Passaged Articular Chondrocytes From the Superficial Zone and Deep Zone Can Regain Zone-Specific Properties After Redifferentiation.

BACKGROUND: Bioengineered cartilage is a developing therapeutic to repair cartilage defects. The matrix must be rich in collagen type II and aggrecan and mechanically competent, withstanding compressive and shearing loads. Biomechanical properties in native articular cartilage depend on the zonal architecture consisting of 3 zones: superficial, middle, and deep. The superficial zone chondrocytes produce lubricating proteoglycan-4, whereas the deep zone chondrocytes produce collagen type X, which allows for integration into the subchondral bone. Zonal and chondrogenic expression is lost after cell number expansion. Current cell-based therapies have limited capacity to regenerate the zonal structure of native cartilage. HYPOTHESIS: Both passaged superficial and deep zone chondrocytes at high density can form bioengineered cartilage that is rich in collagen type II and aggrecan; however, only passaged superficial zone-derived chondrocytes will express superficial zone-specific proteoglycan-4, and only passaged deep zone-derived chondrocytes will express deep zone-specific collagen type X. STUDY DESIGN: Controlled laboratory study. METHODS: Superficial and deep zone chondrocytes were isolated from bovine joints, and zonal subpopulations were separately expanded in 2-dimensional culture. At passage 2, superficial and deep zone chondrocytes were seeded, separately, in scaffold-free 3-dimensional culture within agarose wells and cultured in redifferentiation media. RESULTS: Monolayer expansion resulted in loss of expression for proteoglycan-4 and collagen type X in passaged superficial and deep zone chondrocytes, respectively. By passage 2, superficial and deep zone chondrocytes had similar expression for dedifferentiated molecules collagen type I and tenascin C. Redifferentiation of both superficial and deep zone chondrocytes led to the expression of collagen type II and aggrecan in both passaged chondrocyte populations. However, only redifferentiated deep zone chondrocytes expressed collagen type X, and only redifferentiated superficial zone chondrocytes expressed and secreted proteoglycan-4. Additionally, redifferentiated deep zone chondrocytes produced a thicker and more robust tissue compared with superficial zone chondrocytes. CONCLUSION: The recapitulation of the primary phenotype from passaged zonal chondrocytes introduces a novel method of functional bioengineering of cartilage that resembles the zone-specific biological properties of native cartilage. CLINICAL RELEVANCE: The recapitulation of the primary phenotype in zonal chondrocytes could be a possible method to tailor bioengineered cartilage to have zone-specific expression.

Senescent response in inner annulus fibrosus cells in response to TNFα, H2O2, and TNFα-induced nucleus pulposus senescent secretome.

Senescence, particularly in the nucleus pulposus (NP) cells, has been implicated in the pathogenesis of disc degeneration, however, the mechanism(s) of annulus fibrosus (AF) cell senescence is still not well understood. Both TNFα and H2O2, have been implicated as contributors to the senescence pathways, and their levels are increased in degenerated discs when compared to healthy discs. Thus, the objective of this study is to identify factor(s) that induces inner AF (iAF) cell senescence. Under TNFα exposure, at a concentration previously shown to induce senescence in NP cells, bovine iAF cells did not undergo senescence, indicated by their ability to continue to proliferate as demonstrated by Ki67 staining and growth curves and lack of expression of the senescent markers, p16 and p21. The lack of senescent response occurred even though iAF express higher levels of TNFR1 than NP cells. Interestingly, iAF cells showed no increase in intracellular ROS or secreted H2O2 in response to TNFα which contrasted to NP cells that did. Following TNFα treatment, only iAF cells had increased expression of the superoxide scavengers SOD1 and SOD2 whereas NP cells had increased NOX4 gene expression, an enzyme that can generate H2O2. Treating iAF cells with low dose H2O2 (50 µM) induced senescence, however unlike TNFα, H2O2 did not induce degenerative-like changes as there was no difference in COL2, ACAN, MMP13, or IL6 gene expression or number of COL2 and ACAN immunopositive cells compared to untreated controls. The latter result suggests that iAF cells may have distinct degenerative and senescent phenotypes. To evaluate paracrine signalling by senescent NP cells, iAF and TNFα-treated NP cells were co-cultured. In contact co-culture the NP cells induced iAF senescence. Thus, senescent NP cells may secrete soluble factors that induce degenerative and senescent changes within the iAF. This may contribute to a positive feedback loop of disc degeneration. It is possible these factors may include H2O2 and cytokines (such as TNFα). Further studies will investigate if human disc cells respond similarly.

