Stimulation Of Matrix Synthesis Research Articles

Mechanical stimuli activate gene expression via a cell envelope stress sensing pathway

Mechanosensitive mechanisms are often used to sense damage to tissue structure, stimulating matrix synthesis and repair. While this kind of mechanoregulatory process is well recognized in eukaryotic systems, it is not known whether such a process occurs in bacteria. In Vibrio cholerae, antibiotic-induced damage to the load-bearing cell wall promotes increased signaling by the two-component system VxrAB, which stimulates cell wall synthesis. Here we show that changes in mechanical stress within the cell envelope are sufficient to stimulate VxrAB signaling in the absence of antibiotics. We applied mechanical forces to individual bacteria using three distinct loading modalities: extrusion loading within a microfluidic device, direct compression and hydrostatic pressure. In all cases, VxrAB signaling, as indicated by a fluorescent protein reporter, was increased in cells submitted to greater magnitudes of mechanical loading, hence diverse forms of mechanical stimuli activate VxrAB signaling. Reduction in cell envelope stiffness following removal of the endopeptidase ShyA led to large increases in cell envelope deformation and substantially increased VxrAB response, further supporting the responsiveness of VxrAB. Our findings demonstrate a mechanosensitive gene regulatory system in bacteria and suggest that mechanical signals may contribute to the regulation of cell wall homeostasis.

Mechanical Signal-Tailored Hydrogel Microspheres Recruit and Train Stem Cells for Precise Differentiation.

The aberrant mechanical microenvironment in degenerated tissues induces misdirection of cell fate, making it challenging to achieve efficient endogenous regeneration. Herein, a hydrogel microsphere-based synthetic niche with integrated cell recruitment and targeted cell differentiation properties via mechanotransduction was constructed for enhanced endogenous regeneration. Through the incorporation of microfluidics and photo-polymerization strategies, we prepared fibronectin (Fn) modified methacrylated gelatin (GelMA) microspheres with the independently tunable elastic modulus(1-10Kpa) and ECM ligand density (2 and 10μg/ml), allowing a wide range of cytoskeleton modulation to trigger the corresponding mechanobiological signaling to direct cell fate. The combination of the soft matrix (2Kpa) and low ligand density (2μg/ml) could support the nucleus pulposus (NP)-like differentiation of intervertebral disc (IVD) progenitor/stem cells by translocating Yes-associated protein (YAP), without the addition of inducible biochemical factors. Meanwhile, platelet-derived growth factor-BB was loaded onto Fn-GelMA microspheres (PDGF@Fn-GelMA) via the heparin-binding domain of Fn and exhibited continuous release for over 28 days to initiate endogenous cell recruitment. In in vivo experiments, hydrogel microsphere-niche maintained the IVD structure and stimulated ECM synthesis, thereby enhancing the endogenous regeneration of NP. Overall, this synthetic niche with cell recruiting and mechanical training capabilities offered a promising novel strategy for endogenous tissue regeneration. This article is protected by copyright. All rights reserved.

Injectable bioorthogonal hydrogel (BIOGEL) accelerates tissue regeneration in degenerated intervertebral discs.

Intervertebral disc (IVD) degeneration is a leading cause of back pain and precursor to more severe conditions, including disc herniation and spinal stenosis. While traditional growth factor therapies (e.g., TGFβ) are effective at transiently reversing degenerated disc by stimulation of matrix synthesis, it is increasingly accepted that bioscaffolds are required for sustained, complete IVD regeneration. Current scaffolds (e.g., metal/polymer composites, non-mammalian biopolymers) can be improved in one or more IVD regeneration demands: biodegradability, noninvasive injection, recapitulated healthy IVD biomechanics, predictable crosslinking, and matrix repair induction. To meet these demands, tetrazine-norbornene bioorthogonal ligation was combined with gelatin to create an injectable bioorthogonal hydrogel (BIOGEL). The liquid hydrogel precursors remain free-flowing across a wide range of temperatures and crosslink into a robust hydrogel after 5-10min, allowing a human operator to easily inject the therapeutic constructs into degenerated IVD. Moreover, BIOGEL encapsulation of TGFβ potentiated histological repair (e.g., tissue architecture and matrix synthesis) and functional recovery (e.g., high water retention by promoting the matrix synthesis and reduced pain) in an in vivo rat IVD degeneration/nucleotomy model. This BIOGEL procedure readily integrates into existing nucleotomy procedures, indicating that clinical adoption should proceed with minimal difficulty. Since bioorthogonal crosslinking is essentially non-reactive towards biomolecules, our developed material platform can be extended to other payloads and degenerative injuries.

