Numerical assessment on the effective mechanical stimuli for matrix-associated metabolism in chondrocyte-seeded constructs

The self-regeneration capacity of articular cartilage is limited, due to its avascular and aneural nature. Loaded explants and cell cultures demonstrated that chondrocyte metabolism can be regulated via physiologic loading. However, the explicit ranges of mechanical stimuli that correspond to favourable metabolic response associated with extracellular matrix (ECM) synthesis are elusive. Unsystematic protocols lacking this knowledge produce inconsistent results. This study aims to determine the intrinsic ranges of physical stimuli that increase ECM synthesis and simultaneously inhibit nitric oxide (NO) production in chondrocyte-agarose constructs, by numerically re-evaluating the experiments performed by Tsuang et al. (2008). Twelve loading patterns were simulated with poro-elastic finite element models in ABAQUS. Pressure on solid matrix, von Mises stress, maximum principle stress and pore pressure were selected as intrinsic mechanical stimuli. Their development rates and magnitudes at the steady state of cyclic loading were calculated with MATLAB at the construct level. Concurrent increase in glycosaminoglycan and collagen was observed at 2300 Pa pressure and 40 Pa/s pressure rate. Between 0-1500 Pa and 0-40 Pa/s, NO production was consistently positive with respect to controls, whereas ECM synthesis was negative in the same range. A linear correlation was found between pressure rate and NO production (R = 0.77). Stress states identified in this study are generic and could be used to develop predictive algorithms for matrix production in agarose-chondrocyte constructs of arbitrary shape, size and agarose concentration. They could also be helpful to increase the efficacy of loading protocols for avascular tissue engineering. Copyright (c) 2010 John Wiley \& Sons, Ltd.

The in vitro effects of dexamethasone, insulin and triiodothyronine on degenerative human intervertebral disc cells under normoxic and hypoxic conditions

Degeneration of intervertebral discs (IVD) is one of the main causes of back pain and tissue engineering has been proposed as a treatment. Tissue engineering requires the use of highly expensive growth factors, which might, in addition, lack regulatory approval for human use. In an effort to find readily available differentiation factors, we tested three molecules – dexamethasone, triiodothyronine (T3) and insulin – on human IVD cells isolated after surgery, expanded in vitro and transferred into alginate beads. Triplicates containing 40 ng/ml dexamethasone, 10 nM T3 and 10 µg/ml insulin, together with a positive control (10 ng/mL transforming growth factor (TGF)-beta 1), were sampled weekly over six weeks and compared to a negative control. Furthermore, we compared the results to cultures with optimized chondrogenic media and under hypoxic condition (2% O2). Glycosaminoglycan (GAG) determination by Alcian Blue assay and histological staining showed dexamethasone to be more effective than T3 and insulin, but less than TGF-beta1. DNA quantification showed that only dexamethasone stimulated cell proliferation. qPCR demonstrated that TGF-beta1 and the optimized chondrogenic groups increased the expression of collagen type II, while aggrecan was stimulated in cultures containing dexamethasone. Hypoxia increased GAG accumulation, collagen type II and aggrecan expression, but had no effect on or even lowered cell number. In conclusion, dexamethasone is a valuable and cost-effective molecule for chondrogenic and viability induction of IVD cells under normoxic and hypoxic conditions, while insulin and T3 did not show significant differences.

Bone apposition to a titanium-zirconium alloy implant, as compared to two other titanium-containing implants