Education of the clinical embryology laboratory professional: development of a novel program delivered in a laboratory medicine department.

Clinical embryologists are responsible for the handling, evaluation, and care of human gametes and preimplantation embryos within the context of an assisted reproductive technology laboratory. They are integral members of a team of professionals who provide care for fertility patients. Despite the increasing recognition of clinical embryologists as professionals, training requirements, continuing professional development, and appropriate credentialing have lagged in several countries. In many cases, individuals enter the profession with training limited to technical aspects provided by individual laboratory directors through an apprenticeship model. In this article, we present the rationale for rigorous formal training in clinical embryology, introduce CanEMB competencies for practicing professional clinical embryologists that are founded on CanMEDs role principles, and present a nascent Masters of Health Sciences degree program in Laboratory Medicine with a specialization in clinical embryology. This 2-year program has unique features including a Clinical Embryology Skills Development Laboratory, research capstone project, and 200-hour placement within a practicing assisted reproductive technology laboratory. Importantly, this program is delivered through a university-based Department of Laboratory Medicine and Pathobiology in partnership with a Department of Obstetrics and Gynecology. Thus, this program represents a formal acceptance of clinical embryology as a clinical laboratory science. It can be adopted elsewhere to provide a relevant, robust education that will meet current and future needs of the profession.

Adseverin, an actin-binding protein, modulates hypertrophic chondrocyte differentiation and osteoarthritis progression.

In osteoarthritis (OA), a disease characterized by progressive articular cartilage degradation and calcification, the articular chondrocyte phenotype changes and this correlates with actin cytoskeleton alterations suggesting that it regulates gene expression essential for proper phenotype. This study reports that OA is associated with the loss of adseverin, an actin capping and severing protein. Adseverin deletion (Adseverin-/-) in mice compromised articular chondrocyte function, by reducing F-actin and aggrecan expression and increasing apoptosis, Indian hedgehog, Runx2, MMP13, and collagen type X expression, and cell proliferation. This led to stiffer cartilage and decreased hyaline and increased calcified cartilage thickness. Together, these changes predisposed the articular cartilage to enhanced OA severity in Adseverin-/- mice who underwent surgical induction of OA. Adseverin-/- chondrocyte RNA sequencing and in vitro studies together suggests that adseverin modulates cell viability and prevents mineralization. Thus, adseverin maintains articular chondrocyte phenotype and cartilage tissue homeostasis by preventing progression to hypertrophic differentiation in vivo. Adseverin may be chondroprotective and a potential therapeutic target.

Characterization of Migratory Cells From Bioengineered Bovine Cartilage in a 3D Co-culture Model.

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.

Cost-effectiveness of noninvasive fetal RhD blood group genotyping in nonalloimmunized and alloimmunized pregnancies.

BACKGROUND: We sought to determine the cost-effectiveness of noninvasive fetal RhD blood group genotyping in nonalloimmunized and alloimmunized pregnancies in Canada. STUDY DESIGN AND METHODS: We developed two probabilistic state-transition (Markov) microsimulation models to compare fetal genotyping followed by targeted management versus usual care (i.e., universal Rh immunoglobulin [RhIG] prophylaxis in nonalloimmunized RhD-negative pregnancies, or universal intensive monitoring in alloimmunized pregnancies). The reference case considered a healthcare payer perspective and a 10-year time horizon. Sensitivity analysis examined assumptions related to test cost, paternal screening, subsequent pregnancies, other alloantibodies (e.g., K, Rh c/C/E), societal perspective, and lifetime horizon. RESULTS: Fetal genotyping in nonalloimmunized pregnancies (at per-sample test cost of C$247/US$311) was associated with a slightly higher probability of maternal alloimmunization (22 vs. 21 per 10,000) and a reduced number of RhIG injections (1.427 vs. 1.795) than usual care. It was more expensive (C$154/US$194, 95% Credible Interval [CrI]: C$139/US$175-C$169/US$213) and had little impact on QALYs (0.0007, 95%CrI: -0.01-0.01). These results were sensitive to the test cost (threshold achieved at C$88/US$111), and inclusion of paternal screening. Fetal genotyping in alloimmunized pregnancies (at test cost of C$328/US$413) was less expensive (-C$6280/US$7903, 95% CrI: -C$6325/US$7959 to -C$6229/US$7838) and more effective (0.19 QALYs, 95% CrI 0.17-0.20) than usual care. These cost savings remained robust in sensitivity analyses. DISCUSSION: Noninvasive fetal RhD genotyping saves resources and represents good value for the management of alloimmunized pregnancies. If the cost of genotyping is substantially decreased, the targeted intervention can become a viable option for nonalloimmunized pregnancies.

