Current concepts in osteogenesis imperfecta: bone structure, biomechanics and medical management.

The majority of patients with osteogenesis imperfecta (OI) have mutations in the COL1A1 or COL1A2 gene, which has consequences for the composition of the bone matrix and bone architecture. The mutations result in overmodified collagen molecules, thinner collagen fibres and hypermineralization of bone tissue at a bone matrix level. Trabecular bone in OI is characterized by a lower trabecular number and connectivity as well as a lower trabecular thickness and volumetric bone mass. Cortical bone shows a decreased cortical thickness with less mechanical anisotropy and an increased pore percentage as a result of increased osteocyte lacunae and vascular porosity. Most OI patients have mutations at different locations in the COL1 gene. Disease severity in OI is probably partly determined by the nature of the primary collagen defect and its location with respect to the C-terminus of the collagen protein. The overall bone biomechanics result in a relatively weak and brittle structure. Since this is a result of all of the above-mentioned factors as well as their interactions, there is considerable variation between patients, and accurate prediction on bone strength in the individual patient with OI is difficult. Current treatment of OI focuses on adequate vitamin-D levels and interventions in the bone turnover cycle with bisphosphonates. Bisphosphonates increase bone mineral density, but the evidence on improvement of clinical status remains limited. Effects of newer drugs such as antibodies against RANKL and sclerostin are currently under investigation. This paper was written under the guidance of the Study Group Genetics and Metabolic Diseases of the European Paediatric Orthopaedic Society.

Optimisation of high-quality total ribonucleic acid isolation from cartilaginous tissues for real-time polymerase chain reaction analysis.

OBJECTIVES: Studies which consider the molecular mechanisms of degeneration and regeneration of cartilaginous tissues are seriously hampered by problematic ribonucleic acid (RNA) isolations due to low cell density and the dense, proteoglycan-rich extracellular matrix of cartilage. Proteoglycans tend to co-purify with RNA, they can absorb the full spectrum of UV light and they are potent inhibitors of polymerase chain reaction (PCR). Therefore, the objective of the present study is to compare and optimise different homogenisation methods and RNA isolation kits for an array of cartilaginous tissues. MATERIALS AND METHODS: Tissue samples such as the nucleus pulposus (NP), annulus fibrosus (AF), articular cartilage (AC) and meniscus, were collected from goats and homogenised by either the MagNA Lyser or Freezer Mill. RNA of duplicate samples was subsequently isolated by either TRIzol (benchmark), or the RNeasy Lipid Tissue, RNeasy Fibrous Tissue, or Aurum Total RNA Fatty and Fibrous Tissue kits. RNA yield, purity, and integrity were determined and gene expression levels of type II collagen and aggrecan were measured by real-time PCR. RESULTS: No differences between the two homogenisation methods were found. RNA isolation using the RNeasy Fibrous and Lipid kits resulted in the purest RNA (A260/A280 ratio), whereas TRIzol isolations resulted in RNA that is not as pure, and show a larger difference in gene expression of duplicate samples compared with both RNeasy kits. The Aurum kit showed low reproducibility. CONCLUSION: For the extraction of high-quality RNA from cartilaginous structures, we suggest homogenisation of the samples by the MagNA Lyser. For AC, NP and AF we recommend the RNeasy Fibrous kit, whereas for the meniscus the RNeasy Lipid kit is advised.Cite this article: M. Peeters, C. L. Huang, L. A. Vonk, Z. F. Lu, R. A. Bank, M. N. Helder, B. Zandieh Doulabi. Optimisation of high-quality total ribonucleic acid isolation from cartilaginous tissues for real-time polymerase chain reaction analysis. Bone Joint Res 2016;5:560-568. DOI: 10.1302/2046-3758.511.BJR-2016-0033.R3.

Microsphere size influences the foreign body reaction.

Biodegradable poly-(DL-lactide-co-glycolide) (PLGA) microspheres (MSP) are attractive candidate vehicles for site-specific or systemic sustained release of therapeutic compounds. This release may be altered by the host's foreign body reaction (FBR), which is dependent on the characteristics of the implant, e.g. chemistry, shape or size. In this study, we focused on the characterisation of the influence of MSP size on the FBR. To this end we injected monodisperse MSP of defined size (small 5.8 µm, coefficient of variance (CV) 14 % and large 29.8 µm, CV 4 %) and polydisperse MSP (average diameter 34.1 µm, CV 51 %) under the skin of rats. MSP implants were retrieved at day 7, 14 and 28 after transplantation. The FBR was studied in terms of macrophage infiltration, implant encapsulation, vascularisation and extracellular matrix deposition. Although PLGA MSP of all different sizes demonstrated excellent in vitro and in vivo biocompatibility, significant differences were found in the characteristics of the FBR. Small MSP were phagocytosed, while large MSP were not. Large MSP occasionally elicited giant cell formation, which was not observed after implantation of small MSP. Cellular and macrophage influx and collagen deposition were increased in small MSP implants compared to large MSP. We conclude that the MSP size influences the FBR and thus might influence clinical outcome when using MSP as a drug delivery device. We propose that a rational choice of MSP size can aid in optimising the therapeutic efficacy of microsphere-based therapies in vivo.