Implants made of commercially pure titanium (cpTi) are widely and successfully used in dentistry. For certain indications, diameter-reduced Ti alloy implants with improved mechanical strength are highly desirable. The aim was to compare the osseointegration of titanium-zirconium (TiZr) and cpTi implants with a modified sandblasted and acid-etched (SLActive) surface and with a Ti6Al4V alloy that was sand-blasted and acid-washed. Cylindrical implants with two, 0.75 mm deep, circumferential grooves were placed in the maxilla of miniature pigs and allowed to heal for 1, 2, 4 and 8 weeks. Undecalcified toluidine blue-stained ground sections were produced. Surface topography, area fraction of tissue components, and bone-to-implant contact (BIC) were determined. All materials showed significantly different surface roughness parameters. The amount of new bone within the implant grooves increased over time, without significant differences between materials. However, BIC values were significantly related to the implant material and the healing period. For TiZr and cpTi implants, the BIC increased over time, reaching values of 59.38 % and 76.15 % after 2 weeks, and 74.50 % and 84.67 % after 8 weeks, respectively. In contrast, the BIC for Ti6Al4V implants peaked with 42.29 % after 2 weeks followed by a decline to 28.60 % at 8 weeks. Significantly more surface was covered by multinucleated giant cells on Ti6Al4V implants after 4 and 8 weeks. In conclusion, TiZr and cpTi implants showed faster osseointegration than Ti6Al4V implants. Both chemistry and surface topography might have influenced the results. The use of diameter-reduced TiZr implants in more challenging clinical situations warrants further documentation in long-term clinical studies.

Differential effects of dexamethasone on the chondrogenesis of mesenchymal stromal cells: influence of microenvironment, tissue origin and growth factor

Mesenchymal stromal cells (MSCs), which reside within various tissues, are utilized in the engineering of cartilage tissue. Dexamethasone (DEX)--a synthetic glucocorticoid--is almost invariably applied to potentiate the growth-factor-induced chondrogenesis of MSCs in vitro, albeit that this effect has been experimentally demonstrated only for transforming-growth-factor-beta (TGF-β)-stimulated bone-marrow-derived MSCs. Clinically, systemic glucocorticoid therapy is associated with untoward side effects (e.g., bone loss and increased susceptibility to infection). Hence, the use of these agents should be avoided or limited. We hypothesize that the influence of DEX on the chondrogenesis of MSCs depends upon their tissue origin and microenvironment [absence or presence of an extracellular matrix (ECM)], as well as upon the nature of the growth factor. We investigated its effects upon the TGF-β1- and bone-morphogenetic-protein 2 (BMP-2)-induced chondrogenesis of MSCs as a function of tissue source (bone marrow vs. synovium) and microenvironment [cell aggregates (no ECM) vs. explants (presence of a natural ECM)]. In aggregates of bone-marrow-derived MSCs, DEX enhanced TGF-β1-induced chondrogenesis by an up-regulation of cartilaginous genes, but had little influence on the BMP-2-induced response. In aggregates of synovial MSCs, DEX exerted no remarkable effect on either TGF-β1- or BMP-2-induced chondrogenesis. In synovial explants, DEX inhibited BMP-2-induced chondrogenesis almost completely, but had little impact on the TGF-β1-induced response. Our data reveal that steroids are not indispensable for the chondrogenesis of MSCs in vitro. Their influence is context dependent (tissue source of the MSCs, their microenvironment and the nature of the growth-factor). This finding has important implications for MSC based approaches to cartilage repair.

Electrospun PLLA nanofiber scaffolds and their use in combination with BMP-2 for reconstruction of bone defects

