Linear flow-velocity gradient chromatography-An efficient method for increasing the process efficiency of batch and continuous capture chromatography of proteins.

A new method was proposed for increasing the capture chromatography process efficiency, linear flow-velocity gradient (LFG). The method uses a linear decreasing flow-velocity gradient with time during the sample loading. The initial flow velocity, the final flow velocity and the gradient time are the parameters to be tuned. We have developed a method for determining these parameters by using the total column capacity and the total loaded amount as a function of time. The capacity can be calculated by using the relationships between dynamic binding capacity (DBC) and residence time. By leveraging the capacity, loading amount, and the required conditions, the optimum LFG can be designed. The method was verified by ion-exchange and protein A chromatography of monoclonal antibodies (mAbs). A two-fold increase in the productivity during the sample loading was possible by LFG compared with the constant flow-velocity (CF) operation. LFG was also applied to a 4-column continuous process. The simulation showed that the cost of resin per unit amount of processed mAbs can be reduced by 13% while 1.4 times enhancement in productivity was preserved after optimization by LFG compared to CF. The process efficiency improvement is more pronounced when the isotherm is highly favorable and the loading volume is large.

Physeal Bar Resection Under Guidance With a Navigation System and Endoscopy for Correction of Distal Radial Deformities After Partial Growth Plate Arrest.

Partial growth plate arrest caused by trauma may lead to severe deformity and dysfunction. The Langenskiöld method is a surgical technique that involves resection of the physeal bar causing partial growth plate arrest. However, it is a technically demanding procedure. We used the Langenskiöld method under guidance with a navigation system and endoscopy and obtained good results in 2 cases. We consider that use of these tools can be a helpful adjunct to the carrying out this procedure.

Molecular alterations associated with osteosarcoma development.

Osteosarcoma is the most frequent malignant primary bone tumor characterized by a high potency to form lung metastases which is the main cause of death. Unfortunately, the conventional chemotherapy is not fully effective on osteosarcoma metastases. The progression of a primary tumor to metastasis requires multiple processes, which are neovascularization, proliferation, invasion, survival in the bloodstream, apoptosis resistance, arrest at a distant organ, and outgrowth in secondary sites. Consequently, recent studies have revealed new insights into the molecular mechanisms of metastasis development. The understanding of the mechanism of molecular alterations can provide the identification of novel therapeutic targets and/or prognostic markers for osteosarcoma treatment to improve the clinical outcome.

Relation of low-intensity pulsed ultrasound to the cell density of scaffold-free cartilage in a high-density static semi-open culture system.

BACKGROUND: A scaffold-free cartilage construct, analogous to those found during embryonic precartilage condensation, has received much attention as a novel modality for tissue-engineered cartilage. In the present study, we developed an uncomplicated culture system by which scaffold-free cartilage-like tissues are produced using cell-cell interactions. With this system, we attempted to prevent dedifferentiation and reverse the phenotypic modulations by adjusting the cell density. We investigated whether low-intensity pulsed ultrasound (LIPUS) enhances matrix synthesis of the scaffold-free cartilage construct. METHODS: Rat articular chondrocytes multiplied in monolayers were seeded onto the synthetic porous membrane at stepwise cell densities (i.e., 1.0, 2.0, and 4.0 × 10(7) cells/cm(2)) to allow formation of a scaffold-free cartilage construct via cell-cell interaction. The cartilage constructs were then stimulated by LIPUS for 20 min/day. To investigate the effect of LIPUS stimulation on matrix synthesis, expression of mRNA for cartilage matrix molecules was quantified by a real-time reverse transcription-polymerase chain reaction. Synthesis of type II collagen, type I collagen, and proteoglycan was also assessed histologically. RESULTS: Only the chondrocytes cultured at high cell densities in the 2.0 × 10(7)cells/cm(2) group became concentrated and formed a plate-like construct similar to native articular cartilage by macroscopic and histological assessments. Statistical analysis on the matrix gene expression demonstrated that the levels of type II collagen and aggrecan mRNA of the 2.0 × 10(7)cells/cm(2) group were significantly higher than with the other two cell-density groups. Interestingly, the LIPUS application led to a statistically significant enhancement of aggrecan gene expression only in the 2.0 × 10(7) cells/cm(2) group. CONCLUSIONS: The current study presents a semi-open static culture system that facilitates production of the scaffold-free constructs from monolayer-cultured chondrocytes. It suggests that the LIPUS application enhances matrix production in the construct, and its combination with the scaffold-free construct might become a feasible tool for production of implantable constructs of better quality.