Epinephrine affects gene expression levels and has a complex effect on biofilm formation in Micrococcus luteus strain C01 isolated from human skin

In this study, the effect of epinephrine on the biofilm formation of Micrococcus luteus C01 isolated from human skin was investigated in depth for the first time. This hormone has a complex effect on biofilms in various systems. In a system with polytetrafluoroethylene (PTFE) cubes, treatment with epinephrine at a physiological concentration of 4.9 × 10−9 M increased the total amount of 72-h biofilm biomass stained with crystal violet and increased the metabolic activity of biofilms, but at higher and lower concentrations, the treatment had no significant effect. On glass fiber filters, treatment with the hormone decreased the number of colony forming units (CFUs) and changed the aggregation but did not affect the metabolic activity of biofilm cells. In glass bottom plates examined by confocal microscopy, epinephrine notably inhibited the growth of biofilms. RNA-seq analysis and RT–PCR demonstrated reproducible upregulation of genes encoding Fe–S cluster assembly factors and cyanide detoxification sulfurtransferase, whereas genes encoding the co-chaperone GroES, the LysE superfamily of lysine exporters, short-chain alcohol dehydrogenase and the potential c-di-GMP phosphotransferase were downregulated. Our results suggest that epinephrine may stimulate matrix synthesis in M. luteus biofilms, thereby increasing the activity of NAD(H) oxidoreductases. Potential c-di-GMP pathway proteins are essential in these processes.

Effect of BMP-2 with Microfracture on Osteochondral Defect in a Rabbits Talus

Category:Ankle, Basic Sciences/BiologicsIntroduction/Purpose:Microfracture is a technically safe, simple and cost-effective treatment option for osteochondral defect of the talus. However, fibrous tissue and fibrocartilage represent the predominant repairing tissues derived from these procedures and these tissues types do not exhibit the mechanical properties of native hyaline cartilage, long time for cartilage reconstruction and long-term failure results. Bone morphogenetic protein-2 (BMP-2) has been shown to stimulate matrix synthesis and increase in synthesis of cartilage-specific collagen type IIB thereby enhance regeneration of articular cartilage in vitro. The purpose of this study is to evaluate the treatment ability of combining BMP-2 with microfracture on osteochondral defect in rabbit talus.Methods:Full-thickness chondral defects (3 x 3 x 2 mm) was created in the center talus dome. Twenty four male white New Zealand rabbits were divided into four groups of 6 animals into each group dependent on treatment method; Group I (control, no treatment), II (microfracture only), III (10 µL rhBMP-2 treatment), IV (microfracture combine with 10 µL rhBMP-2 treatment). The animals were sacrificed at 2, 4, 6 weeks post operatively. The macroscopic assessment of the repaired tissue was evaluated using the International Cartilage Repair Society (ICRS) Macroscopic Score, which considers the degree of defect repair, the integration to the border zone, and the macroscopic appearance. Subchondral bone regeneration on defect was evaluated by micro-CT scan and the histological findings were scored by using a modified version of the established scoring system for osteochondral repair described by Wakitani, immunohistochemical staining was used for analysis of collagen type I and II.Results:: Excessive bone formation that originated from the subchondral bone region was not observed in any sample. Micro-CT shows higher subchondral bone regeneration on defect in group III, IV than group I, II at 4 and 6 weeks, respectively. According to macroscopic and histological results, group V shows higher quality of cartilage and faster cartilage regeneration compare with another group. In immunohistochemical analysis, group IV shows stronger positive staining for collagen type II in the area of the defect.Conclusion:These findings indicate that the combination BMP-2 with microfracture is effective at accelerating and improving quality of full-thickness cartilage defect repair in a rabbit talus model.

