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Background: Chondrocyte migration in native cartilage is limited and has been implicated as one of the reasons for the poor integration of native implants. Through use of an in vitro integration model, it has previously been shown that cells from bioengineered cartilage can migrate into the native host cartilage during integration. Platelet-rich plasma (PRP) treatment further enhanced integration of bioengineered cartilage to native cartilage in vitro. However, it is not known how PRP treatment of the bioengineered construct promotes this. Hypothesis: PRP supports cell migration from bioengineered cartilage and these migratory cells have the ability to accumulate cartilage-like matrix. Study Design: Controlled laboratory study. Methods: Osteochondral-like constructs were generated by culturing primary bovine chondrocytes on the top surface of a porous bone substitute biomaterial composed of calcium polyphosphate. After 1 week in culture, the constructs were submerged in PRP and placed adjacent, but 2 mm distant, to a native bovine osteochondral plug in a co-culture model for 2 weeks. Cell migration was monitored using phase-contrast imaging. Cell phenotype was determined by evaluating the gene expression of matrix metalloprotease 13 (MMP-13), Ki67, and cartilage matrix molecules using quantitative polymerase chain reaction. When tissue formed, it was assessed by histology, immunohistochemistry, and quantification of matrix content. Results: PRP treatment resulted in the formation of a fiber network connecting the bioengineered cartilage and native osteochondral plug. Cells from both the bioengineered cartilage and the native osteochondral tissue migrated onto the PRP fibers and formed a tissue bridge after 2 weeks of culture. Migratory cells on the tissue bridge expressed higher levels of collagen types II and I ( COL2, COL1), Ki67 and MMP-13 mRNA compared with nonmigratory cells in the bioengineered cartilage. Ki67 and MMP-13–positive cells were found on the edges of the tissue bridge. The tissue bridge accumulated COL1 and COL2 and aggrecan and contained comparable collagen and glycosaminoglycan content to the bioengineered cartilage matrix. The tissue bridge did not reliably develop in the absence of cells from the native osteochondral plug. Conclusion: Bioengineered cartilage formed by bovine chondrocytes contains cells that can migrate on PRP fibers and form cartilaginous tissue. Clinical Relevance: Migratory cells from bioengineered cartilage may promote cartilage integration. Further studies are required to determine the role of migratory cells in integration in vivo.

A tissue-engineered biological disk replacement has been proposed as a promising approach for the treatment of degenerative disk disease. A perfusion bioreactor would be a logical consideration to facilitate this scale-up as such reactors have been shown to improve nutrient delivery and provide beneficial mechanical forces that support the cultivation of large three-dimensional constructs. It was hypothesized that perfusion culture of tissue-engineered intervertebral disk (IVD) tissues would be capable of generating outer annulus fibrosus (oAF) and nucleus pulposus (NP) tissues comparable with established spinner reactor or static cultures, respectively, without compromising cellular viability, nutrient delivery, and tissue formation. In this study, the perfusion grown oAF and NP tissues did not show a significant difference in extracellular matrix (ECM) quantity or cellular phenotype when compared with their control conditions. In addition, they maintained cellular viability at the center core of the tissues and received enhanced diffusion of medium throughout the tissue when compared with static conditions. This study lays the groundwork for future studies to grow an entire IVD tissue to a physiologically relevant size.

Background The COVID-19 pandemic highlighted the need for evidence-based approaches to decontamination and reuse of N95 filtering facepiece respirators (FFRs). We sought to determine whether vapourized hydrogen peroxide (VHP) reduced SARS-CoV-2 bioburden on FFRs without compromising filtration efficiency. We also investigated coronavirus HCoV-229E as a surrogate for decontamination validation testing. Methods N95 FFRs were laced with SARS-CoV-2 or HCoV-229E and treated with VHP in a hospital reprocessing facility. After sterilization, viral burden was determined using viral outgrowth in a titration assay, and filtration efficiency of FFRs was tested against ATSM F2299 and NIOSH TEB-STP-APR-0059. Results Viable SARS-CoV-2 virus was not detected after VHP treatment. One replicate of the HCoV-229E laced FFRs yielded virus after processing. Unexpired N95 FFRs retained full filtration efficiency after VHP processing. Expired FFRs failed to meet design-specified filtration efficiency and therefore are unsuitable for reprocessing. Discussion In-hospital VHP is an effective decontaminant for SARS-CoV-2 on FFRs. Further, filtration efficiency of unexpired respirators is not affected by this decontamination process. Conclusions VHP is effective in inactivating SARS-CoV-2 on FFRs without compromising filtration efficiency. HCoV-229E is a suitable surrogate for SARS-CoV-2 for disinfection studies.

Transforming growth factor beta (TGFβ) signaling is required for in vitro chondrogenesis. In animal models of osteoarthritis (OA), TGFβ receptor alterations are detected in chondrocytes in severe O...

Additive manufacturing (AM) is a promising method for fabricating customized and anatomically correct surgical implants. Advancement in design and the AM of joint replacement implants has mainly targeted implants for large joint replacement while progress in small joint replacements has been limited. This study describes a method for fabrication of porous biodegradable ceramic “templates” for use in preparing small joint implants such as the proximal interphalangeal (PIP) finger joint by combining powder-bed binder jetting (PBBJ) AM plus post-process sintering. A proposed PIP template design was selected using finite element analysis (FEA) to predict a suitable design for reliable fixation of the implant to host bone through bone ingrowth while providing necessary mechanical properties to avoid implant fracture during anticipated in vivo functional loading. Calcium polyphosphate (CPP) ceramic powder was selected for fabrication of the porous biodegradable ceramic templates intended to degrade in vivo in time to be replaced by bone while retaining a mature articular cartilage layer anchored to the intended joint bearing surface thereby achieving whole joint regeneration. For the AM build-up, density and mechanical strength of porous AM-made samples were optimized by varying the binder saturation level in the PBBJ process. Using the preferred design and processing conditions, PIP joint templates were made and subjected to preliminary fatigue testing to demonstrate survival under anticipated in vivo functional loading conditions. The study has demonstrated the potential of this novel approach for the preparation of implants for use in small digit joint regeneration.

Inorganic polyphosphates (polyP) are polymers composed of phosphate residues linked by energy-rich phosphoanhydride bonds. As polyP can bind calcium, the hypothesis of this study is that polyP enters chondrocytes and exerts its anabolic effect by calcium influx through calcium channels. PolyP treatment of cartilage tissue formed in 3D culture by bovine chondrocytes showed an increase in proteoglycan accumulation but only when calcium was also present at a concentration of 1.5 mM. This anabolic effect could be prevented by treatment with either ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid or the calcium channel inhibitors gadolinium and nifedipine. Calcium and polyP cotreatment of chondrocytes in monolayer culture resulted in calcium oscillations that were polyP chain length specific and were inhibited by gadolinium and nifedipine. The calcium influx resulted in increased gene expression of sox9, collagen type II, and aggrecan which was prevented by treatment with either calphostin, an inhibitor of protein kinase C, and W7, an inhibitor of calmodulin; suggesting activation of the protein kinase C-calmodulin pathway. Tracing studies using 4',6-diamidino-2-phenylindole, Mitotracker Red, and/or Fura-AM staining showed that polyP was detected in the nucleus, mitochondria, and intracellular vacuoles suggesting that polyP may also enter the cell. PolyP colocalizes with calcium in mitochondria. This study demonstrates that polyP requires the influx of calcium to regulate chondrocyte matrix production, likely via activating calcium signaling. These findings identify the mechanism regulating the anabolic effect of polyP in chondrocytes which will help in its clinical translation into a therapeutic agent for cartilage repair.