Treatment of spontaneous anterior interosseous nerve palsy.

OBJECTIVE: The purpose of this study was to clarify the clinical outcomes of spontaneous anterior interosseous nerve palsy (AINP) treated nonsurgically or surgically. METHODS: The authors retrospectively evaluated the clinical course of 27 patients affected with AINP, treated nonsurgically or surgically. Thirteen patients underwent surgical treatment (interfascicular neurolysis), and 14 patients underwent conservative nonsurgical treatment. The mean patient age at the onset of symptoms was 49 years (range 17-77 years). The mean follow-up duration from onset to the latest follow-up examination was 23 months (range 12-38 months). RRESULTS: In 12 of 14 patients receiving conservative treatment, signs of recovery from the palsy were obtained within 6 months. These patients showed a recovery of manual muscle test (MMT) grade ≥ 3. In contrast, 2 patients who took more than 12 months from symptom onset to initial recovery showed poor recovery (MMT grade ≤ 2). Surgical treatment was performed in 13 patients because of no sign of recovery from palsy. The mean period from symptom onset to the operation was 8.4 months (range 6-14 months). Ten of 13 patients who underwent surgical treatment within 8 months after symptom onset showed good recovery, with MMT grade ≥ 4. However, 3 patients who underwent surgical treatment more than 12 months after onset showed recovery with MMT grade ≤ 3. CONCLUSIONS: Conservative treatment for AINP may be continued when patients show signs of recovery within 6 months after symptom onset. In contrast, surgical treatment may be performed within 8 months from the onset of symptoms when the patients show no recovery signs for 6 months. ABBREVIATIONS: AIN = anterior interosseous nerve; AINP = anterior interosseous nerve palsy; FDP1 = flexor digitorum profundus of the index finger; FPL = flexor pollicis longus; MMT = manual muscle test; NSG = nonsurgical treatment group; SG = surgical treatment group.

Effect of dynamic compressive loading and its combination with a growth factor on the chondrocytic phenotype of 3-dimensional scaffold-embedded chondrocytes.

BACKGROUND AND PURPOSE: Three-dimensionally (3D-) embedded chondrocytes have been suggested to maintain the chondrocytic phenotype. Furthermore, mechanical stress and growth factors have been found to be capable of enhancing cell proliferation and ECM synthesis. We investigated the effect of mechanical loading and growth factors on reactivation of the 3D-embedded chondrocytes. METHODS: Freshly isolated chondrocytes from rat articular cartilage were grown in monolayer cultures and then in collagen gel. Real-time RT-PCR and histological analysis for aggrecan and type II and type I collagen was performed to evaluate their chondrocytic activity. Then, the 3D-embedded chondrocytes were cultured under either mechanical loading alone or in combination with growth factor. The dynamic compression (5% compression, 0.33 Hz) was loaded for 4 durations: 0, 10, 60, and 120 min/day. The growth factor administered was either basic fibroblast growth factor (bFGF) or bone morphogenetic protein-2 (BMP-2). RESULTS: Mechanical loading statistically significantly reactivated the aggrecan and type II collagen expression with loading of 60 min/day as compared to the other durations. The presence of BMP-2 and bFGF clearly enhanced the aggrecan and type II collagen expression of 3D-embedded chondrocytes. Unlike previous reports using monolayer chondrocytes, however, BMP-2 or bFGF did not augment the chondrocytic phenotype when applied together with mechanical loading. INTERPRETATION: Dynamic compression effectively reactivated the dedifferentiated chondrocytes in 3D culture. However, the growth factors did not play any synergistic role when applied with dynamic compressive loading, suggesting that growth factors should be administered at different time points during regeneration of the transplantation-ready cartilage.

Conditioned media from mouse osteosarcoma cells promote MC3T3-E1 cell proliferation using JAKs and PI3-K/Akt signal crosstalk.