Introduction Adequate migration and differentiation of mesenchymal stem cells is essential for regeneration of large bone defects. To achieve this, modern graft materials are becoming increasingly important. Among them, electrospun nanofiber scaffolds are a promising approach, because of their high physical porosity and potential to mimic the extracellular matrix (ECM). Materials and Methods The objective of the present study was to examine the impact of electrospun PLLA nanofiber scaffolds on bone formation in vivo, using a critical size rat calvarial defect model. In addition we analyzed whether direct incorporation of bone morphogenetic protein 2 (BMP-2) into nanofibers could enhance the osteoinductivity of the scaffolds. Two critical size calvarial defects (5 mm) were created in the parietal bones of adult male Sprague-Dawley rats. Defects were either (1) left unfilled, or treated with (2) bovine spongiosa, (3) PLLA scaffolds alone or (4) PLLA/BMP-2 scaffolds. Cranial CT-scans were taken at fixed intervals in vivo. Specimens obtained after euthanasia were processed for histology, histomorphometry and immunostaining (Osteocalcin, BMP-2 and Smad5). Results PLLA scaffolds were well colonized with cells after implantation, but only showed marginal ossification. PLLA/BMP-2 scaffolds showed much better bone regeneration and several ossification foci were observed throughout the defect. PLLA/BMP-2 scaffolds also stimulated significantly faster bone regeneration during the first eight weeks compared to bovine spongiosa. However, no significant differences between these two scaffolds could be observed after twelve weeks. Expression of osteogenic marker proteins in PLLA/BMP-2 scaffolds continuously increased throughout the observation period. After twelve weeks osteocalcin, BMP-2 and Smad5 were all significantly higher in the PLLA/BMP-2 group than in all other groups. Conclusion Electrospun PLLA nanofibers facilitate colonization of bone defects, while their use in combination with BMP-2 also increases bone regeneration in vivo and thus combines osteoconductivity of the scaffold with the ability to maintain an adequate osteogenic stimulus.

β1-integrin/matrix interactions support blood-brain barrier integrity

Brain microvascular endothelium forms an active permeability barrier, the blood-brain barrier (BBB). In neurologic disorders, barrier properties of the BBB are often lost indicating their dependance on molecular cues of the brain microenvironment. In this issue, Osada et al demonstrate that the endothelial extracellular matrix (ECM) provides one of these cues. Their study shows that β1-integrin-mediated adhesion of brain endothelial cells to the surrounding ECM is critical for stabilizing claudin-5 in BBB tight junctions (TJs) and BBB integrity. These observations point to a novel intracellular signaling pathway from β1-integrin/ECM endothelial adhesions to BBB TJs contributing to BBB integrity.

Single application of low-dose mycophenolate mofetil-OX7-immunoliposomes ameliorates experimental mesangial proliferative glomerulonephritis

IgA nephropathy, one of the most frequent forms of glomerulonephritis, characterized by mesangial hypercellularity and glomerular extracellular matrix (ECM) expansion, often leads to end-stage renal disease over a prolonged period. We investigated whether antiproliferative treatment in a single low dose specifically targeted to the glomerular mesangium by immunoliposomes (ILs) results in an amelioration of mesangial proliferative glomerulonephritis in rats (anti-Thy1.1 nephritis). Mycophenolate mofetil (MMF) containing ILs was generated that targets the Thy1.1 antigen (OX-7) in rat mesangial cells. Treatment benefit of a single intravenous dose of these ILs given 2 days after disease induction was investigated by stereology, immunohistochemistry, and functional analyses (creatinine, albuminuria) until day +9 and was compared among untreated and free MMF-treated rats using six male Wistar rats per group. MMF-loaded OX7-IL prevented creatinine increase and albuminuria. Stereological analyses of MMF OX7-IL-treated animals yielded 30% reduction of mesangial cells on day +9 and a 40% reduction of glomerular ECM volume on day +5, compared with all of the other nephritic animals. Furthermore, at days +5 and +9 we observed decreased ECM content and decreased glomerular volume (day +5) in the MMF-OX7-IL-treated group compared with the nephritic group treated with free MMF. In conclusion, MMF-OX7-IL-based directed drug delivery represents a novel approach for treating mesangial cell-mediated forms of glomerulonephritis.