Background: Autologous chondrocyte implantation, which uses passaged chondrocytes, commonly leads to the formation of fibrocartilage. When chondrocytes are passaged to increase cell numbers, they lose their phenotype and ability to form hyaline cartilage. The use of transforming growth factor β (TGFβ) to redifferentiate passaged chondrocytes has been validated in vitro; however, it is unknown if redifferentiated chondrocytes will enhance defect repair when implanted in vivo. Furthermore, fibrin gel is used in orthopaedic surgery as a fixative and scaffold and could be an appropriate carrier to enhance retention of cells in the repair site. Purpose: To investigate if passaged redifferentiated chondrocytes in fibrin gel have the ability to form cartilage tissue and if these redifferentiated cells will enhance the formation of hyaline cartilage in vivo when implanted into critical-size osteochondral defects. Study Design: Controlled laboratory study. Methods: Rabbit and human chondrocytes were serially passaged twice in monolayer culture. Twice-passaged cells were used directly (dedifferentiated) or redifferentiated in high-density culture with TGFβ3. Dedifferentiated or redifferentiated cells were mixed with fibrin gel to form fibrin clots, which were cultured in vitro to assess the use of fibrin gel as a scaffold or implanted in vivo in a critical-size osteochondral defect in New Zealand White rabbit knee joints. Rabbits were sacrificed 6 weeks after implantation, and tissues were assessed histologically and by immunohistochemistry. Results: Redifferentiation of passaged chondrocytes by means of 3-dimensional culture in the presence of TGFβ3 improved the formation of cartilaginous tissues in vitro, and culture in fibrin gel did not affect the cell phenotype. Implantation of dedifferentiated cells in vivo resulted in fibrocartilaginous repair tissues. Redifferentiated chondrocyte implants resulted in granulation tissues containing the hyaline cartilage marker collagen type 2. Conclusion: Redifferentiated chondrocytes will maintain their chondrogenic differentiation in fibrin clots. Implanted redifferentiated chondrocytes show a different reparative response than dedifferentiated chondrocytes and do not appear to enhance repair at an early time point. Another study of longer duration is required to assess tissue maturation over time. Clinical Relevance: Redifferentiation of passaged chondrocytes with TGFβ3 before implantation does not improve defect repair in the first 6 weeks.

Intervertebral disc degeneration is an irreversible process associated with accumulation of senescent nucleus pulposus (NP) cells. This study investigates the hypothesis that Tumor necrosis factor-α (TNF-α)-treated senescent NP cells propagate senescence of neighboring healthy cells via a paracrine effect that involves p-Stat3 signaling and the cytokine interleukin-6 (IL-6). NP cells isolated from bovine caudal intervertebral disc (IVD) were treated with TNF-α to induce senescence which was confirmed by demonstrating upregulation of senescence-associated β-galactosidase and p16. This was correlated with downregulation of NP-associated markers, Aggrecan, Col2A1, and Sox9. Direct contact and non-contact co-culture of healthy and senescent cells showed that TNF-α-treated cells increased the senescence in healthy cells via a paracrine effect. The senescent cells have a secretory phenotype as indicated by increased gene and protein levels of IL-6. Phosphorylated Signal Transducer and Activator of Transcription 3 (pStat3) levels were also high in treated cells and appeared to upregulate IL-6 as inhibition of Stat3 phosphorylation by StatticV downregulated IL-6 mRNA expression in cells and protein levels in the culture media. All trans retinoic acid, an IL-6 inhibitor, also decreased the secretion of IL-6 and reduced the paracrine effect of senescent cells on healthy cells. Decreased pStat3 levels and inhibition of IL-6 secretion did not fully restore NP gene expression of Col2A1 but importantly, appeared to cause senescent cells to undergo apoptosis and cell death. This study demonstrated the paracrine effect of senescent NP cells which involves Stat3 and IL-6 and may explain why senescent NP cells accumulate in IVD with age. The role of pSTAT3 and IL-6 in mediating NP senescence requires further study as it may be a novel strategy for modulating the senescent-inducing effects of TNF-α.

Introduction Ubiquitously found in all life forms, inorganic polyphosphates (polyP) are linear polymers of repeated orthophosphate units. Present in intervertebral disc tissue, polyP was previously shown to increase extracellular matrix production in nucleus pulposus (NP) cells. However, the effects of polyP on human annulus fibrosus (hAF) cell metabolism is not known. Methods and Results Here, hAF cells cultured in the presence of 0.5 to 1 mM polyP, chain length 22 (polyP‐22), showed an increase in glycosaminoglycan content, proteoglycan and collagen synthesis, and aggrecan and collagen type 1 gene expression. Gene expression level of matrix metalloproteinases 1 was reduced while matrix metalloproteinases 3 level was increased in hAF cells treated with 1 mM polyP. Adenosine triphosphate (ATP) synthesis was also significantly increased in hAF cell culture 72 hours after the exposure to 1 mM polyP‐22. Conclusions PolyP thus has both anabolic and bioenergetic effects in AF cells, similar to that observed in NP cells. Together, these results suggest polyP as a potential energy source and a metabolic regulator of disc cells., The current study investigates the effect of inorganic polyphosphates (polyPs) on human annulus fibrosus (AF) cell biology. This is a first study of its kind to investigate the effect of polyPs on human AF cells. polyPs, rich in polyanhydride bonds and known donors of phosphate molecule for ATP synthesis, showed increase in matrix production and ATP generation in human AF cells and overall promoted anabolism.

Transforming growth factor beta (TGFβ) signaling is required for

Porous calcium polyphosphate (CPP) is being investigated for use as a biodegradable bone substitute and for repair of osteochondral defects. The necessary requirements for these applications, particularly in load-bearing sites, include sufficient strength to withstand functional forces prior to bone ingrowth and substitution of the initial porous CPP template with new bone and cartilage (for osteochondral implants) in a timely and efficacious manner. The present study explored the effects of Na+ doping and processing to form porous structures of both higher strength and faster degradation than previously reported for ‘pure’ (non-doped) CPP structures of similar geometry. Compressive and tensile strengths were determined before and after 30-day in vitro degradation (PBS, pH 7.1 at 37 °C) and degradation rates assessed. Scanning electron microscopy (SEM), x-ray diffraction (XRD) and solid state nuclear magnetic resonance (31P SS NMR) were used to evaluate ‘pure’ and Na-doped CPP samples before and after degradation. The results indicated that the different processing protocols required to prepare samples of similar volume % porosity (a 2-step procedure with a Step-1 sintering temperatures equal to 575 °C being used with the Na-doped samples versus a 585 °C Step-1 treatment for ‘pure’ CPP) resulted in an approximate 1.5- to 2-fold increase in strength (tensile & compressive respectively) and 2-fold increase in degradation rate of Na-doped CPP compared with ‘pure’ CPP. This difference was attributed to the different Step-1 sintering temperatures used for sample processing.

