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Papers by Joseph De Croos

Cell Calcium, 2010

A single application of cyclic compression (1 kPa, 1 Hz, 30 min) to bioengineered cartilage resul... more A single application of cyclic compression (1 kPa, 1 Hz, 30 min) to bioengineered cartilage results in improved tissue formation through sequential catabolic and anabolic changes mediated via cell shape changes that are regulated by α5β1 integrin and membrane-type metalloprotease (MT1-MMP). To determine if calcium was involved in this process, the role of calcium in regulating cell shape changes, MT1-MMP expression and integrin activity in response to mechanical stimulation was examined. Stimulation-induced changes in cell shape and MT1-MMP expression were abolished by chelation of extracellular calcium, and this effect was reversed by re-introduction of calcium. Spreading was inhibited by blocking stretch-activated channels (with gadolinium), while retraction was prevented by blocking the L-Type voltage-gated channel (with nifedipine); both compounds inhibited MT1-MMP upregulation. Calcium A23187 ionophore restored cellular response further supporting a role for these channels. Calcium regulated the integrin-mediated signalling pathway, which was facilitated through Src kinase. Both calcium- and integrin-mediated pathways converged on ERK-MAPK in response to stimulation. While both integrins and calcium signalling mediate chondrocyte mechanotransduction, calcium appears to play the major regulatory role. Understanding the underlying molecular mechanisms involved in chondrocyte mechanotransduction may lead to the development of improved bioengineered cartilage.

Osteoarthritis and Cartilage, 2007

To determine if membrane type-1 matrix metalloproteinase (MT1-MMP) will respond to cyclic compres... more To determine if membrane type-1 matrix metalloproteinase (MT1-MMP) will respond to cyclic compression of chondrocytes grown in vitro and the regulatory mechanisms underlying this response.Cyclic compression (30 min, 1 kPa, 1 Hz) was applied to bovine chondrocytes (6–9-month-old animals) grown on top of a biodegradable substrate within 3 days of initiating culture. Luciferase assays using bovine articular chondrocytes were undertaken to demonstrate the mechanosensitivity of MT1-MMP. Semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR) and western blot analysis were used to establish the time course of gene and protein upregulation in response to cyclic compression. The regulation of MT1-MMP was assessed by electrophoretic mobility shift assays, RT-PCR and western blot analysis. As well, an MT1-MMP decoy oligonucleotide and an extracellular signal-regulated kinase 1/2 (ERK1/2) pharmacological inhibitor were utilized to further characterize MT1-MMP regulation.After cyclic compression, MT1-MMP showed a rapid and transient increase in gene expression. Elevated protein levels were detected within 2 h of stimulation which returned to baseline by 6 h. During cyclic compression, phosphorylation of the mitogen activated protein kinase ERK1/2 increased significantly. This was followed by increased gene and protein expression of the transcription factor; early growth factor-1 (Egr-1) and Egr-1 binding to the MT1-MMP promoter. Blocking Egr-1 DNA binding with a decoy MT1-MMP oligonucleotide, downregulated MT1-MMP gene expression. The ERK1/2 inhibitor U0126 also reduced Egr-1 DNA binding activity to MT1-MMP promoter sequences and subsequent transcription of MT1-MMP.These data suggest that cyclic compression of chondrocytes in vitro upregulates MT1-MMP via ERK1/2 dependent activation of Egr-1 binding. Delineation of the regulatory pathways activated by mechanical stimulation will further our understating of the mechanisms influencing tissue remodeling.

Tissue Engineering Part A, 2009

The inferior biomechanical properties of in vitro-formed tissue remain a significant obstacle in ... more The inferior biomechanical properties of in vitro-formed tissue remain a significant obstacle in bioengineering articular cartilage tissue. We have previously shown that cyclic compression (30 minutes, 1 kPa, 1 Hz) of chondrocytes isolated from full-thickness cartilage can induce greater matrix synthesis, although articular cartilage is composed of different subpopulations of chondrocytes, and their individual contribution to enhanced tissue formation has not been fully characterized. This study examines the contribution of chondrocyte subpopulations to this response. Bovine articular chondrocytes were isolated from superficial to mid zones (SMZs) or deep zones (DZs), placed in three-dimensional culture, and subjected to cyclic compression. DZ chondrocytes on calcium polyphosphate substrates formed thicker tissue than those from SMZs. Compression increased matrix accumulation in SMZ chondrocytes while decreasing accumulation in DZ chondrocytes. The SMZ and DZ chondrocytes also differed in their type 1 membrane-bound matrix metalloproteinase (MMP) and MMP-13 expression, enzymes that play a crucial role in mediating the response to mechanical stimulation. In addition, the duration of the culture period was important in determining the DZ response, raising the possibility that matrix accumulation plays a role in the response to stimulation. Understanding the cellular response to mechanical stimulation during tissue formation will facilitate our understanding of tissue growth and allow for further optimization of cartilage tissue formation in vitro.