The maintenance of bone mass requires a strict balance between bone formation by osteoblasts and bone resorption by osteoclasts. In tumoral bone environment, tumor cells frequently disturb this balance by interaction with bone cells to create a favorable site for tumor growth, and promote pathological bone changes. Thus, elucidation of the mechanisms underlying interaction between tumor cells and bone cells might eventually lead to a more rational strategy for therapeutic intervention for bone tumors and better understanding of bone biology. In the present study, the effects of mouse osteosarcoma cells on mouse preosteoblastic cells were determined by assessment of cell viability, osteoblastic differentiation and signal transduction pathways. MOS-J/POS-1 conditioned media (CM) significantly induced MC3T3-E1 cell proliferation in a dose-dependent manner and reduced both alkaline phosphatase activity and mineralized nodule formation. Piceatannol, AG490, LY294002 and rapamycin significantly abrogated this up-regulated cell proliferation; however, UO126 and STAT3 inhibitor peptide did not affect this up-regulated cell proliferation. MOS-J/POS-1 CM activated ERK 1/2, STAT3 and Akt signal transduction pathways; however, pro-proliferating signal induced by MOS-J/POS-1 CM was transmitted via Akt not ERK 1/2 and STAT3 pathways. Furthermore, Western blot analyses clearly revealed novel signal crosstalk between JAKs and PI3-K/Akt in osteoblastic cells. The specific factor(s) involved in MOS-J/POS-1 CM-induced MC3T3-E1 cell proliferation that use JAKs/PI3-K/Akt/mTOR pathway remain(s) to be determined. Determination of the specific factor(s) responsible for JAKs and PI3-K/Akt signal crosstalk that results in up-regulated preosteoblast proliferation will offer new insight into the pathology of osteosarcoma as well as other bone-related diseases.

RANKL/RANK/OPG: key therapeutic target in bone oncology.

Cancer is one of the major leading causes of death all over the world. Primary and secondary bone tumors can significantly deteriorate the quality of life (QOL) and the activity of daily living (ADL) of the patients. These unwelcome diseases become a social and economic burden seriously. Thus, more effective therapies for both primary and secondary bone tumors are actually required. Bone homeostasis depends on the strictly balanced activities between bone formation by osteoblasts and bone resorption by osteoclasts. Imbalance of bone formation and resorption results in various bone diseases. Both primary and secondary bone tumors develop in the unique environment bone, it is therefore necessary to understand bone cell biology in tumoral bone environment. Recent findings strongly revealed the significant involvement of the receptor activator of nuclear factor kappaB ligand (RANKL)/RANK/osteoprotegerin (OPG) triad, the key regulators of bone remodeling in bone oncology. Indeed, RANKL/RANK blocking successfully prevented the development of bone metastases. Furthermore, some cancer cells express RANK which is involved in tumor cell migration. Thus, the regulation of this triad will be a rational, encouraged therapeutic hot spot in bone oncology. In this review, we summarize the accumulating knowledge of the RANKL/RANK/OPG triad and discuss about its therapeutic capability in primary and secondary bone tumors.

The Intrinsic and Extrinsic Implications of RANKL/RANK Signaling in Osteosarcoma: From Tumor Initiation to Lung Metastases.

Background: Osteosarcoma is the most frequent form of malignant pediatric bone tumor. Despite the current therapeutic arsenal, patient life-expectancy remains low if metastases are detected at the time of diagnosis, justifying research into better knowledge at all stages of osteosarcoma ontogenesis and identification of new therapeutic targets. Receptor Activator of Nuclear factor κB (RANK)expression has been reported in osteosarcoma cells, raising the question of Receptor Activator of Nuclear factor κB Ligand (RANKL)/RANK signaling implications in these tumor cells (intrinsic), in addition to previously reported implications through osteoclast activation in the tumor microenvironment (extrinsic). Methods: Based on in vitro and in vivo experimentations using human and mouse osteosarcoma cell lines, the consequences on the main cellular processes of RANK expression in osteosarcoma cells were analyzed. Results: The results revealed that RANK expression had no impact on cell proliferation and tumor growth, but stimulated cellular differentiation and, in an immune-compromised environment, increased the number of lung metastases. The analysis of RANKL, RANK and osteoprotegerin (OPG) expressions in biopsies of a cohort of patients revealed that while RANK expression in osteosarcoma cells was not significantly different between patients with or without metastases at the time of diagnosis, the OPG/RANK ratio decreased significantly. Conclusion: Altogether, these results are in favor of RANKL-RANK signaling inhibition as an adjuvant for the treatment of osteosarcoma.

Near-infrared spectroscopic study of the physical and mechanical properties of wood with meso- and micro-scale anatomical observation.