Background. Mesenchymal stromal cells (MSCs) promote wound healing, including after radiotherapy (RT) and surgery. The use of MSCs in regenerative medicine in the context of malignancy, such as to enhance wound healing post-RT/surgery in patients with soft tissue sarcomas (STSs), requires safety validation. The aim of this study was to determine the effects of human MSCs on STS growth in vitro and local recurrence and metastasis in vivo. Methods. Human primary STS and HT-1080 fibrosarcoma lines were transduced to express luciferase/eGFP (enhanced green fluorescent protein). Sarcoma cells were co-cultured or co-injected with bone marrow–derived MSCs for growth studies. Xenograft tumor models were established with STS lines in NOD/SCID/γcnull mice. To emulate a clinical scenario, subcutaneous tumors were treated with RT/surgery prior to MSC injection into the tumor bed. Local and distant tumor recurrence was studied using histology and bioluminescence imaging. Results. MSCs did not promote STS proliferation upon co-culture in vitro, which was consistent among MSCs from different donors. Co-injection of MSCs with sarcoma cells in mice exhibited no significant tumor-stimulating effect, compared with control mice injected with sarcoma cells alone. MSC administration after RT/surgery had no effect on local recurrence or metastasis of STS. Discussion. These studies are important for the establishment of a safety profile for MSC administration in patients with STS. Our data suggest that MSCs are safe in STS management after standard of care RT/surgery, which can be further investigated in early-phase clinical trials to also determine the efficacy of MSCs in reducing morbidity and to mitigate wound complications in these patients.

Generating the best possible bioengineered cartilage from passaged chondrocytes requires culture condition optimization. In this study, the use of adherent agarose mold (adAM) cultures to support redifferentiation of passaged twice (P2) chondrocytes and serve as a scalable platform to assess the effect of growth factor combinations on proteoglycan accumulation by cells was examined. By 2 days in adAM culture, bovine P2 cells were partially redifferentiated as demonstrated by regression of actin-based dedifferentiation signalling and fibroblast matrix and contractile gene expression. By day 10, aggrecan and type II collagen gene expression were significantly increased in adAM cultured cells. At day 20, a continuous layer of cartilage tissue was observed. There was no evidence of tissue contraction by P2 cells in adAM cultures. The matrix properties of the resultant tissue as well as proteoglycan 4 (PRG4) secreted by the cells were dependent on the initial cell seeding density. AdAM cultures were scalable and culture within small 3 mm diameter adAM allowed for multi-factorial assessment of growth factors on proteoglycan accumulation by human P2 chondrocytes. Although there was a patient specific response in proteoglycan accumulation to the various cocktail combinations, the cocktail consisting of 2 ng/ml TGFβ1, 10 ng/ml FGF2, and 250 ng/ml FGF18 resulted in a consistent increase in alcian blue tissue staining. Additional studies will be required to identify the optimal conditions to bioengineer articular cartilage tissue for clinical use. However, the results to date suggest that adAM cultures may be suitable to use for high throughput assessment. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2392-2405, 2018.

Osteoarthritis (OA) is a degenerative disease that initially manifests as loss of the superficial zone (SZ) of articular cartilage. SZ chondrocytes (SZC) differ in morphology from other chondrocytes as they are elongated and oriented parallel to the tissue surface. Proteoglycan 4 (PRG4) and tenascin C (TNC) are molecules expressed by SZC, which have been shown to be chondroprotective. Identification of the signalling pathway(s) regulating expression of SZ molecules may lead to a therapeutic target that can be used to delay or prevent the onset of OA. The hypothesis of this study is that expression of SZ molecules are regulated in part, by the CDC42-actin-myocardin-related transcription factor-A (MRTF-A) signaling pathway. SZC from bovine metacarpal-phalangeal joints were isolated and grown in monolayer culture. Each target in the CDC42-actin-MRTF-A pathway was inhibited and the effect on cell shape, actin cytoskeleton status, and expression of PRG4 and TNC were determined. Treatment with the CDC42 inhibitor ML141 decreased PRG4 and TNC expression, and correlated with increased cell circularity and G-/F-actin ratio. PRG4 and TNC expression were differentially regulated by actin depolymerizing agents, latrunculin B and cytochalasin D. Chemical inhibition of MRTF-A resulted in decreased expression of both PRG4 and TNC; however, specific knockdown by small interfering RNA only decreased expression of TNC indicating that TNC, but not PRG4, is regulated by MRTF-A. Although PRG4 and TNC expression are both regulated by CDC42 and actin, it appears to occur through different downstream signaling pathways. Further study is required to elucidate the pathway regulating PRG4. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2421-2430, 2018.

The annulus fibrosus (AF) of the intervertebral disc (IVD) has a zonal distribution of phenotypically distinct cells. The outer AF (OAF) cells produce an extracellular matrix (ECM) rich in type I collagen with little proteoglycans, whereas the ECM of the inner AF (IAF) has abundant type II collagen and proteoglycans. The inhomogeneous distribution of the ECM in the AF may reflect the complex mechanical forces that the IVD experiences. A bioengineered AF tissue should recapitulate both the inner and outer zones in order to have proper functionality. The aim of this study is to generate multi-lamellated OAF and IAF tissues with ECM compositions that resemble their zonal origin using polycarbonate urethane (PU) scaffolds. It was observed that supplementation of the media with insulin-transferrin-selenium (ITS) and proline yielded tissues with good cellularity. However, IAF cells accumulated only type I collagen, similar to OAF cells. Addition of dexamethasone and sodium pyruvate induced the accumulation of IAF tissues rich in type II collagen and aggrecan, without altering the accumulation of type I collagen in OAF tissues. Dexamethasone stimulated mitochondrial membrane potential in both tissues in the presence of sodium pyruvate, suggesting a relationship between the mitochondrial aerobic respiratory state and dexamethasone signalling during the in vitro-tissue formation by OAF and IAF cells. Inhibition of the glucocorticoid receptor blocked the stimulation of mitochondrial membrane potentials and type II collagen accumulation. In summary, biologically distinct multi-lamellated OAF and IAF tissues can be generated, which will facilitate advancement towards the goal of engineering a biological IVD replacement. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1346-1355, 2018.

Integration of in vitro -formed cartilage on a suitable substrate to form tissue-engineered implants for osteochondral defect repair is a considerable challenge. In healthy cartilage, a zone of calcified cartilage (ZCC) acts as an intermediary for mechanical force transfer from soft to hard tissue, as well as an effective interlocking structure to better resist interfacial shear forces. We have developed biphasic constructs that consist of scaffold-free cartilage tissue grown in vitro on, and interdigitated with, porous calcium polyphosphate (CPP) substrates. However, as CPP degrades, it releases inorganic polyphosphates (polyP) that can inhibit local mineralization, thereby preventing the formation of a ZCC at the interface. Thus, we hypothesize that coating CPP substrate with a layer of hydroxyapatite (HA) might prevent or limit this polyP release. To investigate this we tested both inorganic or organic sol-gel processing methods, as a barrier coating on CPP substrate to inhibit polyP release. Both types of coating supported the formation of ZCC in direct contact with the substrate, however the ZCC appeared more continuous in the tissue formed on the organic HA sol gel coated CPP. Tissues formed on coated substrates accumulated comparable quantities of extracellular matrix and mineral, but tissues formed on organic sol-gel (OSG)-coated substrates accumulated less polyP than tissues formed on inorganic sol-gel (ISG)-coated substrates. Constructs formed with OSG-coated CPP substrates had greater interfacial shear strength than those formed with ISG-coated and non-coated substrates. These results suggest that the OSG coating method can modify the location and distribution of ZCC and can be used to improve the mechanical integrity of tissue-engineered constructs formed on porous CPP substrates. Statement of Significance Articular cartilage interfaces with bone through a zone of calcified cartilage. This study describes a method to generate an “osteochondral-like” implant that mimics this organization using isolated deep zone cartilage cells and a sol-gel hydroxyapatite coated bone substitute material composed of calcium polyphosphate (CPP). Developing a layer of calcified cartilage at the interface should contribute to enhancing the success of this “osteochondral-like” construct following implantation to repair cartilage defects.