Clinical Orthopaedics and Related Research

Background We developed a tissue-engineered biphasic cartilage bone substitute construct which ha... more Background We developed a tissue-engineered biphasic cartilage bone substitute construct which has been shown to integrate with host cartilage and differs from autologous osteochondral transfer in which integration with host cartilage does not occur. Questions/purposes (1) Develop a reproducible in vitro model to study the mechanisms regulating tissue-engineered cartilage integration with host cartilage, (2) compare the integrative properties of tissue-engineered cartilage with autologous cartilage and (3) determine if chondrocytes from the in-vitro formed cartilage migrate across the integration site. Methods A biphasic construct was placed into host bovine osteochondral explant and cultured for up to 8 weeks (n = 6 at each time point). Autologous osteochondral implants served as controls (n = 6 at each time point). Integration was evaluated histologically, ultrastructurally, biochemically and biomechanically. Chondrocytes used to form cartilage in vitro were labeled with carboxyfluorescein diacetate which allowed evaluation of cell migration into host cartilage. Results Histologic assessment demonstrated that tissue-engineered cartilage integrated over time, unlike autologous osteochondral implant controls. Biochemically there was an increase in collagen content of the tissue-engineered implant over time but was well below that for native cartilage. Integration strength increased between 4 and 8 weeks as determined by a pushout test. Fluorescent cells were detected in the host cartilage up to 1.5 mm from the interface demonstrating chondrocyte migration. Conclusions Tissue-engineered cartilage demonstrated improved integration over time in contrast to autologous osteochondral implants. Integration extent and strength increased with culture duration. There was chondrocyte migration from tissue-engineered cartilage to host cartilage. Clinical Relevance This in vitro integration model will allow study of the mechanism(s) regulating cartilage integration. Understanding this process will facilitate enhancement of cartilage repair strategies for the treatment of chondral injuries.

Matrix Biology, 2006

Overcoming the limited ability of articular cartilage to self-repair may be possible through tiss... more Overcoming the limited ability of articular cartilage to self-repair may be possible through tissue engineering. However, bioengineered cartilage formed using current methods does not match the physical properties of native cartilage. In previous studies we demonstrated that mechanical stimulation improved cartilage tissue formation. This study examines the mechanisms by which this occurs. Application of uniaxial, cyclic compression (1 kPa, 1 Hz, 30 min) significantly increased matrix metalloprotease (MMP)-3 and MMP-13 gene expression at 2 h compared to unstimulated cells. These returned to constitutive levels by 6 h. Increased MMP-13 protein levels, both pro- and active forms, were detected at 6 h and these decreased by 24 h. This was associated with tissue degradation as more proteoglycans and collagen had been released into the culture media at 6 h when compared to the unstimulated cells. This catabolic change was followed by a significant increase in type II collagen and aggrecan gene expression at 12 h post-stimulation and increased synthesis and accumulation of these matrix molecules at 24 h. Mechanical stimulation activated the MAP kinase pathway as there was increased phosphorylation of ERK1/2 and JNK as well as increased AP-1 binding. Mechanical stimulation in the presence of the JNK inhibitor, SP600125, blocked AP-1 binding preventing the increased gene expression of MMP-3 and -13 at 2 h and type II collagen and aggrecan at 12 h as well as the increased matrix synthesis and accumulation. Given the sequence of changes, cyclic compressive loading appears to initiate a remodelling effect involving MAPK and AP-1 signalling resulting in improved in vitro formation of cartilage.

