Supplementation with platelet-rich plasma improves the in vitro formation of tissue-engineered cartilage with enhanced mechanical properties.

PURPOSE: This study aimed to determine the effects of platelet-rich plasma (PRP) on the histologic, biochemical, and biomechanical properties of tissue-engineered cartilage. METHODS: Chondrocytes isolated from bovine metacarpal-phalangeal articular cartilage were seeded on top of a porous ceramic substrate (calcium polyphosphate [CPP]). Cultures were supplemented with fetal bovine serum (FBS), PRP, or platelet-poor plasma (PPP) at 5%. On day 5, the concentration was increased to 20%. PRP and PPP were obtained through centrifugation of whole blood withdrawn from a mature cow. After 2 weeks, samples (n = 8) were analyzed histologically, biochemically, and biomechanically. Data were analyzed using the Wilcoxon test (significance, P < .05). RESULTS: Chondrocytes cultured in 20% PRP formed thicker cartilage tissue (1.6 ± 0.2 mm) than did cells grown in 20% FBS (0.7 ± 0.008 mm; P = .002) and 20% PPP (0.8 ± 0.2 mm; P = .03). Cartilage tissue generated in the presence of 20% PRP had a greater equilibrium modulus of 38.1 ± 3.6 kPa versus 15.6 ± 1.5 kPa (P = .0002) for 20% PPP and 20.4 ± 3.5 kPa (P = .007) for 20% FBS. Glycosaminoglycan (GAG) content was increased in tissues formed in 20% PRP (176 ± 18.8 µg GAG/mg) compared with those grown in 20% FBS (112 ± 10.6 µg GAG/mg; P = .01) or 20% PPP (131.5 ± 14.8 µg GAG/mg; P = .11). Hydroxyproline content was similar whether the media was supplemented with 20% PRP (8.7 ± 0.9 µg/mg), 20% FBS (7.6 ± 0.9 µg/mg; P = .37), or 20% PPP (6.4 ± 1 µg/mg; P = .28). DNA content was similar in all tissues whether formed in 20% PRP (11.9 ± 3.5 µg/mg), 20% FBS (9.3 ± 2.5 µg/mg; P = .99), or 20% PPP (7.2 ± 1.3 µg/mg; P = .78). Immunostained samples showed prevalence of type II collagen in tissues formed in the presence of 20% PRP. CONCLUSIONS: The presence of PRP in the culture media enhances the in vitro formation of cartilage, with increased GAG content and greater compressive mechanical properties, while maintaining characteristics of hyaline phenotype. CLINICAL RELEVANCE: Understanding the in vitro effects of PRP on tissue-engineered cartilage may lead to the creation of engineered cartilage tissue with enhanced properties suitable for cartilage repair.

Enhanced levels of non-enzymatic glycation and pentosidine crosslinking in spontaneous osteoarthritis progression.

OBJECTIVE: To test the hypothesis that heightened advanced glycation endproducts (AGEs) content in cartilage accelerates the progression of spontaneous osteoarthritis (OA) in the Hartley guinea pig (HGP) model. METHODS: Twenty-eight male, 3-month-old HGPs were used. Eight were left untreated as a baseline control group and sacrificed at 3 months of age (n = 4) and 9 months of age (n = 4; age-matched controls). The other 20 HGPs received intra-articular knee injections in the right knee whereas the left knees acted as contra-lateral non-injected controls. Injections consisted of 100 µl phosphate buffered saline (PBS; n = 10) or PBS+2.0 M D-(-)-Ribose (n = 10). Injections were given once weekly for 24 weeks. At the end of the treatment period, the tibiae were fixed with formalin, scanned with microCT for sub-chondral bone mineral density, and then histological slides were prepared, stained with Safranin-O with Fast Green counter stain and scored using the OARSI-HISTOgp scheme. Cartilage pentosidine (established biomarker for AGEs) content, collagen content (% dry mass), glucosaminoglycan GAG-to-collagen ratio (µg/µg), GAG-to-DNA ratio and DNA-to-collagen ratio were measured. RESULTS: Pentosidine content increased greatly due to PBS + Ribose injection (P < 0.0001) and reached levels found in cartilage from 80-year-old humans. Surprisingly, mean OARSI-HISTOgp scores for both the injected and contra-lateral controls in the PBS + Ribose group were not detectably different, nor were they different from the mean score for the age-matched control group. CONCLUSION: AGEs accumulation due to intra-articular ribose-containing injections in the HGP model of spontaneous knee OA did not enhance disease progression.