Current therapies for cartilage repair can be limited by an inability of the repair tissue to integrate with host tissue. Thus there is interest in developing approaches to enhance integration. We have previously shown that platelet-rich plasma (PRP) improves cartilage tissue formation. This raised the question as to whether PRP could promote cartilage integration. Chondrocytes were isolated from cartilage harvested from bovine joints, seeded on a porous bone substitute, and grown in vitro to form an osteochondral-like implant. After 7 days, the biphasic construct was soaked in PRP for 30 minutes prior to implantation into the core of a donut-shaped biphasic explant of native cartilage and bone. Controls were not soaked in PRP. The implant-explant construct was cultured for 2-4 weeks. PRP-soaked bio-engineered implants integrated with host tissue in 73% of samples, whereas controls only integrated in 19% of samples. The integration strength, as determined by a push-out test, was significantly increased in the PRP-soaked implant group (219 ± 35.4 kPa) compared to controls (72.0 ± 28.5 kPa). This correlated with an increase in glycosaminoglycan and collagen accumulation in the region of integration in the PRP-treated implant group, compared to untreated controls. Immunohistochemical studies revealed that the integration zone contained collagen type II and aggrecan. The cells at the zone of integration in the PRP soaked group had a 3.5 fold increase in matrix metalloproteinase-13 gene expression compared to controls. These results suggest that PRP-soaked bio-engineered cartilage implants may be a better approach for cartilage repair due to enhanced integration.

Summary Ideally, disease modeling using patient‐derived induced pluripotent stem cells (iPSCs) enables analysis of disease initiation and progression. This requires any pathological features of the patient cells used for reprogramming to be eliminated during iPSC generation. Hutchinson–Gilford progeria syndrome (HGPS) is a segmental premature aging disorder caused by the accumulation of the truncated form of Lamin A known as Progerin within the nuclear lamina. Cellular hallmarks of HGPS include nuclear blebbing, loss of peripheral heterochromatin, defective epigenetic inheritance, altered gene expression, and senescence. To model HGPS using iPSCs, detailed genome‐wide and structural analysis of the epigenetic landscape is required to assess the initiation and progression of the disease. We generated a library of iPSC lines from fibroblasts of patients with HGPS and controls, including one family trio. HGPS patient‐derived iPSCs are nearly indistinguishable from controls in terms of pluripotency, nuclear membrane integrity, as well as transcriptional and epigenetic profiles, and can differentiate into affected cell lineages recapitulating disease progression, despite the nuclear aberrations, altered gene expression, and epigenetic landscape inherent to the donor fibroblasts. These analyses demonstrate the power of iPSC reprogramming to reset the epigenetic landscape to a revitalized pluripotent state in the face of widespread epigenetic defects, validating their use to model the initiation and progression of disease in affected cell lineages.

Cell-based therapies, defined here as the delivery of cells in vivo to treat disease, have recently gained increasing public attention as a potentially promising approach to restore structure and function to musculoskeletal tissues. Although cell-based therapy has the potential to improve the treatment of disorders of the musculoskeletal system, there is also the possibility of misuse and misrepresentation of the efficacy of such treatments. The medical literature contains anecdotal reports and research studies, along with web-based marketing and patient testimonials supporting cell-based therapy. Both the American Society for Bone and Mineral Research (ASBMR) and the Orthopaedic Research Society (ORS) are committed to ensuring that the potential of cell-based therapies is realized through rigorous, reproducible, and clinically meaningful scientific discovery. The two organizations convened a multidisciplinary and international Task Force composed of physicians, surgeons, and scientists who are recognized experts in the development and use of cell-based therapies. The Task Force was charged with defining the state-of-the art in cell-based therapies and identifying the gaps in knowledge and methodologies that should guide the research agenda. The efforts of this Task Force are designed to provide researchers and clinicians with a better understanding of the current state of the science and research needed to advance the study and use of cell-based therapies for skeletal tissues. The design and implementation of rigorous, thorough protocols will be critical to leveraging these innovative treatments and optimizing clinical and functional patient outcomes. In addition to providing specific recommendations and ethical considerations for preclinical and clinical investigations, this report concludes with an outline to address knowledge gaps in how to determine the cell autonomous and nonautonomous effects of a donor population used for bone regeneration. © 2020 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:485-502, 2020.

Chondral or osteochondral (OC) lesions of the knee are common. They are present in over 60% of knees undergoing arthroscopy. These lesions can be painful, often limiting function in young, active individuals in their productive years. At present, the surgical treatment of articular cartilage defects remains a challenging area with poor long-term results. In order to prevent the need for joint replacement to treat the osteoarthritis (OA) that can develop, there has been great interest in developing biological methods for joint repair. This approach results in tissue that can respond to loading and can remodel as required. This chapter reviews current and future approaches for cartilage repair and summarizes the rationale and clinical studies underlying the use of human cells and tissue engineering for the treatment of chondral and osteochondral lesions.

ENMD-2076, an aurora-A kinase inhibitor with anti-angiogenic properties, has shown activity in solid and hematologic malignancies. We investigated oral ENMD-2076 in an open-label, single-arm phase II study using 275 mg daily on a 28-day cycle in patients with advanced soft-tissue sarcomas (STS) receiving ≤1 line of prior therapy. Primary endpoint was 6-month progression-free survival (PFS) with ≤15% indicating no interest, and ≥40% indicating further interest in ENMD-2076. Secondary/exploratory endpoints included clinical benefit (CBR ≥6-months) and objective response (ORR) rates, PFS, OS, safety, and whole-exome sequencing (WES) for potentially associated biomarkers. Overall, 23/25 (92%) patients receiving ENMD-2076 were efficacy evaluable with median follow-up of 14 months (range 2.2–39.5). Common subtypes were leiomyosarcoma (n = 10), undifferentiated pleomorphic sarcoma (n = 3), angiosarcoma (n = 3), and alveolar soft-part sarcoma (n = 3). The 6-month PFS was 20.8% (95% CI:3.2–38.4) with a CBR of 17% (95% CI:1.55–33.23) and ORR of 9% (95% CI:3.08–20.46). Median PFS was 2.5 months (95% CI:2.20–4.47) and OS was 14.1 months (95% CI:6.07–20.07). The most common high-grade treatment-related adverse event was hypertension (60%). WES identified PTPRB mutations in 3/4 patients (p = 0.018) benefiting from ENMD-2076. Although this study failed to meet its primary endpoint, occasional responses and prolonged stable disease was noted. ENMD-2076 evaluation in PTPRB mutated tumors and/or angiosarcoma is warranted.

