6 and 4T1 cells. These results suggest that the inhibition of RhoA/C-YAP pathway by rhosin could be an extremely useful therapeutic approach in patients with melanoma and breast cancer.

A detailed understanding of biomechanical characteristics of healthy articular cartilage and the effect of mechanobiology on cartilage is needed to prevent joint degeneration with biological-based treatment strategies. In vitro and in vivo experiments of cartilage repair enhanced by mechanical stress have been able to guide rehabilitation protocols after surgery treatments of the cartilage defect site, including microfracturing, autologous chondrocyte implantation (ACI), and osteochondral autograft transplantation (OAT) in our practice. However, it remains unclear how loads produced in the course of postoperative rehabilitation affect the facilitative cartilage repair. This chapter will review how joint loading influences healthy cartilage structure and how mechanical loading mediates cellular responses that are either chondroprotective or catabolic, contributing to the initiation and progression of osteoarthritis. Examples of the application of mechanical loads to improve cartilage repair will also be highlighted.

Historically, anterior cruciate ligament (ACL) suture repair mostly resulted in failure because of intra-articular hypovascularity and poor intrinsic healing capacity of ACL. ACL reconstruction was therefore deemed the gold standard with a high success rate because of more evolved surgical technique. There are, however, clinical and subclinical disadvantages of reconstruction; low rate in full recovery to sports, donor harvest morbidity, tunnel enlargement, and incomplete microscopic healing of the graft. Recent experimental and clinical studies on biological augmentation of mesenchymal stem cells, platelet-rich plasma, or the other biologic agents with scaffold suggested potential feasibility of positive effects by such bio-therapies for both ACL repair and reconstruction. Biological augmentation of ACL surgery is still in the exploratory stages and more evidence from preclinical and clinical studies is required for implementation in clinical practice.

Purpose Chondrocyte -based tissue engineering has been a promising option for the treatment of cartilage lesions. In previous literature, TD198946 has been shown to promote chondrogenic differentiation which could prove useful in cartilage regeneration therapies. Our study aimed to investigate the effects of TD198946 in generating engineered cartilage using dedifferentiated chondrocyte-seeded collagen scaffolds treated with TD198946. Methods Articular chondrocytes were isolated from mini pig knees and expanded in 2-dimensional cell culture and subsequently used in the experiments. 3-D pellets were then cultured for two weeks. Cells were also cultured in a type I collagen scaffolds for four weeks. Specimens were cultured with TD198946, BMP-2, or both in combination. Outcomes were determined by gene expression levels of RUNX1, SOX9, ACAN, COL1A1, COL2A1 and COL10A1, the glycosaminoglycan content, and characteristics of histology and immunohistochemistry. Furthermore, the maturity of the engineered cartilage cultured for two weeks was evaluated through subcutaneous implantation in nude mice for four weeks. Results Addition of TD198946 demonstrated the upregulation of gene expression level except for ACAN, type II collagen and glycosaminoglycan synthesis in both pellet and 3D scaffold cultures. TD198946 and BMP-2 combination cultures showed higher chondrogenic differentiation than TD198946 or BMP-2 alone. The engineered cartilage maintained its extracellular matrices for four weeks post implantation. In contrast, engineered cartilage treated with either TD198946 or BMP-2 alone was mostly absorbed. Conclusions Our results indicate that TD198946 could improve quality of engineered cartilage by redifferentiation of dedifferentiated chondrocytes pre-implantation and promoting collagen and glycosaminoglycan synthesis.

Background: Cartilage restoration is a desperately needed bridge for patients with symptomatic cartilage lesions. Chondral lesion is a pathology with high prevalence, reaching as much as 63% of general population and 36% among athletes. Despite autologous chondrocyte implantation versatility, it still fails to fully reproduce hyaline articular cartilage characteristics. Mesenchymal stem cells (MSCs) may be isolated from various known tissues, including discarded fragments at arthroscopy such as synovial membrane. Choice of harvesting site is motivated by MSCs' abilities to modulate immunologic and inflammatory response through paracrine communication. Synovial MSCs have a greater proliferation and strong chondrogenic potential than bone and adipose MSCs and a less hypertrophic differentiation than bone MSCs. Good manufacturing practice (GMP) laboratory techniques for human clinical trials are still novel. To our knowledge, there are only two clinical trials in humans published since today. Purpose: Therefore, this work aimed to isolate and characterize synovial MSCs and evaluated their differentiation properties according to GMP standards. Materials and Methods: One-gram tissue sample from three patients of synovia was harvested at the beginning of arthroscopy surgery. MSCs were isolated, expanded, and characterized by flow cytometry. Results: It was possible to isolate and expand MSCs cultures from synovia, characterize MSCs by flow cytometry using proper monoclonal antibodies, and differentiate MSCs by coloring technique after chondrogenic, adipogenic, and osteogenic differentiations. Cartilage treatment may benefit from these tissue engineering protocols since arthroscopic procedures are routinely performed for different purposes in a previous stage and a favorable chondronegic differentiation cell lineage may be collected and stored in a less invasive way. Conclusion: Laboratory protocols established according to presented GMP were able to isolate and characterize MSCs obtained from synovia. Impact Statement Articular cartilage restoration is a desperately needed bridge for patients with symptomatic cartilage lesions and it rises as a socioeconomic issue with a considerable economic burden. Synovial mesenchymal stem cells (MSCs) have a greater proliferation rate and strong chondrogenic potential than bone and adipose MSCs and a less hypertrophic differentiation than bone MSCs. To our knowledge, there are only two human clinical trials with good manufacturing practice laboratory techniques for synovial MSCs harvesting and differentiation. Cartilage treatment may benefit from these tissue engineering protocols since arthroscopic procedures are routinely performed for different purposes in a previous stage.