Long term cyclic compression of articular cartilage alters extracellular matrix turnover

Purpose: Osteoarthritis (OA) is a disease characterized by progressive degradation of articular cartilage extracellular matrix (ECM), Mechanical factors play a large role in the initiation and progression of OA, where mechanical compression at physiologic levels of cartilage is shown to have chondroprotective effects, activating anabolic processes and stimulating matrix synthesis. Previous studies suggest that mechanical loading of cartilage increases aggrecan and type II collagen gene expression, and reduce matrix metalloprotease formation. Biomarkers specifically targeting aggrecan and type II collagen turnover, provides a unique opportunity to study the dynamic effects of mechanical loading on cartilage ECM turnover. A better understanding of these processes might aid the development of novel DMOADs. The aim of this study was to investigate the effects of long-term cyclic compression on the cartilage ECM turnover, through measurement of specific cartilage turnover biomarkers, using a bovine articular cartilage ex vivo model. Methods: Full depth bovine cartilage explants (BEX) were cultured for 2 weeks. The explants were treated 3 times a week with either OSM [10 ng/mL] and TNF-a [2 ng/mL] (O+T), or TGF-B1 [50 ng/mL]. Untreated samples were included as negative controls (w/o). For each condition two groups were established; an unloaded group and a group compressed 3 times a week. Compression was applied using Electroforce 5500® (TA Instruments), in a sine wave with a maximum load per cycle of 1 MPa, at 1 Hz frequency for 1200 cycles. Metabolic activity was measured once a week using AlamarBlue. Biomarkers released to the supernatant were assessed using the following well-described ELISAs: type II collagen was measured by ProC2, MMP-degradation of type II collagen was measured by C2M, and aggrecanase-mediated degradation of aggrecan was measured by AGNx1. Sulfated glycosaminoglycans (GAGs) released to media was measured using a 1.9-dimethylmethylene blue (DMB) assay. Results: Compression of BEX significantly decreased the MMP-degradation of type II collagen measured by C2M at day 14 with 48% and 42% compared to the unloaded samples in the untreated and the O-T treated groups respectively (Figure 1c). The compressed samples release similar levels of AGNx1 and ProC2 to the unloaded explants (Figure 1b+d). The metabolic activity similarly appears to be unaltered by compression (Figure 1a). Compression significantly increased the GAG release in the w/o group by 195% and by 152% in the TGF-B group. Conclusions: Compression of cartilage, and compared to standard cultures, resulted in significant effect on cartilage tissue turnover and metabolic profile. Compression reduced the degradation of type II collagen, which indicates an inhibition of the involved catabolic mechanisms. Furthermore, type II collagen formation was unaltered, which in combination with the reduced degradation may result in an elevated deposition of collagen in the explants. While the AGNx1 release was unchanged by compression, the total amount of GAG was increased in the supernatants, suggesting an increase in proteoglycan turnover in the explants. In conclusion, compression of cartilage resulted in altered cartilage turnover, which was quantified by molecular cartilage turnover biomarkers. This approach may be essential for translation science, selecting and validating the best approaches to regenerate cartilage or block degradation.

Short Link N Stimulates Intervertebral Disc Repair in a Novel Long-Term Organ Culture Model that Includes the Bony Vertebrae.

Link N (DHLSDNYTLDHDRAIH) is a peptide that occurs naturally in the intervertebral discs (IVDs) and cartilage as a result of proteolytic cleavage of Link protein. Several studies have identified Link N as a growth factor capable of stimulating matrix synthesis in these tissues. We have recently discovered that annulus fibrosus cells can release an enzyme (possibly cathepsin K) that can further cleave Link N resulting in an eight amino acid peptide, we called short Link N (sLink N). Separately, we recently developed and validated an organ culture model that has the vertebrae attached (vIVDs; IVD with intact vertebrae). The aims of this study were (i) to examine if sLink N has the potential to repair early degenerate discs and (ii) to determine if this new model can be used to test potential drugs for disc repair. To determine if sLink N was able to stimulate repair of the degenerate disc, vIVDs with trypsin-induced degeneration (DG) were used. After 4 weeks of culture, the proteoglycan content measured as glycosaminoglycans was stimulated by sLink N in the degenerated discs, and the staining of proteoglycan was observed throughout the tissue irrespective of its proximity to the cells. The quantity of extractable type II collagen and aggrecan was also increased when the degenerate discs were treated with sLink N. Taken together, the results suggest that sLink N can increase key disc matrix molecules, namely type II collagen and aggrecan. Thus sLink N is an attractive peptide for tissue engineering and regeneration of the disc due to its anabolic effects. Finally, we show the feasibility of using the long-term whole organ culture system with adjacent intact vertebrae for studying the DG and regeneration of the IVD.