The nucleus pulposus (NP) is composed of NP and notochord cell. It is a paucicellular tissue and if it is to be used as a source of cells for tissue engineering the cell number will have to be expanded by cell passaging. The hypothesis of this study is that passaged NP and notochordal cells grown in three-dimensional (3D) culture in the presence of transforming growth factor β (TGFβ) will show enhanced NP tissue formation compared with cells grown in the absence of this growth factor. Bovine NP cells isolated by sequential enzymatic digestion from caudal intervertebral discs were either placed directly in 3D culture (P0) or serially passaged up to passage 3 (P3) prior to placement in 3D culture. Serial cell passage in monolayer culture led to de-differentiation, increased senescence and oxidative stress and decreases in the gene expression of NP and notochordal associated markers and increases in de-differentiation markers. The NP tissue regeneration capacity of cells in 3D culture decreases with passaging as indicated by diminished tissue thickness and total collagen content when compared with tissues formed by P0 cells. Immunohistochemical studies showed that type II collagen accumulation appeared to decrease. TGFβ1 or TGFβ3 treatment enhanced the ability of cells at each passage to form tissue, in part by decreasing cell death. However, neither TGFβ1 nor TGFβ3 were able to restore the notochordal phenotype. Although TGFβ1/3 recovered NP tissue formation by passaged cells, to generate NP in vitro that resembles the native tissue will require identification of conditions facilitating retention of notochordal cell differentiation. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:438-449, 2020.

Objective. Inorganic polyphosphates (polyP) play a multitude of roles in mammalian biology. PolyP research is hindered by the lack of a simple and sensitive quantification method. The aim of this study was to develop a robust method for quantifying the low levels of polyP in mammalian tissue such as cartilage, which is rich in macromolecules that interfere with its determination. Design. Native and in vitro formed tissues were digested with proteinase K to release sequestrated polyP. The tissue digest was loaded on to silica spin columns, followed by elution of bound polyP and various treatments were assessed to minimize non-polyP fluorescence. The eluent was then quantified for polyP content using fluorometry based on DAPI (4′,6-diamidino-2-phenylindole) fluorescence shift occurring with polyP. Results. Proteinase K pretreatment reduced the inhibitory effect of proteins on polyP recovery. The eluent was contaminated with nucleic acids and glycosaminoglycans, which cause extraneous fluorescence signals. These were then effectively eliminated by nucleases treatment and addition of concentrated Tris buffer. PolyP levels were quantified and recovery ratio determined using samples spiked with a known amount of polyP. This silica spin column method was able to recover at least 80% of initially loaded polyP, and detect as little as 10−10 mol. Conclusions. This sensitive, reproducible, easy to do method of quantifying polyP will be a useful tool for investigation of polyP biology in mammalian cells and tissues. Although the protocol was developed for mammalian tissues, this method should be able to quantify polyP in most biological sources, including fluid samples such as blood and serum.

Context.— In osteosarcoma treated with neoadjuvant chemotherapy the extent of tumor necrosis on resection is considered an indicator of treatment response, and this has been shown to correlate with survival in most but not all studies. Objective.— To identify additional histologic variables of prognostic significance in high-grade osteosarcoma. Design.— Slides of pretreatment biopsy and primary postneoadjuvant chemotherapy resections from 165 patients with high-grade osteosarcoma were reviewed. Univariate (Kaplan-Meier) and multivariate (Cox regression) analyses were performed to identify clinical and histomorphologic attributes associated with overall survival. Results.— Univariate analyses confirmed the prognostic significance of metastatic status on presentation, primary tumor size, anatomic site, and histologic subtype. Additionally, the identification of lymphovascular invasion, 10% or more residual viable tumor, and 10 or more mitoses per 10 high-powered fields assessed in posttreatment resections were associated with poor survival, retaining significance in multivariate analyses. Based on results from multivariate analysis, we developed a prognostic index incorporating primary tumor size and site, and significant histologic features assessed on resection (ie, lymphovascular invasion status, mitotic rate, and extent of viable tumor). This scoring system segregates patients into 3 risk categories with significant differences in overall survival and retained significance in an independent validation set of 42 cases. Conclusions.— The integration of clinical and microscopic features improves prognostication of patients with osteosarcoma.

Objectives (1) To provide recommendations regarding the choice of surgery, radiation therapy (RT), or the combination of surgery plus RT in patients with localized Ewing's sarcoma of bone following neoadjuvant chemotherapy. (2) To determine the appropriate surgical planning imaging (pre-chemotherapy magnetic resonance imaging [MRI] or post-chemotherapy MRI) to identify optimal resection margins in patients with localized Ewing's sarcoma who undergo surgery following neoadjuvant chemotherapy. Methods MEDLINE, EMBASE, the Cochrane Library (1999 to February 2015), main guideline websites, and relevant annual meeting abstracts (2012 to January 2015) were searched. Internal and external reviews were conducted. Recommendations 1. Recommendation (1) – In patients with localized Ewing's sarcoma of bone following neoadjuvant chemotherapy: (a) Surgery alone or RT alone are two reasonable treatment options; the combination of surgery plus RT is not recommended as an initial treatment option. (b) The local treatment for an individual patient should be decided by a multidisciplinary tumour board together with the patient after consideration of the following: (1) patient characteristics (e.g., age, tumour location, tumour size, response to neoadjuvant chemotherapy, and existing comorbidities), (2) the potential benefit weighed against the potential complications from surgery and/or toxicities associated with RT, and (3) patient preferences. 2. Recommendation (2) – In patients with localized Ewing's sarcoma who will undergo surgery: (a) Both pre-chemotherapy and post-chemotherapy MRI scans should be taken into consideration for surgical planning. In certain anatomic locations with good chemotherapy response, the post-chemotherapy MRI may be the appropriate imaging modality to plan surgical resection margins.

Disc degeneration is associated with low back pain for which currently there is no optimal therapy so there is a great need to identify new treatment approaches. Inorganic polyphosphates (polyP) are linear polymers of orthophosphate units varying in chain length and present in many cell types. As polyP has anabolic effects on chondrocytes, we hypothesized that polyP treatment would enhance matrix accumulation by nucleus pulposus (NP) cells. NP cells isolated from bovine caudal discs were grown in 3D culture under normoxic or in select experiments under hypoxic conditions, in the presence or absence of various concentrations and sizes of polyP. Gene expression was determined using RT-PCR. Matrix accumulation was quantified by measuring proteoglycan and collagen contents. DAPI fluorescence shift was used to stain for polyP in tissue. DAPI staining showed polyP present predominantly in the pericellular region of in vitro formed tissue. PolyP treatment enhanced matrix accumulation in a concentration and chain length dependant manner. NP cells exposed to polyP-22 (22 phosphate units length) showed an increase in gene expression of aggrecan, Collagen II, Sox 9, and MMP-13 which was maintained for the 14 days of culture. This suggests that polyP may enhance NP tissue formation in vitro by upregulating the expression of matrix genes. As polyP enhances proteoglycan accumulation even under hypoxic conditions, this raises the possibility that polyP may be a novel treatment to induce NP regeneration. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:41-50, 2017.