Background: Articular cartilage has limited healing capacity, owing in part to poor vascularity and innervation. Once injured, it cannot be repaired, typically leading to high risk for developing osteoarthritis. Thus, cell-based and/or tissue-engineered approaches have been investigated; however, no approach has yet achieved safety and regenerative repair capacity via a simple implantation procedure. Purpose: To assess the safety and efficacy of using a scaffold-free tissue-engineered construct (TEC) derived from autologous synovial membrane mesenchymal stem cells (MSCs) for effective cartilage repair. Study Design: Case series; Level of evidence, 4. Methods: Five patients with symptomatic knee chondral lesions (1.5-3.0 cm2) on the medial femoral condyle, lateral femoral condyle, or femoral groove were included. Synovial MSCs were isolated from arthroscopic biopsy specimens and cultured to develop a TEC that matched the lesion size. The TECs were then implanted into chondral defects without fixation and assessed up to 24 months postoperatively. The primary outcome was the safety of the procedure. Secondary outcomes were self-assessed clinical scores, arthroscopy, tissue biopsy, and magnetic resonance image–based estimation of morphologic and compositional quality of the repair tissue. Results: No adverse events were recorded, and self-assessed clinical scores for pain, symptoms, activities of daily living, sports activity, and quality of life were significantly improved at 24 months after surgery. Secure defect filling was confirmed by second-look arthroscopy and magnetic resonance imaging in all cases. Histology of biopsy specimens indicated repair tissue approaching the composition and structure of hyaline cartilage. Conclusion: Autologous scaffold-free TEC derived from synovial MSCs may be used for regenerative cartilage repair via a sutureless and simple implantation procedure. Registration: 000008266 (UMIN Clinical Trials Registry number).

Purpose Chondral lesion is a pathology with high prevalence, reaching as much as 63% of general population and 36% among athletes. The ability of human Dental Pulp Stem Cells (DPSCs) to differentiate into chondroblasts in vitro suggests that this stem cell type may be useful for tissue bioengineering. However, we have yet to identify a study of large animal models in which DPSCs were used to repair articular cartilage. Therefore, this study aimed to describe a novel treatment for cartilage lesion with DPSCs on a large animal model. Methods Mesenchymal stem cells (MSC) were obtained from deciduous teeth and characterized by flow cytometry. DPSCs were cultured and added to a collagen type I/III biomaterial composite scaffold. Brazilian miniature pig (BR-1) was used. A 6-mm diameter, full-thickness chondral defect was created in each posterior medial condyle. The defects were covered with scaffold alone or scaffold + DPSCs on the contralateral side. Animals were euthanized 6 weeks post-surgery. Cartilage defects were analyzed macroscopically and histology according to modified O’Driscoll scoring system. Results Flow cytometry confirmed characterization of DPSCs as MSCs. Macroscopic and histological findings suggested that this time period was reasonable for evaluating cartilage repair. To our knowledge, this study provides the first description of an animal model using DPSCs to study the differentiation of hyaline articular cartilage in vivo. Conclusion The animals tolerated the procedure well and did not show clinical or histological rejection of the DPSCs, reinforcing the feasibility of this descriptive miniature pig model for pre-clinical studies.