Adipose-derived stem cells improve the viability of nucleus pulposus cells in degenerated intervertebral discs.

Patients with degenerative disc disease (DDD) experience serious clinical symptoms, including chronic low back pain. A series of therapies have been developed to treat DDD, including physical therapy and surgical treatment. However, the therapeutic effect of such treatments has remained insufficient. Recently, stem cell‑based therapy, in which stem cells are injected into the nucleus pulposus in degenerated intervertebral disc tissue, has appeared to be effective in the treatment of DDD. In the present study, the effect of adipose‑derived stem cells on degenerated nucleus pulposus cells was investigated using a co‑culture system to evaluate the biological activity of degenerated nucleus pulposus cells. Human degenerated nucleus pulposus tissue was obtained from surgical specimens and the adipose‑derived stem cells were derived from adipose tissue. The degenerated nucleus pulposus cells were cultured in a mono‑culture or in a co‑culture with adipose‑derived stem cells using 0.4‑µm Transwell inserts. The results indicated that adipose‑derived stem cells were able to stimulate matrix synthesis and the cell proliferation of degenerated nucleus pulposus cells, promoting the restoration of nucleus pulposus cells in the degenerated intervertebral disc.

Nitric Oxide Stimulates Matrix Synthesis and Deposition by Adult Human Aortic Smooth Muscle Cells Within Three-Dimensional Cocultures

Vascular diseases are characterized by the over-proliferation and migration of aortic smooth muscle cells (SMCs), and degradation of extracellular matrix (ECM) within the vessel wall, leading to compromise in cell-cell and cell-matrix signaling pathways. Tissue engineering approaches to regulate SMC over-proliferation and enhance healthy ECM synthesis showed promise, but resulted in low crosslinking efficiency. Here, we report the benefits of exogenous nitric oxide (NO) cues, delivered from S-Nitrosoglutathione (GSNO), to cell proliferation and matrix deposition by adult human aortic SMCs (HA-SMCs) within three-dimensional (3D) biomimetic cocultures. A coculture platform with two adjacent, permeable 3D culture chambers was developed to enable paracrine signaling between vascular cells. HA-SMCs were cultured in these chambers within collagen hydrogels, either alone or in the presence of human aortic endothelial cells (HA-ECs) cocultures, and exogenously supplemented with varying GSNO dosages (0-100 nM) for 21 days. Results showed that EC cocultures stimulated SMC proliferation within GSNO-free cultures. With increasing GSNO concentration, HA-SMC proliferation decreased in the presence or absence of EC cocultures, while HA-EC proliferation increased. GSNO (100 nM) significantly enhanced the protein amounts synthesized by HA-SMCs, in the presence or absence of EC cocultures, while lower dosages (1-10 nM) offered marginal benefits. Multi-fold increases in the synthesis and deposition of elastin, glycosaminoglycans, hyaluronic acid, and lysyl oxidase crosslinking enzyme (LOX) were noted at higher GSNO dosages, and coculturing with ECs significantly furthered these trends. Similar increases in TIMP-1 and MMP-9 levels were noted within cocultures with increasing GSNO dosages. Such increases in matrix synthesis correlated with NO-stimulated increases in endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) expression within EC and SMC cultures, respectively. Results attest to the benefits of delivering NO cues to suppress SMC proliferation and promote robust ECM synthesis and deposition by adult human SMCs, with significant applications in tissue engineering, biomaterial scaffold development, and drug delivery.

Mechanism of parathyroid hormone-mediated suppression of calcification markers in human intervertebral disc cells

In degenerative intervertebral discs (IVD), type X collagen (COL X) expression (associated with hypertrophic differentiation) and calcification has been demonstrated. Suppression of COL X expression and calcification during disc degeneration can be therapeutic. In the present study we investigated the potential of human parathyroid hormone 1-34 (PTH) in suppressing indicators of calcification potential (alkaline phosphatase (ALP), Ca(2+), inorganic phosphate (Pi)), and COL X expression. Further, we sought to elucidate the mechanism of PTH action in annulus fibrosus (AF) and nucleus pulposus (NP) cells from human lumbar IVDs with moderate to advanced degeneration. Mitogen activated protein kinase (MAPK) signalling and alterations in the markers of calcification potential were analysed. PTH increased type II collagen (COL II) expression in AF (~200 %) and NP cells (~163 %) and decreased COL X levels both in AF and NP cells (~75 %). These changes in the expression of collagens were preceded by MAPK phosphorylation, which was increased in both AF and NP cells by PTH after 30 min. MAPK signalling inhibitor U0126 and protein kinase-A inhibitor H-89 DCH attenuated PTH stimulated COL II expression in both cell types. PTH decreased ALP activity and increased Ca(2+) release only in NP cells. The present study demonstrates that PTH can potentially retard IVD degeneration by stimulating matrix synthesis and suppressing markers of calcification potential in degenerated disc cells via both MAPK and PKA signalling pathways. Inhibition of further mineral deposition may therefore be a viable therapeutic option for improving the status of degenerating discs.