Development of a Perfusion Reactor for Intervertebral Disk Regeneration.

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.

The anabolic effect of inorganic polyphosphate on chondrocytes is mediated by calcium signalling.

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(ß-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.

Inorganic polyphosphates stimulates matrix production in human annulus fibrosus cells.

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.

Induced senescence of healthy nucleus pulposus cells is mediated by paracrine signaling from TNF-α-activated cells.

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 ß-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.

Vapourized hydrogen peroxide decontamination in a hospital setting inactivates SARS-CoV-2 and HCoV-229E without compromising filtration efficiency of unexpired N95 respirators.

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.

Comparative evaluation of four hydrogen peroxide-based systems to decontaminate N95 respirators.

Objective: Protocols designed to facilitate N95 filtering facepiece respirator (FFR) decontamination by commercial sterilization devices do not recommend that operators verify the device's performance against pathogens deposited on FFRs. Here, we compared the treatment efficacy of 4 hydrogen peroxide-based systems that were authorized for N95 decontamination during the COVID-19 pandemic. Methods: Suspensions prepared from S. aureus ATCC 29213 and 44300, B. subtilis ATCC 6633, a vancomycin-resistant E. faecium isolate (VRE), E. coli ATCC 25922, and P. aeruginosa ATCC 27853 colonies were inoculated onto nine 1-cm2 areas on a 3M 1805, 1860, 1860S, 1870+, 8210, 8110S, or 9105S FFR. Contaminated respirators were treated according to protocols recommended by the STERRAD 100NX, Bioquell Z-2, Sterizone VP4, or Clean Works Mini systems. Decontamination efficacy was determined by comparing colony counts cultured from excised segments of treated and untreated FFR. Results: All devices achieved a 6-log reduction in bacterial burden and met FDA sterilization criteria. The Bioquell Z-2 device demonstrated 100% efficacy against both gram-positive and gram-negative organisms with all FFRs tested. Colonies of S. aureus ATCC 29213 and 44300 and VRE were cultivable from up to 9 (100%) of 9 STERRAD 100NX- and Sterizone VP4-treated segments. Viable B. subtilis ATCC 6633 organisms were recovered from 76.0% of STERRAD 100NX-treated FFR segments. Conclusions: Variability in decontamination efficacy was noted across devices and FFR types. gram-positive organisms were more difficult to completely eliminate than were gram-negative organisms. Prior to initiating FFR decontamination practices, institutions should verify the effectiveness of their devices and the safety of treated FFR.

Endoglin Level Is Critical for Cartilage Tissue Formation In Vitro by Passaged Human Chondrocytes.

Transforming growth factor beta (TGFß) signaling is required for in vitro chondrogenesis. In animal models of osteoarthritis (OA), TGFß receptor alterations are detected in chondrocytes in severe OA cartilage. It is not known whether such changes are dependent on the grade of human OA and if they affect chondrogenesis. Thus, the purpose of this study was to determine if human OA chondrocytes obtained from low-grade or high-grade disease could form cartilage tissue and to assess the role of the co-receptors, endoglin (ENG) and TGFß receptor 3 (TGFBRIII), in the regulation of this tissue generation in vitro. We hypothesized that the grade of OA disease would not affect the ability of cells to form cartilage tissue and that the TGFß co-receptor, ENG, would be critical to regulating tissue formation. Chondrocytes isolated from low-grade OA or high-grade OA human articular cartilage (AC) were analyzed directly (P0) or passaged in monolayer to P2. Expression of the primary TGFß receptor ALK5, and the co-receptors ENG and TGFßRIII, was assessed by image flow cytometry. To assess the ability to form cartilaginous tissue, cells were placed in three-dimensional culture at high density and cultured in chondrogenic media containing TGFß3. ENG knockdown was used to determine its role in regulating tissue formation. Overall, grade-specific differences in expression of ALK5, ENG, and TGFßRIII in primary or passaged chondrocytes were not detected; however, ENG expression increased significantly after passaging. Despite the presence of ALK5, P0 cells did not form cartilaginous tissue. In contrast, P2 cells derived from low-grade and high-grade OA AC formed hyaline-like cartilaginous tissues of similar quality. Knockdown of ENG in P2 cells inhibited cartilaginous tissue formation compared to controls indicating that the level of ENG protein expression is critical for in vitro chondrogenesis by passaged articular chondrocytes. This study demonstrates that it is not the grade of OA, but the levels of ENG in the presence of ALK5 that influences the ability of human passaged articular chondrocytes to form cartilaginous tissue in vitro in 3D culture. This has implications for cartilage repair therapies. Impact statement These findings are important clinically, given the limited availability of osteoarthritis (OA) cartilage tissue. Being able to use cells from all grades of OA will increase our ability to obtain sufficient cells for cartilage repair. In addition, it is possible that endoglin (ENG) levels, in the presence of ALK5 expression, may be suitable to use as biomarkers to identify cells able to produce cartilage.