Estimation of the density along with the tensile strength of wood within both the elastic and plastic deformation ranges, represented as modulus of elasticity (MOE) and ultimate tensile stress (UTS), respectively, were performed using near-infrared (NIR) spectroscopy. A partial least squares (PLS) analysis was applied to the measurements of density, MOE, and UTS, and resulted in a high accuracy of prediction, independent of wood species. The correlation coefficient between the NIR spectra and criterion variables, and the regression vector resulting from the PLS analysis, suggested that the characteristic absorption bands were strongly related to the predictability of each property. In the case of softwood, absorption bands due to intra-molecular hydrogen-bonded OH groups in the crystalline regions of cellulose, which are oriented preferentially in a direction parallel to the cellulose chain, might strongly affect the tensile strength of softwood. Hardwoods have much more complex and variable structures than softwoods; therefore, it was supposed that the key factor governing the tensile strength in hardwood would be the interaction between the three principal constituents (i. e., cellulose, hemicellulose, and lignin) of wood.

Extracellular and intracellular mechanisms of mechanotransduction in three-dimensionally embedded rat chondrocytes.

PURPOSE: Articular cartilage homeostasis involves modulation of chondrocyte matrix synthesis in response to mechanical stress (MS). We studied extracellular and intracellular mechanotransduction pathways mediating this response. METHODS: We first confirmed rapid up-regulation of the putative chondro-protective cytokine, interleukin (IL)-4, as an immediate response to MS. We then studied the role of IL-4 by investigating responses to exogenous IL-4 or a specific IL-4 inhibitor, combined with MS. Next we investigated the intracellular second messengers. Since chondrocyte phenotype alters according to the extracellular environment, we characterized the response to mechanotransduction in 3-dimensionally embedded chondrocytes. RESULTS: Expression of aggrecan and type II collagen was significantly up-regulated by exogenous IL-4 whereas MS-induced matrix synthesis was inhibited by an IL-4 blocker. Further, MS-induced matrix synthesis was completely blocked by a p38 MAPK inhibitor, while it was only partially blocked by inhibitors of other putative second messengers. CONCLUSION: IL-4 mediates an extracellular pathway of mechanotransduction, perhaps via an autocrine/paracrine loop, while p38 mediates an intracellular pathway prevalent only in a 3-dimensional environment.

Novel RANK antagonists for the treatment of bone-resorptive disease: theoretical predictions and experimental validation.

Receptor activator of nuclear factor-κB (RANK) and RANK ligand (RANKL) play a pivotal role in bone metabolism, and selective targeting of RANK signaling has become a promising therapeutic strategy in the management of resorptive bone diseases. Existing antibody-based therapies and novel inhibitors currently in development were designed to target the ligand, rather than the membrane receptor expressed on osteoclast precursors. We describe here an alternative approach to designing small peptides able to specifically bind to the hinge region of membrane RANK responsible for the conformational change upon RANKL association. A nonapeptide generated by this method was validated for its biological activity in vitro and in vivo and served as a lead compound for the generation of a series of peptide RANK antagonists derived from the original sequence. Our study presents a structure- and knowledge-based strategy for the design of novel effective and affordable small peptide inhibitors specifically targeting the receptor RANK and opens a new therapeutic opportunity for the treatment of resorptive bone disease.

Current therapeutic strategies and novel approaches in osteosarcoma.

Osteosarcoma is the most frequent malignant primary bone tumor and a main cause of cancer-related death in children and adolescents. Although long-term survival in localized osteosarcoma has improved to about 60% during the 1960s and 1970s, long-term survival in both localized and metastatic osteosarcoma has stagnated in the past several decades. Thus, current conventional therapy consists of multi-agent chemotherapy, surgery and radiation, which is not fully adequate for osteosarcoma treatment. Innovative drugs and approaches are needed to further improve outcome in osteosarcoma patients. This review describes the current management of osteosarcoma as well as potential new therapies.

Mifamurtide for the treatment of nonmetastatic osteosarcoma.

INTRODUCTION: The standard treatment for osteosarcoma requires both macroscopic surgical wide resection and postoperative multi-drug chemotherapy in neoadjuvant and adjuvant settings. However, the 5-year event-free survival has remained at a plateau of 60-70% of patients with nonmetastatic osteosarcoma for more than 30 years. AREAS COVERED: Mifamurtide (liposomal muramyl tripeptide phosphatidylethanolamine; L-MTP-PE) is a new agent. L-MTP-PE is a nonspecific immunomodulator, which is a synthetic analog of a component of bacterial cell walls. L-MTP-PE activates macrophages and monocytes as a potent activator of immune response in addition to standard chemotherapy. It also improves the overall survival from 70 to 78% and results in a one-third reduction in the risk of death from osteosarcoma. This review summarizes the most recent findings about L-MTP-PE and its therapeutic application for nonmetastatic osteosarcoma. EXPERT OPINION: Recently, L-MTP-PE has been approved in Europe for the treatment of nonmetastatic osteosarcoma with chemotherapy. L-MTP-PE in combination with traditional treatment is expected to go mainstream and to be beneficial for patients with osteosarcoma. Information about potential benefit regarding mifamurtide use in the neoadjuvant setting (i.e., before surgery) and/or usefulness of L-MTP-PE in metastatic in relapsed and metastatic osteosarcoma requires analysis of expanded access and/or future clinical trials of L-MTP-PE in high-burden and low-burden situations.