Transient Supplementation of Anabolic Growth Factors Rapidly Stimulates Matrix Synthesis in Engineered Cartilage

The purpose of the presented work is to examine the response of engineered cartilage to a transient, 2-week application of anabolic growth factors compared to continuous exposure in in vitro culture. Immature bovine chondrocytes were suspended in agarose hydrogel and cultured for 28 days (Study 1) or 42 days (Study 2) in chondrogenic media with TGF-β1, TGF-β3, or IGF-I either added for only the first 14 days in culture or added to the media for the entire study period. In both studies, there were no statistical differences in tissue mechanical or biochemical properties between the growth factors on day 14. In Study 1, growth factor removal led to a significant and drastic increase in Young's modulus and glycosaminoglycans content compared to continuously exposed controls on day 28. In Study 2, both TGF-β1 and β3 led to significantly higher mechanical properties and collagen content vs. IGF-I on day 42. These results indicate that the rapid rise in tissue properties (previously observed with TGF-β3 only) is not dependent on the type but rather the temporal application of the anabolic growth factor. These findings shed light on possible techniques to rapidly develop engineered cartilage tissue for the future treatment of osteoarthritis.

In Vivo Low-Intensity Pulsed Ultrasound (LIPUS) Following Tendon Injury Promotes Repair During Granulation but Suppresses Decorin and Biglycan Expression During Remodeling

Bench research, cross-sectional. To determine if the effects of low-intensity pulsed ultrasound (LIPUS) on matrix synthesis change at different stages of tendon healing. LIPUS is effective in promoting tendon healing by stimulation of matrix synthesis. The timing of initiation and duration of LIPUS treatment have been shown to affect its effectiveness to promote tendon healing, suggesting a change of tissue responses to LIPUS stimulation. Understanding how the cellular responses to LIPUS stimulation change during tendon healing is thus important. In a rat model of patellar tendon donor site injury, a single sonication of LIPUS or mock sonication was delivered to the injured knee of the rats on the fourth, 14th or 28th day postinjury. Tendon samples were harvested at 4 hours and 24 hours after sonication and the mRNA expression of COL1A1, COL3A1, decorin, biglycan, and TGF-beta 1 was analyzed. The results showed that a single sonication of LIPUS increased COL1A1 and COL3A1 mRNA in healing patellar tendons when administered on the fourth or 14th day postinjury, but not when administered on the 28th day postinjury. Both decorin and biglycan mRNA were decreased by treatment with LIPUS on the 28th day postinjury. Our results showed that LIPUS enhanced collagen synthesis in vivo only during the granulation phase. Matrix remodeling may be affected by LIPUS with the suppressed expression of decorin and biglycan. Our findings suggest that LIPUS should be applied during the granulation phase but not during the remodeling phase, to promote tendon healing.

Simvastatin Stimulates Chondrogenic Phenotype of Intervertebral Disc Cells Partially Through BMP-2 Pathway