Integration of tissue-engineered cartilage with host cartilage: an in vitro model.

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.

Calcium regulates cyclic compression-induced early changes in chondrocytes during in vitro cartilage tissue formation.

A single application of cyclic compression (1kPa, 1Hz, 30min) to bioengineered cartilage results in improved tissue formation through sequential catabolic and anabolic changes mediated via cell shape changes that are regulated by α5ß1 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.

Solid freeform fabrication and characterization of porous calcium polyphosphate structures for tissue engineering purposes.

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 microm 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.

Improved bioengineered cartilage tissue formation following cyclic compression is dependent on upregulation of MT1-MMP.

The generation of bioengineered cartilage tissue suitable for transplantation is a potential therapy to treat damaged cartilage. We have shown previously that the physical and biomechanical properties of bioengineered cartilage can be improved by the application of 30 min of cyclic compression by a mechanism involving sequential upregulation of gene and protein levels of membrane type-1 matrix metalloproteinase (MT1-MMP) and MMP-13. In the current study, we demonstrated that MT1-MMP is critical to this response, as blocking the upregulation of MT1-MMP prevented the improvement in tissue formation. MT1-MMP seems to act by inducing tissue remodeling as evidenced by the presence of aggrecan degradation products by Western blot analysis and increased release of matrix molecules into the media. Release of these molecules was diminished when MT1-MMP upregulation was prevented. This matrix degradation was likely due to MT1-MMP, as under conditions where MMP-13 expression is maintained (stimulation in the presence of MT1-MMP siRNA) the release of these matrix molecules into the media was still prevented. It also appears that MT1-MMP does not regulate MMP-13 gene expression, as MT1-MMP-siRNA pretreatment had no effect on MMP-13 expression following mechanical stimulation. Further analysis of the anabolic genes and proteins involved in mechanically stimulated cartilage will lead to better understanding of the mechanism(s) underlying tissue formation yielding improved bioengineered cartilage.

Articular cartilage subpopulations respond differently to cyclic compression in vitro.

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.

AP-1 DNA binding activity regulates the cartilage tissue remodeling process following cyclic compression in vitro.

Generating bioengineered cartilage yields tissue with physical qualities inferior to that of native tissue. Application of cyclic compression (30 min, 1 kPa, 1 Hz) to cartilage cells (chondrocytes) seeded on calcium polyphosphate substrates significantly increases the accumulation of collagens and proteoglycans by 24 hours, thus improving the tissue generated. The mechanism for this increase is not fully known but seems to follow a remodeling pathway of sequential catabolic and anabolic changes. The initial catabolic event involves increased transcription of matrix metalloproteinase (MMP)-3 and MMP-13 two hours after the end of cyclic compression. As MMP-3 and MMP-13 promoters contain activating protein-1 (AP-1) DNA binding sites, we investigated the effect of inhibiting DNA binding through the use of modified decoy oligodeoxynucleotides (ODN). Mechanical stimulation in the presence of the ODN blocked AP-1 DNA binding as detected by electrophoretic mobility shift assay and prevented the increased transcription of MMP-3 and MMP-13. As well the increased accumulation of collagens and proteoglycans by 24 hours in mechanically stimulated samples was prevented. The data suggests that the mechano-induction of MMP-3 and MMP-13 may be regulated at the AP-1 DNA binding site and that upregulation of these metalloproteases is a necessary component of the matrix remodeling initiated by cyclic compression.