Putative functions of extracellular matrix glycoproteins in secondary palate morphogenesis

Cleft palate is a common birth defect in humans. Elevation and fusion of paired palatal shelves are coordinated by growth and transcription factors, and mutations in these can cause malformations. Among the effector genes for growth factor signaling are extracellular matrix (ECM) glycoproteins. These provide substrates for cell adhesion (e.g., fibronectin, tenascins), but also regulate growth factor availability (e.g., fibrillins). Cleft palate in Bmp7 null mouse embryos is caused by a delay in palatal shelf elevation. In contrast, palatal shelves of Tgf-β3 knockout mice elevate normally, but a cleft develops due to their failure to fuse. However, nothing is known about a possible functional interaction between specific ECM proteins and Tgf-β/Bmp family members in palatogenesis. To start addressing this question, we studied the mRNA and protein distribution of relevant ECM components during secondary palate development, and compared it to growth factor expression in wildtypewild type and mutant mice. We found that fibrillin-2 (but not fibrillin-1) mRNA appeared in the mesenchyme of elevated palatal shelves adjacent to the midline epithelial cells, which were positive for Tgf-β3 mRNA. Moreover, midline epithelial cells started expressing fibronectin upon contact of the two palatal shelves. These findings support the hypothesis that fibrillin-2 and fibronectin are involved in regulating the activity of Tgf-β3 at the fusing midline. In addition, we observed that tenascin-W (but not tenascin-C) was misexpressed in palatal shelves of Bmp7-deficient mouse embryos. In contrast to tenascin-C, tenascin-W secretion was strongly induced by Bmp7 in embryonic cranial fibroblasts in vitro. These results are consistent with a putative function for tenascin-W as a target of Bmp7 signaling during palate elevation. Our results indicate that distinct ECM proteins are important for morphogenesis of the secondary palate, both as downstream effectors and as regulators of Tgf-β/Bmp activity.

Evaluation of chemically modified SLA implants (modSLA) biofunctionalized with integrin (RGD)- and heparin (KRSR)-binding peptides

Enhancing osseointegration through surface immobilization of multiple short peptide sequences that mimic extracellular matrix (ECM) proteins, such as arginine-glycine-aspartic acid (RGD) and lysine-arginine-serine-arginine (KRSR), has not yet been extensively explored. Additionally, the effect of biofunctionalizing chemically modified sandblasted and acid-etched surfaces (modSLA) is unknown. The present study evaluated modSLA implant surfaces modified with RGD and KRSR for potentially enhanced effects on bone apposition and interfacial shear strength during early stages of bone regeneration. Two sets of experimental implants were placed in the maxillae of eight miniature pigs, known for their rapid wound healing kinetics: bone chamber implants creating two circular bone defects for histomorphometric analysis on one side and standard thread configuration implants for removal torque testing on the other side. Three different biofunctionalized modSLA surfaces using poly-L-lysine-graft-poly(ethylene glycol) (PLL-g-PEG) as a carrier minimizing nonspecific protein adsorption [(i) 20 pmol cm⁻² KRSR alone (KRSR); or in combination with RGD in two different concentrations; (ii) 0.05 pmol cm⁻² RGD (KRSR/RGD-1); (iii) 1.26 pmol cm⁻² RGD (KRSR/RGD-2)] were compared with (iv) control modSLA. Animals were sacrificed at 2 weeks. Removal torque values (701.48-780.28 N mm), bone-to-implant contact (BIC) (35.22%-41.49%), and new bone fill (28.58%-30.62%) demonstrated no significant differences among treatments. It may be concluded that biofunctionalizing modSLA surfaces with KRSR and RGD derivatives of PLL-g-PEG polymer does not increase BIC, bone fill, or interfacial shear strength.

Low-dose oral rapamycin treatment reduces fibrogenesis, improves liver function, and prolongs survival in rats with established liver cirrhosis