In vitro experiment to study the effect of simvastatin on rat intervertebral disc (IVD) cells. To evaluate the time-course effect of simvastatin on the gene expression of bone morphogenetic protein-2 (BMP-2), aggrecan, and collagen type II in rat IVD cells cultured in alginate bead. Role of BMP-2 on the simvastatin-induced chondrogenesis of IVD cells was also investigated. Growth factors including BMP-2 have been found to improve anabolism of IVD cells and have shown promise for the treatment of disc degeneration. Statins is known to increase BMP-2 expression in vitro and stimulate bone formation in vivo. However, it is still unknown whether statins can also increase BMP-2 expression and in turn, stimulate matrix synthesis by IVD cells. Rat IVD cells (harvested from nucleus pulpusos and inner annular fibrosus) cultured in alginate beads were exposed to different doses of simvastatin. DMMB, and real-time polymerase chain reaction were used to quantify proteoglycan and gene expression of BMP-2, aggrecan and collagen type II, respectively. Noggin or mevalonate was used to investigate the mechanism of the effect of simvastatin on rat IVD cells. Simvastatin significantly upregulated BMP-2 mRNA expression, followed by aggrecan and type II collagen gene expression and proteoglycan content in rat IVD cells. Moderate dose (500 ng/mL) of noggin completely hindered the expression of aggrecan and collagen type II induced by simvastatin on day 7, but not on day 14. The upregulated type II collagen expression was blocked with 3 mug/mL of noggin on day 14, whereas aggrecan levels remained unchanged. Lastly, simvastatin appeared to facilitate BMP-2, aggrecan, and type II collagen gene expression by inhibiting the production of mevalonate as evidenced that the anabolic effect was completely reversed with the addition of mevalonate. Simvastatin drives a mechanism for promoting chondrogenesis of IVD cells partially mediated by upregulated BMP-2 through the inhibition of mevalonate pathway.

Effects of platelet rich plasma and acellular bone marrow on gene expression patterns and DNA content of equine suspensory ligament explant cultures

Suspensory ligament (SL) desmitis is a common source of lameness. The results of this study will determine if blood-derived products stimulate SL matrix synthesis and have potential as regenerative therapies for SL desmitis To determine if various blood-based biological products including plasma, blood, PRP, platelet poor plasma (PPP) and ABM aspirate stimulates anabolic and/or catabolic pathways in suspensory ligaments (SL). The body of the SL was harvested from 6 horses and used to establish explant cultures. Explants were cultured in plasma, blood, PRP, PPP or ABM at concentrations of 10, 50 or 100%. Anabolic responses were assessed by use of quantitative PCR for collagens type I and III, cartilage oligomeric matrix protein (COMP) and decorin. Total DNA and collagen protein content were also measured. Catabolic reactions were measured by quantitative PCR for matrix metalloproteinases 3 and 13 (MMP-3, MMP-13). Acellular bone marrow aspirate at 100% stimulated decorin and COMP mRNA synthesis more than all other treatments at all concentrations. No treatment at any concentration stimulated the catabolic gene MMP-13; only 50% ABM stimulated MMP-13 mRNA expression. Acellular bone marrow is indicated, and might be preferred to plasma, blood or PPP, as a blood-based biological source for SL tissue regenerative therapy. Long-term, placebo controlled case studies are indicated to determine if ABM aids in recovery from SL desmitis. Bone marrow aspirate is an autogenous, readily available biological source for SL regenerative therapy where the aim is to stimulate matrix synthesis.

Gentherapie für traumatische Defekte des Gelenkknorpels

Recent advances in gene transfer technology have provided novel tools for its therapeutic use in the regeneration of focal articular cartilage defects. Current strategies aiming at improving chondrogenic pathways in the new repair tissue that fills such defects include the stimulation of chondrocyte proliferation and maturation as well as the stimulation of matrix synthesis via direct or ex vivo approaches. To target gene vectors to cartilage defects, a localized intraarticular delivery into the defect of therapeutic gene constructs is necessary. This review outlines the basic scientific aspects of cartilage defect repair, defines the regenerative options that might be applied, summarizes the progress that has been made to date, and analyses some of the unique obstacles that need to be resolved before a clinical translation of gene therapy for cartilage defects may be considered.

Prevention of disc degeneration with growth factors

Clinically, a large number of patients have persistent low back pain attributable to intervertebral disc (IVD) degeneration. After the concept of biologically regenerating the degenerated IVD using growth factor injection was first proposed in early 1990, the advancement of molecular technology to produce recombinant proteins, including growth factors, on an industrial scale accelerated research in this field. The purpose of this review is to summarize the most recent findings of the in vitro and in vivo effects of growth factors on the IVD and, further, to discuss the limitations of growth factor therapy and its clinical implications. In vitro data showed that stimulation of matrix synthesis by growth factors alters the balance of homeostasis by shifting cellular metabolism to the anabolic state. In vivo data using small animals has shown the possibility of using growth factors as a "structural modifying therapy". Based on in vitro and in vivo data previously reported, the clinical application of growth factors by direct injection of protein into the nucleus pulposus or anulus fibrosus was shown to be feasible as a new therapeutic intervention for treatment of disc degeneration. Stimulation of the biological repair process will create a new category of therapy to treat disc degeneration, where no active treatment currently exists, between conservative therapies and more aggressive therapies such as fusion or disc replacement. However, it should be noted that there are several important factors to be taken into consideration. In a relatively advanced degenerative condition, the supply of nutrients is disturbed and stimulation of cellular activity by growth factors may result in an increased demand for nutrients, eventually inducing an adverse event. Further investigations of the optimal environment for growth factor stimulation should be pursued. Growth factor therapy, which has experimental evidence supporting it to be a "structural modifying therapy", may not be a "symptom modifying therapy" that is able to resolve the symptoms associated with pathologic changes. Therefore, further studies on the effect of growth factor therapy on pain are essential to shed light on its therapeutic usefulness for degenerative disc disease.