RAS and BRAF-mutated colorectal cancers are associated with resistance to chemotherapy and poor prognosis, highlighting the need for new therapeutic strategies. Although these cancers sometimes respond to mitogen activated protein kinase kinase (MEK) inhibitor treatment, they often acquire resistance via mechanisms, which are poorly understood. Here, we investigated the mechanism of MEK inhibitor resistance in primary- and acquired-resistant cells. Cell viability was examined using the trypan blue dye exclusion assay. Protein expression was analyzed by western blotting. Somatic mutations in colorectal cancer cells were investigated using the polymerase chain reaction array. PD0325901 and trametinib induced cell death in LoVo and Colo-205 cells but not in DLD-1 and HT-29 cells, which have a PIK3CA mutation constitutively activating Akt and NF-&kappa, B. Treatment with PD0325901 and trametinib suppressed ERK1/2 activation in all four cell lines but only induced Akt and NF-&kappa, B activation in DLD-1 and HT-29 cells. Inhibition of Akt but not NF-&kappa, B, overcame MEK inhibitor resistance in DLD-1 and HT-29 cells. Acquired-resistant LoVo/PR, Colo-205/PR and LoVo/TR cells have constitutively active Akt due to a M1043V mutation in the kinase activation loop of PIK3CA and Akt inhibitor resensitized these cells to MEK inhibitor. These results demonstrate that the overactivation of Akt plays a critical role in MEK inhibitor primary and acquired resistance and implicate combined Akt/MEK inhibition as a potentially useful treatment for RAS/BRAF-mutated colorectal cancer.

Background: Intra-articular injection of adipose-derived stem cells (ASCs) has shown promise for improving symptoms and cartilage quality in the treatment of osteoarthritis (OA). However, while most preclinical studies have been performed with plastic-adherent ASCs, most clinical trials are being conducted with the stromal vascular fraction (SVF), prepared from adipose tissue without prior culture. Purpose: To directly compare clinical outcomes of intra-articular injection with ASCs or SVF in patients with knee OA. Study Design: Cohort study; Level of evidence, 3. Methods: The authors retrospectively compared 6-month outcomes in 42 patients (59 knees) receiving intra-articular injection with 12.75 million ASCs and 38 patients (69 knees) receiving a 5-mL preparation of SVF. All patients had Kellgren-Lawrence grade 2, 3, or 4 knee OA and had failed standard medical therapy. The visual analog scale (VAS) pain score and Knee injury and Osteoarthritis Outcome Score (KOOS) at baseline and 1, 3, and 6 months after injection were considered as outcomes. Outcome Measures in Rheumatology–Osteoarthritis Research Society International (OMERACT-OARSI) criteria were also used to assess positive response. A repeated measures analysis of variance was used for comparison between the treatment groups. Results: No major complications occurred in either group. The SVF group had a higher frequency of knee effusion (SVF 8%, ASC 2%) and minor complications related to the fat harvest site (SVF 34%, ASC 5%). Both groups reported improvements in pain VAS and KOOS domains. Specifically, in the ASC group, symptoms improved earlier (by 3 months; P < .05) and pain VAS decreased to a greater degree (55%; P < .05) compared with the SVF group (44%). The proportion of OMERACT-OARSI responders in the ASC group was slightly higher (ASCs, 61%; SVF, 55%; P = .25). Conclusion: It was observed that both ASCs and SVF resulted in clinical improvement in patients with knee OA, but that ASCs outperform SVF in the early reduction of symptoms and pain with less comorbidity.

Vibration acceleration through whole body vibration has been reported to promote fracture healing. However, the mechanism responsible for this effect remains unclear. Purpose of this study was to determine whether vibration acceleration directly affects cells around the fracture site and promotes endochondral ossification. Four-week-old female Wistar Hannover rats were divided into two groups (vibration [V group] and control [C group]). The eighth ribs on both sides were cut vertically using scissors. From postoperative day 3 to 11, vibration acceleration using Power Plate® (30 Hz, low amplitude [30-Low], 10 min/day) was applied in the V group. Mature calluses appeared earlier in the V group than in the C group by histological analysis. The GAG content in the fracture callus on day 6 was significantly higher in the V group than in the C group. The mRNA expressions of SOX-9, aggrecan, and Col-II in the fracture callus on day 6 and Col-X on day 9 were significantly higher in the V group than in the C group. For in vitro analysis, four different conditions of vibration acceleration (30 or 50 Hz with low or high amplitude [30-Low, 30-High, 50-Low, and 50-High], 10 min/day) were applied to a prechondrogenic cell (ATDC5) and an undifferentiated cell (C3H10T1/2). There was no significant difference in cell proliferation between the control and any of the four vibration conditions for both cell lines. For both cell lines, alcian blue staining was greater under 30-Low and 50-Low conditions than under control as well as 30-High and 50-High conditions on days 7 and 14. Vibration acceleration under 30-L condition upregulated chondrogenic gene expressions of SOX-9, aggrecan, Col-II, and Col-X. Low-amplitude vibration acceleration can promote endochondral ossification in the fracture healing in vivo and chondrogenic differentiation in vitro.