Journal of Biomedical Materials Research Part B-applied Biomaterials, 2010

Solid freeform fabrication (SFF) enables the fabrication of anatomically shaped porous components... more Solid freeform fabrication (SFF) enables the fabrication of anatomically shaped porous components required for formation of tissue engineered implants. This article reports on the characterization of a three-dimensional-printing method, as a powder-based SFF technique, to create reproducible porous structures composed of calcium polyphosphate (CPP). CPP powder of 75–150 μm was mixed with 10 wt % polyvinyl alcohol (PVA) polymeric binder, and used in the SFF machine with appropriate settings for powder mesh size. The PVA binder was eliminated during the annealing procedure used to sinter the CPP particles. The porous SFF fabricated components were characterized using scanning electron microscopy, micro-CT scanning, X-ray diffraction, and mercury intrusion porosimetry. In addition, mechanical testing was conducted to determine the compressive strength of the CPP cylinders. The 35 vol % porous structures displayed compressive strength on average of 33.86 MPa, a value 57% higher than CPP of equivalent volume percent porosity made through conventional gravity sintering. Dimensional deviation and shrinkage analysis was conducted to identify anisotropic factors required for dimensional compensation during SFF sample formation and subsequent sintering. Cell culture studies showed that the substrate supported cartilage formation in vitro, which was integrated with the top surface of the porous CPP similar to that observed when chondrocytes were grown on CPP formed by conventional gravity sintering methods as determined histologically and biochemically. © 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2010

Gene, 2008

Natural freezing survival by the wood frog, Rana sylvatica, involves multiple organ-specific chan... more Natural freezing survival by the wood frog, Rana sylvatica, involves multiple organ-specific changes in gene expression. The present study used differential display PCR to find cold-responsive genes in wood frog skin. A cDNA was retrieved from skin that was in higher amounts in cold-versus warm-acclimated frogs. The cDNA was used to probe a wood frog liver cDNA library and retrieve a long sequence that, after the further application of 5′RACE, was shown to encode the full sequence of the ribosomal large subunit protein 7 (RPL7) (GenBank accession number AF175983). Wood frog RPL7 contained 246 amino acids and shared 90% identity with Xenopus laevis RPL7, 82-83% with chicken and zebrafish homologues, and 79% with mammalian RPL7. Multiple binding domains found in human RPL7 showed differing degrees of conservation in the frog protein.

Journal of Bioenergetics and Biomembranes, 2004

Screening of a cDNA library prepared from liver of the freeze-tolerant wood frog (Rana sylvatica)... more Screening of a cDNA library prepared from liver of the freeze-tolerant wood frog (Rana sylvatica) identified a freeze-responsive clone containing a 1370-nt sequence with an open reading frame of 360 amino acids. Sequence analysis revealed 84-86% identity with the mammalian inorganic phosphate carrier (PiC) that spans the inner mitochondrial membrane. Northern blot analysis showed that pic transcript levels increased over a time course of freezing, reaching 60-fold upregulation after 24-h frozen. Transcript levels were also assessed under freezing-related stresses with results showing a strong increase in pic transcript levels in response to dehydration (elevated 9.0-fold in 40% dehydrated frogs) but not under anoxia. Western blotting revealed elevated PiC protein over a time course of freeze-thaw whereas other mitochondrial carriers (dicarboxylate carrier, oxoglutarate transporter) of the same family were not affected by freezing. This modulation of PiC protein levels may play a role in mitochondrial ionic and/or osmotic balance during freeze-induced cell volume reduction.

Mycological Research, 2001

The entomopathogenic fungus Metarhizium anisopliae is generally considered to be mesophylic, alth... more The entomopathogenic fungus Metarhizium anisopliae is generally considered to be mesophylic, although some isolates have the ability to grow at 8 °C. In two cold-active isolates (DAT-1 from Tasmania and CLB2-1vi from Ontario, Canada) and one non-cold-active isolate (Ma 2575 from South Carolina), we (1) analyzed cold-induced intracellular proteins, using two-dimensional (2D) gel electrophoresis, and (2) analyzed cold-active transcripts, using a polymerase chain reaction (PCR) based subtractive hybridization technique. Protein differences, as observed by 2D gel electrophoresis, were observed in cold active isolates grown at 8° when compared with 25°, but such differences were not observed in the non-cold active isolate. Fungi were also subjected to various stresses (45°, pH, and salinity) to determine how proteins induced under these conditions compared to proteins induced under low temperature growth. Proteins induced were either specific to cold-active growth or were general stress proteins and differences were also observed in 2D patterns between the two cold-active isolates. Transcripts upregulated during growth at 8° and isolated by a PCR based subtractive hybridization technique also differed between the two cold-active isolates. Analysis of the transcripts showed several novel sequences but also included transcripts with similarities to actin, NADPH quinone oxidoreductase, a thiamine biosynthesis protein and a yeast-like membrane protein. The potential role of these proteins in cold-active growth is discussed.

Canadian Journal of Microbiology, 1999