Mesangial autoantigens in IgA nephropathy: matrix synthesis and localization

Primary IgA nephropathy, a chronic nephritis with variable prognosis, is characterized by mesangial immunoglobulin A, frequently with codeposition of other immunoglobulin isotypes and complement components accompanying matrix expansion typically preceding glomerular scarring. Glomerular immunoglobulin G, when present, is localized to the mesangial periphery found variably in repeat biopsies. IgG anti-mesangial cell autoantibodies (IgG-MESCA) in sera of patients with IgA nephropathy, specific by F(ab')(2) binding to 48- and 55-kD autoantigen(s) could account for these deposits, but their in vivo localization, and the functional role in promoting scarring is unknown. A specific monoclonal antibody raised previously to these human mesangial cell autoantigen fractions, in this study localized to similar glomerular sites, reinforcing the view that immunoglobulin G deposition in vivo is a result of antibody-autoantigen binding. The propensity for immunoglobulin G more than other isotypes to enhance inflammation prompted study of its functional role in vitro. Using cultured human mesangial cells in a complement-free tritiated glycosaminoglycan synthesis single outcome assay, purified IgG fractions from patient sera increased matrix production in a dose-dependent manner compared with controls. At a constant total IgG concentration, matrix synthesis was proportional to the titre of IgG-MESCA. Autoreactive IgG stimulated matrix synthesis when compared with controls or IgA fractions. These findings are consistent with IgG-MESCA autoantibodies enhancing mesangial matrix synthesis in vitro, which suggests that in IgA nephropathy, similar prosclerotic autoimmune mechanisms might operate. Recombinant TGFbeta(1) also induced matrix synthesis, raising the possibility that both autoimmune mechanisms and those TGFbeta(1)-dependent are functional or inter-related. The pathogenesis of glomerular scarring and loss in IgA nephropathy may include, in part, these mechanisms.

Fenofibrate modifies human vascular smooth muscle proteoglycans and reduces lipoprotein binding

Vascular disease in type 2 diabetes is associated with an up-regulation of atherogenic growth factors, which stimulate matrix synthesis including proteoglycans. We have examined the direct actions of fenofibrate on human vascular smooth muscle cells (VSMCs) and have specifically investigated proteoglycan synthesis and binding to LDL. Proteoglycans synthesised by human VSMCs treated with fenofibrate (30 micromol/l) were assessed for binding to human LDL using a gel mobility shift assay, metabolically labelled with [(35)S]-sulphate and quantitated by cetylpyridinium chloride. They were then assessed for electrophoretic mobility by SDS-PAGE, for size by gel filtration, for sulphation pattern by fluorophore-assisted carbohydrate electrophoresis, and for glycosaminoglycan (GAG) composition by enzyme digestion. Proteoglycans synthesised in the presence of fenofibrate showed an increase in the half-maximum saturation concentration of LDL from 36.8+/-12.4 microg/ml to 77.7+/-17 microg/ml under basal conditions, from 24.9+/-4.6 microg/ml to 39.1+/-6.1 microg/ml in the presence of TGF-beta1, and from 9.5+/-4.4 microg/ml to 31.1+/-3.4 microg/ml in the presence of platelet-derived growth factor/insulin. Fenofibrate treatment in the presence of TGF-beta1 inhibited the incorporation of [(35)S]-sulphate into secreted and cell-associated proteoglycans synthesised by human VSMCs by 59.2% (p<0.01) and 39.8% (p<0.01) respectively. The changes in sulphate incorporation following treatment with fenofibrate were associated with a concentration-related increase in the electrophoretic mobility due to a reduction in GAG length. There was no change in the sulphation pattern; however, there was an alteration in the disaccharide composition of the GAGs. Fenofibrate modifies the structure of vascular proteoglycans by reducing the length of the GAG chains and GAG composition, resulting in reduced binding to human LDL, a mechanism which may lead to a reduction of atherosclerosis and cardiovascular disease in people with diabetes treated with fenofibrate.