Chondroitin sulfate for the treatment of hip and knee osteoarthritis: current status and future trends.

AIMS: Osteoarthritis (OA) is a common joint disorder and a major socio-economic burden. Chondroitin sulfate (CS), which has chondroprotective properties, is a promising candidate for the therapeutic treatment of OA. Here, we summarize current knowledge as well as future trends of CS for the treatment of hip and knee OA. MAIN METHODS: We retrospectively reviewed pharmacokinetics, pharmacodynamics, clinical efficacy, safety and tolerability of CS for the treatment of OA. KEY FINDINGS: The safety and tolerability of CS are confirmed. CS is effective, at least in part, for the treatment of OA, and its therapeutic benefits occur through three main mechanisms: 1) stimulation of extracellular matrix production by chondrocytes; 2) suppression of inflammatory mediators; and 3) inhibition of cartilage degeneration. SIGNIFICANCE: CS is a safe and tolerable therapeutic agent for the management of OA. Its effects include benefits that are not achieved by current medicines and include chondroprotection and the prevention of joint space narrowing. Such positive effects of CS represent a breakthrough in the treatment of hip and knee OA.

Liposomal muramyl tripeptide phosphatidyl ethanolamine: a safe and effective agent against osteosarcoma pulmonary metastases.

Osteosarcoma is the most common form of primary malignant bone tumor. The use of chemotherapy drugs with many side effects, including high-dose methotrexate, doxorubicin, cisplatin and ifosfamide, has greatly improved osteosarcoma survival compared with surgery alone. However, for 20 years, overall survival remained at a plateau of 60-70% in nonmetastatic disease and 20-30% in metastatic osteosarcoma owing to lung metastases. Liposomal muramyl tripeptide phosphatidyl ethanolamine (L-MTP-PE) is a new agent that improves overall osteosarcoma survival (chemotherapy without L-MTP-PE 70% versus with L-MTP-PE 78%; p = 0.03). L-MTP-PE offers additional benefit for osteosarcoma treatment in combination with chemotherapy, particularly ifosfamide-containing regimens. Clinical experience indicates that side effects such as fever are temporary and controlled or prevented with ibuprofen and/or acetoaminophen premedication; severe side effects are rare. Although surgery will remain the main approach for osteosarcoma treatment of lung metastases, L-MTP-PE combined with other modalities, including chemotherapy, appears to be of benefit in these patients as well.

Exogenous collagen-enhanced recruitment of mesenchymal stem cells during rabbit articular cartilage repair.

BACKGROUND: Despite the well-known effect of type-I collagen in promoting cartilage repair, the mechanism still remains unknown. In this study we investigated this mechanism using a rabbit model of cartilage defects. ANIMALS AND METHODS: 5-mm-diameter full-thickness defects were created on both patellar grooves of 53 Japanese white rabbits (approximately 13 weeks old). The left defect was filled with collagen gel and the right defect was left empty. The rabbits were killed and examined morphometrically until the twenty-fourth postoperative week, by (1) evaluation of matrix production, (2) enumeration of the total number of cells engaged in cartilage repair, (3) enumeration of the proliferating cells, (4) localization of mesenchymal stem cells, and (v) localization of apoptotic cells. RESULTS: We found that type-I collagen enhances cell recruitment, and thereby increases the number of proliferating cells. A considerable proportion of the proliferating cells were identified as bone marrow-derived mesenchymal stem cells. However, type-I collagen does not prevent the chondrocyte precursors from undergoing apoptotic disengagement from the chondrogenic lineage. INTERPRETATION: Type-I collagen promotes cartilage repair by enhancing recruitment of bone marrow-derived mesenchymal stem cells. Additional use of agent(s) that sustain mesenchymal stem cells along the chondrogenic path of differentiation may constitute an appropriate environment for cartilage repair.