Cold acclimation-induced up-regulation of the ribosomal protein L7 gene in the freeze tolerant wood frog, Rana sylvatica.

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. Transcript levels of rpl7 were elevated up to 4-fold in skin of cold-acclimated frogs as compared with warm-acclimated animals. Organ-specific responses by rpl7 transcripts also occurred when frogs were given survivable freezing exposures. Transcripts rose by 1.8-3.3 fold in brain and skeletal muscle during freezing but were unaffected in central organs such as liver and heart. Up-regulation of rpl7 also occurred in brain of anoxia-exposed frogs and RPL7 protein levels increased strongly in heart under both freezing and dehydration stresses. Cold- and freezing-responsive up-regulation of the rpl7 gene and RPL7 protein in selected organs suggests that targeted changes in selected ribosomal proteins may be an integral part of natural freeze tolerance.

Membrane type-1 matrix metalloproteinase is induced following cyclic compression of in vitro grown bovine chondrocytes.

OBJECTIVE: 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. METHODS: Cyclic compression (30min, 1kPa, 1Hz) 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. RESULTS: After cyclic compression, MT1-MMP showed a rapid and transient increase in gene expression. Elevated protein levels were detected within 2h of stimulation which returned to baseline by 6h. 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. CONCLUSIONS: 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.

Cyclic compressive mechanical stimulation induces sequential catabolic and anabolic gene changes in chondrocytes resulting in increased extracellular matrix accumulation.

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.

Upregulation of the mitochondrial phosphate carrier during freezing in the wood frog Rana sylvatica: potential roles of transporters in freeze tolerance.

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.

Polymer hydrogels usable for nervous tissue repair.

The implantation of non-resorbable biocompatible polymer hydrogels into defects in the central nervous system can reduce glial scar formation, bridge the lesion and lead to tissue regeneration within the hydrogel. We implanted hydrogels based on crosslinked poly hydroxyethyl-methacrylate (pHEMA) and poly N-(2-hydroxypropyl)-methacrylamide (pHPMA) into the rat cortex and evaluated the cellular invasion into the hydrogels by means of immunohistochemical methods and tetramethylammonium diffusion measurements. Astrocytes and NF160-positive axons grew similarly into both types of hydrogels. We found no cell types other than astrocytes in the pHEMA hydrogels. In the pHPMA hydrogels, we found a massive ingrowth of connective tissue elements. These changes were accompanied by corresponding changes in the extracellular space volume fraction and tortuosity of the hydrogels.

Habitat association in two genetic groups of the insect-pathogenic fungus metarhizium anisopliae: uncovering cryptic species?

Strains of insect-pathogenic fungi with high virulence toward certain pest insects have great potential for commercial biological control applications. Identifying such strains has been a central theme in using fungi for biological control. This theme is supported by a persistent paradigm in insect pathology which suggests that the host insect is the predominant influence on the population genetics of insect-pathogenic fungi. In this study, a population genetics analysis of the insect-pathogenic fungus Metarhizium anisopliae from forested and agricultural habitats in Ontario, Canada, showed a nonrandom association of alleles between two distinct, reproductively isolated groups (index of multilocus association = 1.2). Analyses of the mitochondrial DNA showed no differences between the groups. The two groups were associated with different habitat types, and associations with insect hosts were not found. The group from forested areas showed an ability for cold-active growth (i.e., 8 degrees C), while the group from the agricultural area showed an ability for growth at high temperatures (i.e., 37 degrees C) and resilience to UV exposure. These results represent a significant paradigm shift; habitat selection, not host insect selection, drives the population structure of these insect-pathogenic deuteromycetous fungi. With each group we observed recombining population structures as well as clonally reproducing lineages. We discuss whether these groups may represent cryptic species. Worldwide, M. anisopliae may be an assembly of cryptic species, each adapted to certain environmental conditions. The association of fungal genotypes with habitat but not with host insects has implications on the criteria for utility of this, and perhaps other, fungal biocontrol agents.