Dual transduction of insulin-like growth factor-I and interleukin-1 receptor antagonist protein controls cartilage degradation in an osteoarthritic culture model

This study evaluated the potential of gene induced synoviocyte expression of a combination of insulin-like growth factor-I (AdIGF-I) and interleukin-1 receptor antagonist protein (AdIL-1Ra) to control articular cartilage degradation in vitro. Cartilage explants and synovial membrane were harvested from young mature horses. Synovial monolayers were established and either (1) maintained as untransduced controls; (2) transduced with AdIGF-I at 200 MOI in 500 μl serum-free medium; (3) transduced with AdIL-1Ra at 100 MOI; or (4) transduced with a combination of AdIGF-I (200 MOI) and AdIL-1Ra (100 MOI). Following transduction, cartilage explants were exposed to the synovial monolayer medium using co-culture inserts. Cultures were maintained for 6 days in either serum-free medium or medium containing 10 ng/ml recombinant human interleukin-1β. At termination, synovial cell RNA was isolated for real-time PCR analysis, and cartilage explants were collected for H&E and toluidine blue staining, immunohistochemistry for type II collagen and IGF-I, in situ localization of IGF-I and type II collagen gene expression, and biochemical assays. Synovial monolayers were readily transduced with both AdIGF-I and AdIL-1Ra. IGF-I and IL-1Ra protein were secreted at beneficial levels throughout the experiment, having peak concentrations of 94.6 ng/ml and 33.0 ng/ml, respectively. Transduction with IGF-I promoted cartilage production of proteoglycan and type II collagen, suggesting a beneficial role for healing injured cartilage. Transduction with IL-1Ra decreased the synovial expression of IL-1α and IL-1β and matrix metalloproteinases, indicating a mechanism for prevention of matrix degradation. The beneficial effects of the combination of anabolic growth factors and catabolic blockers were evident in improved preservation of proteoglycan content of cartilage explants exposed to the depleting effects of IL-1. These results show that gene therapy combining anabolic growth factors to stimulate matrix synthesis and catabolic blockers to prevent matrix degradation by IL-1, protects and causes partial restoration of cartilage matrix, and suggest a potential benefit of combination gene therapy for cartilage healing.

Expression of bone morphogenetic protein 6 in healthy and osteoarthritic human articular chondrocytes and stimulation of matrix synthesis in vitro.

To elucidate the role of bone morphogenetic protein 6 (BMP-6) in human articular cartilage, we investigated whether BMP-6 is expressed in adult human articular chondrocytes and analyzed the potential stimulatory effects of BMP-6 on these cells. In addition, we investigated whether osteoarthritic (OA) and normal cartilage chondrocytes behave differently. Endogenous expression of the BMP-6 gene was examined by reverse transcription-polymerase chain reaction. BMP-6 protein was detected by Western immunoblotting. Chondrocytes were grown as monolayer cultures for 7 days in a chemically defined serum-free medium, in the absence or presence of recombinant BMP-6. Proteoglycan (PG) synthesis was measured by (35)S-sulfate incorporation into newly synthesized macromolecules. Cell proliferation was assessed by (3)H-thymidine incorporation. BMP-6 was expressed in both healthy and OA chondrocytes at the messenger RNA and protein levels. Total PG synthesis was significantly increased after BMP-6 stimulation of healthy (mean +/- SEM 191 +/- 11%; P < 0.001) and OA (150 +/- 25%; P < 0.03) chondrocyte cultures. A direct comparison between healthy and OA samples revealed no significant difference. The proliferation rates of normal and OA chondrocytes were not affected by BMP-6 treatment. BMP-6 is endogenously expressed in chondrocytes obtained from OA and normal adult human articular cartilage. Furthermore, BMP-6 has the potential to stimulate total PG synthesis in human articular chondrocytes derived from normal as well as OA joints. We conclude that the presence of BMP-6 in adult human articular cartilage indicates a functional role for this growth factor in the maintenance of joint integrity.