Generation of an in vitro model of the outer annulus fibrosus-cartilage interface.

Current treatments for degenerative disc disease do not restore full biological functionality of the intervertebral disc (IVD). As a result, regenerative medicine approaches are being developed to generate a biological replacement that when implanted will restore form and function of the degenerated IVD. Tissue-engineered models to date have focused on the generation of nucleus pulposus and annulus fibrosus IVD components. However, these tissues need to be integrated with a cartilage endplate in order for successful implantation to occur. The purpose of this study was to generate an in vitro annulus fibrosus-cartilage interface model which would enable us to better understand the biological and biomechanical implications of such interfaces. It was hypothesized that in vitro-formed outer annulus fibrosus (OAF) and cartilage tissues would integrate in direct-contact coculture to yield an interface containing extracellular matrix with aspects resembling the native OAF-CEP interface. In vitro-formed tissues were generated using bovine OAF cell-seeded angle-ply, multi-lamellated polycarbonate urethane scaffolds and articular chondrocytes, which were then placed in direct-contact coculture. 2-week old OAF tissues integrated with 3-day old cartilage by 1 week of coculture. Immunohistochemical staining of 2-week interfaces showed that distributions of collagen type I, collagen type II, and aggrecan were similar to the native bovine interface. The apparent tensile strength of the in vitro interface increased significantly between 2 and 4 weeks of coculture. In summary, an annulus fibrosus-cartilage interface model can be formed in vitro which will facilitate the identification of conditions required to generate an entire tissue-engineered disc replacement suitable for clinical use.

An evidence-based guideline on the application of molecular testing in the diagnosis, prediction of prognosis, and selection of therapy in non-GIST soft tissue sarcomas.

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.

American Society for Bone and Mineral Research-Orthopaedic Research Society Joint Task Force Report on Cell-Based Therapies - Secondary Publication.

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.

Superficial and deep zone articular chondrocytes exhibit differences in actin polymerization status and actin-associated molecules in vitro.

Objective: The actin cytoskeleton regulates cell shape and plays a role in regulating chondrocyte phenotype. Most studies investigating regulation of the chondrocyte phenotype by the actin cytoskeleton use chondrocytes isolated from full-thickness (FT) cartilage, which has a heterogeneous cell population. Superficial zone chondrocytes (SZC) have an elongated morphology and account for 10-20% of chondrocytes, while the remaining chondrocytes in the deeper zones appear more rounded. This study characterizes the actin cytoskeleton and expression of actin-associated molecules in SZC and deep zone (DZ) chondrocytes (DZC) in vitro in order to identify molecules differentially expressed by SZC and DZC that may contribute to the observed differences in zonal chondrocyte shapes. Design: SZ, DZ, and FT chondrocytes isolated from bovine metacarpal-phalangeal joints were cultured in monolayer for 48 h. Macroscopic morphology, actin polymerization status, and expression of select actin-associated molecules (adseverin, cofilin, transgelin, vinculin, MRTF-A, and YAP/TAZ) were determined. Results: SZC appeared more elongated and have more filamentous actin compared to DZC, as determined by quantifying cell circularity and G-/F-actin ratio. MRTF-A gene and protein levels were significantly higher in SZC compared to DZC while DZC more highly expressed transgelin and TAZ. Although there was differential gene expression, no significant differences in adseverin, cofilin, vinculin, or YAP protein levels were observed between the two cell populations. Conclusions: This study identifies differences in actin polymerization status and expression of actin-associated molecules in primary SZC and DZC in vitro. These findings further our understanding of candidate actin-related pathways that may be regulating zonal chondrocyte phenotype.

American Society for Bone and Mineral Research-Orthopaedic Research Society Joint Task Force Report on Cell-Based Therapies.

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.

Transforming Growth Factor ß Enhances Tissue Formation by Passaged Nucleus Pulposus Cells In Vitro.

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 ß (TGFß) 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. TGFß1 or TGFß3 treatment enhanced the ability of cells at each passage to form tissue, in part by decreasing cell death. However, neither TGFß1 nor TGFß3 were able to restore the notochordal phenotype. Although TGFß1/3 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.