Cell-based therapies, defined here as the delivery of cells in vivo to treat disease, have recently gained increasing public attention as a potentially promising approach to restore structure and function to musculoskeletal tissues. Although cell-based therapy has the potential to improve the treatment of disorders of the musculoskeletal system, there is also the possibility of misuse and misrepresentation of the efficacy of such treatments. The medical literature contains anecdotal reports and research studies, along with web-based marketing and patient testimonials supporting cell-based therapy. Both the American Society for Bone and Mineral Research (ASBMR) and the Orthopaedic Research Society (ORS) are committed to ensuring that the potential of cell-based therapies is realized through rigorous, reproducible, and clinically meaningful scientific discovery. The two organizations convened a multidisciplinary and international Task Force composed of physicians, surgeons, and scientists who are recognized experts in the development and use of cell-based therapies. The Task Force was charged with defining the state-of-the art in cell-based therapies and identifying the gaps in knowledge and methodologies that should guide the research agenda. The efforts of this Task Force are designed to provide researchers and clinicians with a better understanding of the current state of the science and research needed to advance the study and use of cell-based therapies for skeletal tissues. The design and implementation of rigorous, thorough protocols will be critical to leveraging these innovative treatments and optimizing clinical and functional patient outcomes. In addition to providing specific recommendations and ethical considerations for preclinical and clinical investigations, this report concludes with an outline to address knowledge gaps in how to determine the cell autonomous and nonautonomous effects of a donor population used for bone regeneration. © 2019 American Society for Bone and Mineral Research.

Chondrocytes dedifferentiate as a result of monolayer culture for cell number expansion. This is associated with the development of an elongated shape, increased actin polymerization, development of stress fibres, and expression of contractile molecules. Given the changes in actin status with dedifferentiation, the hypothesis of this study was that adseverin, an actin severing and capping protein, plays a role in regulating chondrocyte phenotype and function. This study reports that serial passaging of articular chondrocytes in monolayer culture resulted in loss of adseverin protein expression as early as Day 14 of culture and remained repressed in Passage 2 (P2) cells. Knockdown of adseverin by siRNA in primary chondrocytes promoted an increase in cell size and an elongated shape, actin stress fibres, decreased G-/F-actin ratio, and increased number of actin-free barbed ends. The cells also showed increased expression of the contractile genes and proteins, vinculin and α-smooth muscle actin, and increased ability to contract collagen gels. These are all features of dedifferentiation. These effects were due to adseverin as adseverin overexpression following transfection of the green fluorescent protein-adseverin plasmid partially reversed all of these changes in P2 chondrocytes. Furthermore, sox9 and aggrecan chondrogenic gene expression was upregulated, and collagen type I genes expression was downregulated with adseverin overexpression. The change in aggrecan mRNA expression had functional consequence as these cells exhibited increased total proteoglycan synthesis. These findings demonstrate that adseverin regulates features indicative of redifferentiation in passaged articular chondrocytes through modulation of the actin cytoskeleton status and potentially may regulate the maintenance of phenotype in primary chondrocytes.

Aims To make recommendations on the indications for molecular testing regarding the diagnosis, prediction of prognosis, and treatment selection in adult patients with s oft tissue sarcomas (STS) excluding gastrointestinal stromal tumour. Materials and methods This guideline was developed by the Cancer Care Ontario’s Program in Evidence-Based Care (PEBC) and the Sarcoma Disease Site Group (DSG). The medline, embase, and Cochrane Library databases, main guideline websites, abstracts of relevant annual meetings, and PROSPERO databases were searched (January 2005 to October 2016). Internal and external reviews were conducted, with final approval by the PEBC and the Sarcoma DSG. Results Based on the available evidence, we made three S trong Recommendations, 14 Recommendations, 9 Qualified Statements, and seven No Recommendations. The three Strong Recommendations include: i) MDM2 amplification by fluorescence in situ hybridization (FISH) is recommended as a sensitive and specific test to differentiate patients with atypical lipomatous tumour/well-differentiated liposarcoma, or dedifferentiated liposarcoma from lipoma or other STS in the differential diagnosis; ii) SS18 (SYT) break-apart by FISH or SS18-SSX (SYT-SSX) fusion by reverse transcription-polymerase chain reaction is recommended as a sensitive and specific test to differentiate patients with synovial sarcoma from other sarcomas; iii) CTNNB1 S45F mutation by polymerase chain reaction is recommended as a prognostic factor for poor recurrence-free survival in patients with desmoid tumours. Conclusion This guideline may serve as a framework for the thoughtful implementation of molecular studies at cancer centres and other jurisdictions. Some of the recommendations may need to be updated when new evidence appears in the future.

This study investigates the characteristics of porous calcium polyphosphate particulates (CPPp) formed using two different processing treatments as bone void fillers in non- or minimally load-bearing sites. The two calcium polyphosphate particulate variants (grades) were formed using different annealing conditions during particulate preparation to yield either more slowly degrading calcium polyphosphate particulates (SD-CPPp) or faster degrading particulates (FD-CPPp) as suggested by a previous degradation study conducted in vitro (Hu et al., Submitted for publication 2016). The two CPPp grades were compared as bone void fillers in vivo by implanting particulates in defects created in rabbit femoral condyle sites (critical size defects). The SD-CPPp and FD-CPPp were implanted for 4- and 16-week periods. The in vivo study indicated a significant difference in amount of new bone formed in the prepared sites with SD-CPPp resulting in more new bone formation compared with FD-CPPp. The lower bone formation characteristic of the FD-CPPp was attributed to its faster degradation rate and resulting higher local concentration of released polyphosphate degradation products. The study results indicate the importance of processing conditions on preparing calcium polyphosphate particulates for potential use as bone void fillers in nonload-bearing sites. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 874-884, 2017.

Nucleofection of chondrocytes has been shown to be an adequate method of transfection. Using Amaxa’s nucleofection system, transfection efficiencies up to 89% were achievable for vector (pmaxGFP) and 98% for siRNA (siGLO) into passaged chondrocytes. However, such methods rely on costly commercial kits with proprietary reagents limiting its use in basic science labs and in clinical translation. Bovine-passaged chondrocytes were plated in serum reduced media conditionsand then nucleofected using various in laboratory-produced buffers. Cell attachment, confluency, viability, and transfection efficiency was assessed following nucleofection. For each parameter the buffers were scored and a final rank for each buffer was determined. Buffer denoted as 1M resulted in no significant difference for cell attachment, confluency, and viability as compared to non-nucleofected controls. Nucleofection in 1M buffer, in the absence of DNA vectors, resulted in increased col2, ki67, ccnd1 mRNA levels, and decreased col1 mRNA levels at 4 days of culture. Flow cytometry revealed that the transfection efficiency of 1M buffer was comparable to that obtained using the Amaxa commercial kit. siRNA designed against lamin A/C resulted in an average reduction of lamin A and C proteins to 19% and 8% of control levels, respectively. This study identifies a cost-effective, efficient method of nonviral nucleofection of bovine-passaged chondrocytes using known buffer formulations. Human-passaged chondrocytes could also be successfully nucleofected in 1M buffer. Thus this method should facilitate cost-efficient gene targeting of cells used for articular cartilage repair in a research setting.