Gene expression analysis of murine and human osteoarthritis synovium reveals elevation of transforming growth factor ß-responsive genes in osteoarthritis-related fibrosis.

OBJECTIVE: Synovial fibrosis is a major contributor to joint stiffness in osteoarthritis (OA). Transforming growth factor ß (TGFß), which is elevated in OA, plays a key role in the onset and persistence of synovial fibrosis. However, blocking of TGFß in OA as a therapeutic intervention for fibrosis is not an option since TGFß is crucial for cartilage maintenance and repair. Therefore, we undertook the present study to seek targets downstream of TGFß for preventing OA-related fibrosis without interfering with joint homeostasis. METHODS: Experiments were performed to determine whether genes involved in extracellular matrix turnover were responsive to TGFß and were elevated in OA-related fibrosis. We analyzed gene expression in TGFß-stimulated human OA synovial fibroblasts and in the synovium of mice with TGFß-induced fibrosis, mice with experimental OA, and humans with end-stage OA. Gene expression was determined by microarray, low-density array, or quantitative polymerase chain reaction analysis. RESULTS: We observed an increase in expression of procollagen genes and genes encoding collagen crosslinking enzymes under all of the OA-related fibrotic conditions investigated. Comparison of gene expression in TGFß-stimulated human OA synovial fibroblasts, synovium from mice with experimental OA, and synovium from humans with end-stage OA revealed that the genes PLOD2, LOX, COL1A1, COL5A1, and TIMP1 were up-regulated in all of these conditions. Additionally, we confirmed that these genes were up-regulated by TGFß in vivo in mice with TGFß-induced synovial fibrosis. CONCLUSION: Most of the up-regulated genes identified in this study would be poor targets for therapy development, due to their crucial functions in the joint. However, the highly up-regulated gene PLOD2, responsible for the formation of collagen crosslinks that make collagen less susceptible to enzymatic degradation, is an attractive and promising target for interference in OA-related synovial fibrosis.

A morphological and functional comparison of proximal tubule cell lines established from human urine and kidney tissue.

Promising renal replacement therapies include the development of a bioartificial kidney using functional human kidney cell models. In this study, human conditionally immortalized proximal tubular epithelial cell (ciPTEC) lines originating from kidney tissue (ciPTEC-T1 and ciPTEC-T2) were compared to ciPTEC previously isolated from urine (ciPTEC-U). Subclones of all ciPTEC isolates formed tight cell layers on Transwell inserts as determined by transepithelial resistance, inulin diffusion, E-cadherin expression and immunocytochemisty. Extracellular matrix genes collagen I and -IV α1 were highly present in both kidney tissue derived matured cell lines (p<0.001) compared to matured ciPTEC-U, whereas matured ciPTEC-U showed a more pronounced fibronectin I and laminin 5 gene expression (p<0.01 and p<0.05, respectively). Expression of the influx carrier Organic Cation Transporter 2 (OCT-2), and the efflux pumps P-glycoprotein (P-gp), Multidrug Resistance Protein 4 (MRP4) and Breast Cancer Resistance Protein (BCRP) were confirmed in the three cell lines using real-time PCR and Western blotting. The activities of OCT-2 and P-gp were sensitive to specific inhibition in all models (p<0.001). The highest activity of MRP4 and BCRP was demonstrated in ciPTEC-U (p<0.05). Finally, active albumin reabsorption was highest in ciPTEC-T2 (p<0.001), while Na(+)-dependent phosphate reabsorption was most abundant in ciPTEC-U (p<0.01). In conclusion, ciPTEC established from human urine or kidney tissue display comparable functional PTEC specific transporters and physiological characteristics, providing ideal human tools for bioartificial kidney development.

Relation between radiological assessment and biomechanical stability of lumbar interbody fusion in a large animal model.

PURPOSE: To relate the progress of vertebral segmental stability after interbody fusion surgery with radiological assessment of spinal fusion. METHODS: Twenty goats received double-level interbody fusion and were followed for a period of 3, 6 and 12 months. After killing, interbody fusion was assessed radiographically by two independent observers. Subsequently, the lumbar spines were subjected to four-point bending and rotational deformation, assessed with an optoelectronic 3D movement registration system. In addition, four caprine lumbar spines were analysed in both the native situation and after the insertion of a cage device, as to mimic the direct post-surgical situation. The range of motion (ROM) in flexion/extension, lateral bending and axial rotation was analysed ex vivo using a multi-segment testing system. RESULTS: Significant reduction in ROM in the operated segments was already achieved with moderate bone ingrowth in flexion/extension (71 % reduction in ROM) and with only limited bone ingrowth in lateral bending (71 % reduction in ROM) compared to the post-surgical situation. The presence of a sentinel sign always resulted in a stable vertebral segment in both flexion/extension and lateral bending. For axial rotation, the ROM was already limited in both native and cage inserted situations, resulting in non-significant differences for all radiographic scores. DISCUSSION: In vivo vertebral segment stability, defined as a significant reduction in ROM, is achieved in an early stage of spinal fusion, well before a radiological bony fusion between the vertebrae can be observed. Therefore, plain radiography underestimates vertebral segment stability.