BACKGROUND/AIMS: Mammalian target of rapamycin (mTOR) signalling is central in the activation of hepatic stellate cells (HSCs), the key source of extracellular matrix (ECM) in fibrotic liver. We tested the therapeutic potential of the mTOR inhibitor rapamycin in advanced cirrhosis. METHODS: Cirrhosis was induced by bile duct-ligation (BDL) or thioacetamide injections (TAA). Rats received oral rapamycin (0.5 mg/kg/day) for either 14 or 28 days. Untreated BDL and TAA-rats served as controls. Liver function was quantified by aminopyrine breath test. ECM and ECM-producing cells were quantified by morphometry. MMP-2 activity was measured by zymography. mRNA expression of procollagen-alpha1, transforming growth factor-beta1 (TGF-beta1) and beta2 was quantified by RT-PCR. RESULTS: Fourteen days of rapamycin improved liver function. Accumulation of ECM was decreased together with numbers of activated HSCs and MMP-2 activity in both animal models. TGF-beta1 mRNA was downregulated in TAA, TGF-beta2 mRNA was downregulated in BDL. 28 days of rapamycin treatment entailed a survival advantage of long-term treated BDL-rats. CONCLUSIONS: Low-dose rapamycin treatment is effectively antifibrotic and attenuates disease progression in advanced fibrosis. Our results warrant the clinical evaluation of rapamycin as an antifibrotic drug.

Oral imatinib treatment reduces early fibrogenesis but does not prevent progression in the long term

BACKGROUND/AIMS: Transactivated hepatic stellate cells (HSCs) represent the key source of extra cellular matrix (ECM) in fibrotic liver. Imatinib, a potent inhibitor of the PDGF receptor tyrosine kinase, reduces HSC proliferation and fibrogenesis when treatment is initiated before fibrosis has developed. We tested the antifibrotic potential of imatinib in ongoing liver injury and in established fibrosis. METHODS: BDL-rats were gavage fed with 20 mg/kg/d imatinib either early (days 0-21) or late (days 22-35) after BDL. Untreated BDL-rats served as controls. ECM and activated HSCs were quantified by morphometry. Tissue activity of MMP-2 was determined by gelatin zymography. mRNA expression of TIMP-1 and procollagen alpha1(I) were measured by RT-PCR. Liver tissue concentration of imatinib was measured by tandem mass spectrometry. RESULTS: Early imatinib reduced ECM formation by 30% (P=0.0455) but left numbers of activated HSCs and procollagen I expression unchanged. MMP-2 activity and TIMP-1 expression were reduced by 50%. Late imatinib treatment did not alter histological or molecular markers of fibrogenesis despite high imatinib tissue levels. CONCLUSIONS: The antifibrotic effectiveness of imatinib is limited to the early phase of fibrogenesis. In ongoing liver injury other mediators most likely compensate for the inhibited PDGF effect.

Influence of diurnal hyperosmotic loading on the metabolism and matrix gene expression of a whole-organ intervertebral disc model

It is generally agreed that the mechanical environment of intervertebral disc cells plays an important role in maintaining a balanced matrix metabolism. The precise mechanism by which the signals are transduced into the cells is poorly understood. Osmotic changes in the extracellular matrix (ECM) are thought to be involved. Current in-vitro studies on this topic are mostly short-term and show conflicting data on the reaction of disc cells subjected to osmotic changes which is partially due to the heterogenous and often substantially-reduced culture systems. The aim of the study was therefore to investigate the effects of cyclic osmotic loading for 4 weeks on metabolism and matrix gene expression in a full-organ intervertebral disc culture system. Intervertebral disc/endplate units were isolated from New Zealand White Rabbits and cultured either in iso-osmotic media (335 mosmol/kg) or were diurnally exposed for 8 hours to hyper-osmotic conditions (485 mosmol/kg). Cell viability, metabolic activity, matrix composition and matrix gene expression profile (collagen types I/II and aggrecan) were monitored using Live/Dead cell viability assay, tetrazolium reduction test (WST 8), proteoglycan and DNA quantification assays and quantitative PCR. The results show that diurnal osmotic stimulation did not have significant effects on proteoglycan content, cellularity and disc cell viability after 28 days in culture. However, hyperosmolarity caused increased cell death in the early culture phase and counteracted up-regulation of type I collagen gene expression in nucleus and annulus cells. Moreover, the initially decreased cellular dehydrogenase activity recovered with osmotic stimulation after 4 weeks and aggrecan gene down-regulation was delayed, although the latter was not significant according to our statistical criteria. In contrast, collagen type II did not respond to the osmotic changes and was down-regulated in both groups. In conclusion, diurnal hyper-osmotic stimulation of a whole-organ disc/endplate culture partially inhibits a matrix gene expression profile as encountered in degenerative disc disease and counteracts cellular metabolic hypo-activity.