Purpose To review the available literature on studies focusing on platelet-rich plasma (PRP)–enhanced scaffolds for cartilage lesion repair in animals and to analyze the clinical outcomes of similar biologically augmented cartilage regeneration techniques in humans. Methods We conducted a literature search and subsequent review investigating the potential of PRP to enhance articular cartilage repair using scaffolds or bioengineered implants. Results Of the 14 animal model studies reviewed, 10 reported positive effects with PRP whereas only 2 showed negative overall effects. The remaining 2 studies reported no significant differences, or neutral results, with the use of PRP. With the addition of PRP, the gross appearance and histologic analysis of repair cartilage were improved or no difference was seen compared with control (11 of 12 studies that looked at this). Human studies of the knee or talar dome showed improvements in clinical assessment scores as soon as 6 months after surgery. There was great variability in the method of PRP preparation, choice of scaffold, and cell source between studies. Conclusions PRP-augmented scaffolds have been shown to be beneficial in the articular cartilage repair process in animals and humans based on macroscopic, histologic, and biochemical analysis and based on clinical outcome scores, respectively. Comparison between studies is difficult because there is great variability in PRP preparation and administration. Level of Evidence Level IV, systematic review of Level III and IV studies.

Summary Objective In healthy joints, a zone of calcified cartilage (ZCC) provides the mechanical integration between articular cartilage and subchondral bone. Recapitulation of this architectural feature should serve to resist the constant shear force from the movement of the joint and prevent the delamination of tissue-engineered cartilage. Previous approaches to create the ZCC at the cartilage–substrate interface have relied on strategic use of exogenous scaffolds and adhesives, which are susceptible to failure by degradation and wear. In contrast, we report a successful scaffold-free engineering of ZCC to integrate tissue-engineered cartilage and a porous biodegradable bone substitute, using sheep bone marrow stromal cells (BMSCs) as the cell source for both cartilaginous zones. Design BMSCs were predifferentiated to chondrocytes, harvested and then grown on a porous calcium polyphosphate substrate in the presence of triiodothyronine (T3). T3 was withdrawn, and additional predifferentiated chondrocytes were placed on top of the construct and grown for 21 days. Results This protocol yielded two distinct zones: hyaline cartilage that accumulated proteoglycans and collagen type II, and calcified cartilage adjacent to the substrate that additionally accumulated mineral and collagen type X. Constructs with the calcified interface had comparable compressive strength to native sheep osteochondral tissue and higher interfacial shear strength compared to control without a calcified zone. Conclusion This protocol improves on the existing scaffold-free approaches to cartilage tissue engineering by incorporating a calcified zone. Since this protocol employs no xenogeneic material, it will be appropriate for use in preclinical large-animal studies.

Current approaches to cartilage tissue engineering require a large number of chondrocytes. Although chondrocyte numbers can be expanded in monolayer culture, the cells dedifferentiate and unless they can be redifferentiated are not optimal to use for cartilage repair. We took advantage of the differential effect of culture conditions on the ability of passaged and primary chondrocytes to form cartilage tissue to dissect out the extracellular matrix (ECM) molecules produced and accumulated in the early stages of passaged cell cartilage tissue formation as we hypothesized that passaged bovine cells that form cartilage accumulate a pericellular matrix that differs from cells that do not form cartilage. Twice passaged bovine chondrocytes (P2) (cartilage forming), or as a control primary chondrocytes (P0) (which do not generate cartilage), were cultured on three-dimensional membrane inserts in serum-free media. P2 redifferentiation was occurring during the first 8 days as indicated by increased expression of the chondrogenic genes Sox9, collagen type II, aggrecan, and COMP, suggesting that this is an appropriate time period to examine the ECM. Mass spectrometry showed that the P2 secretome (molecules released into the media) at 1 week had higher levels of collagen types I, III, and XII, and versican while type II collagen and COMP were found at higher levels in the P0 secretome. There was increased collagen synthesis and retention by P2 cells compared to P0 cells as early as 3 days of culture. Confocal microscopy showed that types XII, III, and II collagen, aggrecan, versican, and decorin were present in the ECM of P2 cells. In contrast, collagen types I, II, and III, aggrecan, and decorin were present in the ECM of P0 cells. As primary chondrocytes grown in serum-containing media, a condition that allows for the generation of cartilage tissue in vitro, also accumulate versican and collagen XII, this study suggests that these molecules may be necessary to provide a microenvironment that supports hyaline cartilage formation. Further study is required to determine if these molecules are also accumulated by passaged human chondrocytes and their role in promoting hyaline cartilage formation.

Low back pain is a major physical and socioeconomic problem. Degeneration of the intervertebral disc and especially that of nucleus pulposus (NP) has been linked to low back pain. In spite of much research focusing on the NP, consensus among the research community is lacking in defining the NP cell phenotype. A consensus agreement will allow easier distinguishing of NP cells from annulus fibrosus (AF) cells and endplate chondrocytes, a better gauge of therapeutic success, and a better guidance of tissue-engineering-based regenerative strategies that attempt to replace lost NP tissue. Most importantly, a clear definition will further the understanding of physiology and function of NP cells, ultimately driving development of novel cell-based therapeutic modalities. The Spine Research Interest Group at the 2014 Annual ORS Meeting in New Orleans convened with the task of compiling a working definition of the NP cell phenotype with hope that a consensus statement will propel disc research forward into the future. Based on evaluation of recent studies describing characteristic NP markers and their physiologic relevance, we make the recommendation of the following healthy NP phenotypic markers: stabilized expression of HIF-1α, GLUT-1, aggrecan/collagen II ratio >20, Shh, Brachyury, KRT18/19, CA12, and CD24.

Osteoarthritis (OA) is a degenerative disease that initially manifests as loss of the superficial zone (SZ) of articular cartilage. SZ chondrocytes (SZC) differ in morphology from other chondrocytes as they are elongated and oriented parallel to the tissue surface. Proteoglycan 4 (PRG4) and tenascin C (TNC) are molecules expressed by SZC, which have been shown to be chondroprotective. Identification of the signalling pathway(s) regulating expression of SZ molecules may lead to a therapeutic target that can be used to delay or prevent the onset of OA. The hypothesis of this study is that expression of SZ molecules are regulated in part, by the CDC42-actin-myocardin-related transcription factor-A (MRTF-A) signaling pathway. SZC from bovine metacarpal-phalangeal joints were isolated and grown in monolayer culture. Each target in the CDC42-actin-MRTF-A pathway was inhibited and the effect on cell shape, actin cytoskeleton status, and expression of PRG4 and TNC were determined. Treatment with the CDC42 inhibitor ML141 decreased PRG4 and TNC expression, and correlated with increased cell circularity and G-/F-actin ratio. PRG4 and TNC expression were differentially regulated by actin depolymerizing agents, latrunculin B and cytochalasin D. Chemical inhibition of MRTF-A resulted in decreased expression of both PRG4 and TNC; however, specific knockdown by small interfering RNA only decreased expression of TNC indicating that TNC, but not PRG4, is regulated by MRTF-A. Although PRG4 and TNC expression are both regulated by CDC42 and actin, it appears to occur through different downstream signaling pathways. Further study is required to elucidate the pathway regulating PRG4. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2421-2430, 2018.