Distribution of lactate dehydrogenase in healthy and degenerative canine stifle joint cartilage

In dogs, degenerative joint diseases (DJD) have been shown to be associated with increased lactate dehydrogenase (LDH) activity in the synovial fluid. The goal of this study was to examine healthy and degenerative stifle joints in order to clarify the origin of LDH in synovial fluid. In order to assess the distribution of LDH, cartilage samples from healthy and degenerative knee joints were investigated by means of light and transmission electron microscopy in conjunction with immunolabeling and enzyme cytochemistry. Morphological analysis confirmed DJD. All techniques used corroborated the presence of LDH in chondrocytes and in the interterritorial matrix of healthy and degenerative stifle joints. Although enzymatic activity of LDH was clearly demonstrated in the territorial matrix by means of the tetrazolium-formazan reaction, immunolabeling for LDH was missing in this region. With respect to the distribution of LDH in the interterritorial matrix, a striking decrease from superficial to deeper layers was present in healthy dogs but was missing in affected joints. These results support the contention that LDH in synovial fluid of degenerative joints originates from cartilage. Therefore, we suggest that (1) LDH is transferred from chondrocytes to ECM in both healthy dogs and dogs with degenerative joint disease and that (2) in degenerative joints, LDH is released from chondrocytes and the ECM into synovial fluid through abrasion of cartilage as well as through enhanced diffusion as a result of increased water content and degradation of collagen.

Follicle-forming cat thyroid cell lines synthesizing extracellular matrix and basal membrane components: a new tool for the study of thyroidal morphogenesis

Interactions between follicular epithelial cells and extracellular matrix (ECM) are supposed to play an important role in the development and maintenance of thyroid tissue architecture. In the present study we have therefore investigated the synthesis of ECM components by a feline thyroid cell line which is able to form follicle-like structures in vitro, and also in v-ras-transfected and control-transfected sublines. Transfections were performed by lipofection with pZSR (viral Harvey ras gene; neo) and pSV2-neo (control, neo only) plasmids. We have adapted a semisolid culture system composed exclusively of polymerized alginate and therefore devoid of ECM components. Feline cells embedded in alginate gels as single cells and cultured for up to 90 days formed cell clusters within 10 days. Follicle-like structures were formed in the original cell lines and also in the v-ras- and control-transfected cells. Differences in proliferation rates were observed, the v-ras-transfected cells growing up to two to three times faster than the non-transfected cells. Immunostaining was done using rabbit first antibodies directed against mouse collagen IV, human fibronectin, laminin (tumor Engelbreth-Holm-Swarm laminin), perlecan and other ECM components. For comparison, immunostaining was also performed on cryosections of nodular goiters of six hyperthyroid cats. The cell lines and their transfected clones stained strongly positive for collagen IV and fibronectin, and positively but less strongly for laminin and perlecan. The cat goiter tissue stained positively for collagen IV, laminin, perlecan, and fibronectin, and positive staining for S-laminin (containing the beta2-chain) was seen in blood vessel walls in this tissue. In conclusion, cat cell lines grow three-dimensionally in alginate beads over several weeks, they form follicle-like structures and express the same ECM components as the native cat goiter tissue. Transfection with v-ras does increase proliferation rate, but does not fundamentally alter formation of follicle-like structures and ECM expression. Alginate gel culture is a promising new tool for the study of follicular morphogenesis, polarity, the expression pattern of ECM components and of the interaction between thyrocytes and ECM. It avoids interference caused by gels composed of ECM components.