Background The molecular pathogenesis of many forms of soft tissue sarcomas (STS) have been rigorously characterized in the medical literature, which may be particularly important for the diagnosis and prediction of prognosis in STS. Methods Electronic databases (2005 to October 2016) were searched. Gastrointestinal stromal tumor and pediatric sarcomas were excluded. The eligible individual study’s risk of bias and the quality of aggregate evidence were assessed. Meta-analyses were performed. Results Of 6674 identified articles, 70 were eligible and analyzed, covering 13 types of STS. Meta-analyses showed that the test of detecting MDM2 amplification by fluorescence in situ hybridization was accurate in differentiating atypical lipomatous tumor/well-differentiated liposarcoma/dedifferentiated liposarcoma from benign tumors (N = 971; sensitivity = 95%, 95% confidence interval [CI] 89–98; specificity = 100%, CI 89–100) or from other STS (N = 347; sensitivity = 99%, CI 72–100; specificity = 90%, CI 78–95); that the test of detecting SS18-SSX fusion by reverse transcription polymerase chain reaction (PCR) was accurate in differentiating synovial sarcoma from other STS (N = 532; sensitivity = 93%, CI 85–96; specificity = 99%, CI 96–100). The presence of a CTNNB1 S45F mutation detected by PCR was a risk factor for decreased recurrence-free survival in desmoid tumors (N = 418; hazard ratio from 3.50 [CI 1.51–8.14] to 6.20 [CI 2.24–17.15]). Conclusions Sarcomas are rare cancers whose molecular pathogenesis is becoming increasingly understood. The current evidence demonstrates that molecular analyses are useful in the diagnosis and prediction of prognosis in some STS.

Generating the best possible bioengineered cartilage from passaged chondrocytes requires culture condition optimization. In this study, the use of adherent agarose mold (adAM) cultures to support redifferentiation of passaged twice (P2) chondrocytes and serve as a scalable platform to assess the effect of growth factor combinations on proteoglycan accumulation by cells was examined. By 2 days in adAM culture, bovine P2 cells were partially redifferentiated as demonstrated by regression of actin-based dedifferentiation signalling and fibroblast matrix and contractile gene expression. By day 10, aggrecan and type II collagen gene expression were significantly increased in adAM cultured cells. At day 20, a continuous layer of cartilage tissue was observed. There was no evidence of tissue contraction by P2 cells in adAM cultures. The matrix properties of the resultant tissue as well as proteoglycan 4 (PRG4) secreted by the cells were dependent on the initial cell seeding density. AdAM cultures were scalable and culture within small 3 mm diameter adAM allowed for multi-factorial assessment of growth factors on proteoglycan accumulation by human P2 chondrocytes. Although there was a patient specific response in proteoglycan accumulation to the various cocktail combinations, the cocktail consisting of 2 ng/ml TGFβ1, 10 ng/ml FGF2, and 250 ng/ml FGF18 resulted in a consistent increase in alcian blue tissue staining. Additional studies will be required to identify the optimal conditions to bioengineer articular cartilage tissue for clinical use. However, the results to date suggest that adAM cultures may be suitable to use for high throughput assessment. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2392-2405, 2018.

The annulus fibrosus (AF) of the intervertebral disc (IVD) has a zonal distribution of phenotypically distinct cells. The outer AF (OAF) cells produce an extracellular matrix (ECM) rich in type I collagen with little proteoglycans, whereas the ECM of the inner AF (IAF) has abundant type II collagen and proteoglycans. The inhomogeneous distribution of the ECM in the AF may reflect the complex mechanical forces that the IVD experiences. A bioengineered AF tissue should recapitulate both the inner and outer zones in order to have proper functionality. The aim of this study is to generate multi-lamellated OAF and IAF tissues with ECM compositions that resemble their zonal origin using polycarbonate urethane (PU) scaffolds. It was observed that supplementation of the media with insulin-transferrin-selenium (ITS) and proline yielded tissues with good cellularity. However, IAF cells accumulated only type I collagen, similar to OAF cells. Addition of dexamethasone and sodium pyruvate induced the accumulation of IAF tissues rich in type II collagen and aggrecan, without altering the accumulation of type I collagen in OAF tissues. Dexamethasone stimulated mitochondrial membrane potential in both tissues in the presence of sodium pyruvate, suggesting a relationship between the mitochondrial aerobic respiratory state and dexamethasone signalling during the in vitro-tissue formation by OAF and IAF cells. Inhibition of the glucocorticoid receptor blocked the stimulation of mitochondrial membrane potentials and type II collagen accumulation. In summary, biologically distinct multi-lamellated OAF and IAF tissues can be generated, which will facilitate advancement towards the goal of engineering a biological IVD replacement. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1346-1355, 2018.

(1) To characterize the effects of mechanical stimulation on the integration of a tissue-engineered construct in terms of histology, biochemistry and biomechanical properties; (2) to identify whether cells of the implant or host tissue were critical to implant integration; and (3) to study cells believed to be involved in lateral integration of tissue-engineered cartilage to host cartilage. We hypothesized that mechanical stimulation would enhance the integration of the repair implant with host cartilage in an in vitro integration model. Articular cartilage was harvested from 6- to 9-month-old bovine metacarpal-phalangeal joints. Constructs composed of tissue-engineered cartilage implanted into host cartilage were placed in spinner bioreactors and maintained on a magnetic stir plate at either 0 (static control) or 90 (experimental) rotations per minute (RPM). The constructs from both the static and spinner bioreactors were harvested after either 2 or 4 weeks of culture and evaluated histologically, biochemically, biomechanically and for gene expression. The extent and strength of integration between tissue-engineered cartilage and native cartilage improved significantly with both time and mechanical stimulation. Integration did not occur if the implant was not viable. The presence of stimulation led to a significant increase in collagen content in the integration zone between host and implant at 2 weeks. The gene profile of cells in the integration zone differs from host cartilage demonstrating an increase in the expression of membrane type 1 matrix metalloproteinase (MT1-MMP), aggrecan and type II collagen. This study shows that the integration of in vitro tissue-engineered implants with host tissue improves with mechanical stimulation. The findings of this study suggests that consideration should be given to implementing early loading (mechanical stimulation) into future in vivo studies investigating the long-term viability and integration of tissue-engineered cartilage for the treatment of cartilage injuries. This could simply be done through the use of continuous passive motion (CPM) in the post-operative period or through a more complex and structured rehabilitation program with a gradual increase in forces across the joint over time.

We set out to determine the optimal treatment options&mdash, surgery, radiation therapy (rt), systemic therapy, or any combinations thereof&mdash, for patients with desmoid tumours once the decision to undergo active treatment has been made (that is, monitoring and observation have been determined to be inadequate). provide clinical-expert consensus opinions on follow-up strategies in patients with desmoid tumours after primary interventional management. This guideline was developed by Cancer Care Ontario&rsquo, s Program in Evidence-Based Care and the Sarcoma Disease Site Group. The medline, embase, and Cochrane Library databases, main guideline Web sites, and abstracts of relevant annual meetings (1990 to September 2012) were searched. Internal and external reviews were conducted, with final approval by the Program in Evidence-Based Care and the Sarcoma Disease Site Group.