Osteoarthritis-related fibrosis is associated with both elevated pyridinoline cross-link formation and lysyl hydroxylase 2b expression.

OBJECTIVE: Fibrosis is a major contributor to joint stiffness in osteoarthritis (OA). We investigated several factors associated with the persistence of transforming growth factor beta (TGF-ß)-induced fibrosis and whether these factors also play a role in OA-related fibrosis. DESIGN: Mice were injected intra-articularly (i.a.) with an adenovirus encoding either TGF-ß or connective tissue growth factor (CTGF). In addition, we induced OA by i.a. injection of bacterial collagenase into the right knee joint of C57BL/6 mice. mRNA was isolated from the synovium for Q-PCR analysis of the gene expression of various extracellular matrix (ECM) components, ECM degraders, growth factors and collagen cross-linking-related enzymes. Sections of murine knee joints injected with Ad-TGF-ß or Ad-CTGF or from experimental OA were stained for lysyl hydroxylase 2 (LH2). The number of pyridinoline cross-links per triple helix collagen in synovium biopsies was determined with high-performance liquid chromatography (HPLC). RESULTS: Expression of collagen alpha-1(I) chain precursor (Col1a1), tissue inhibitor of metalloproteinases 1 (TIMP1) and especially procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2b (Plod2b) were highly upregulated by TGF-ß but not by CTGF. Elevated expression of Plod2b mRNA was associated with high lysyl hydroxylase 2 (LH2) protein staining after TGF-ß overexpression and in experimental OA. Furthermore, in experimental OA the number of hydroxypyridinoline cross-links was significant increased compared to control knee joints. CONCLUSIONS: Our data show that elevated LH2b expression is associated with the persistent nature of TGF-ß-induced fibrosis. Also in experimental OA, LH2b expression as well as the number of hydroxypyridinoline cross-link were significantly upregulated. We propose that LH2b, and the subsequent increase in pyridinoline cross-links, is responsible for the persistent fibrosis in experimental OA.

Site-specific tissue inhibitor of metalloproteinase-1 governs the matrix metalloproteinases-dependent degradation of crosslinked collagen scaffolds and is correlated with interleukin-10.

We have previously shown that the foreign body reaction (FBR) against crosslinked collagen type I (Col-I) differs between subcutaneous and epicardial implantation sites; Col-I was quickly degraded epicardially, whereas degradation was attenuated subcutaneously. The current study set out to dissect the nature and regulation of the MMP-based degradation of implanted Col-I in mice during the FBR. Immunohistochemistry showed that MMP-2, MMP-8 and MMP-13 were present in subcutaneous and epicardial implants, whereas only MMP-9 was also present epicardially. Western blotting showed that MMP-8 and MMP-9 were mainly present in their inactive proform. In contrast, collagenase MMP-13 and gelatinase MMP-2 were the predominant active MMPs at both sites. Interestingly, the major MMP inhibitor TIMP-1 was solely observed in subcutaneous implants, which is why MMP-13 and MMP-2 are not able to degrade the collagen scaffold at the subcutaneous implantation site. Interleukin 10 (IL-10), a potent inducer of TIMP-1 expression, was also mainly detected subcutaneously; giant cells were the main source. Therefore, we surmise that IL-10, through regulation of the balance between MMPs and TIMP-1, suppresses the FBR against implanted biomaterials. Together, our findings would provide cues and clues to improve future therapies in regenerative medicine that are based on the tuned regulation of the degradation of biomaterial scaffolds.

Endo180 and MT1-MMP are involved in the phagocytosis of collagen scaffolds by macrophages and is regulated by interferon-gamma.

Subcutaneously implanted disks of hexamethylenediisocyanate or glutaraldehyde cross-linked sheep collagen (referred to as HDSC and GDSC, respectively) in mice show large differences in degradation rate. Although comparable numbers of macrophages are seen in HDSC and GDSC, phagocytosis of collagen by macrophages occurred only in GDSC. The molecular mechanisms involved in the phagocytosis of collagen by macrophages are essentially unknown. Immunofluorescence and RT-PCR showed that Endo180 was expressed in GDSC only. TissueFaxs showed that Endo180 co-localized with MT1-MMP on F4÷80 positive cells, which is likely responsible for the phagocytosis in GDSC. RT-PCR further showed that Endo180 expression correlated with high levels of IFN-γ mRNA. In vitro, IFN-γ induced the expression Endo180 and MT1-MMP in murine macrophages cultured on collagen type I (although too high levels of IFN-γ dampened the expression of Endo180 and MT1-MMP). Moreover, the expression of Endo180 and MT1-MMP induced by IFN-γ can be inhibited through IL-10. The differences in microenvironment between GDSC and HDSC (high IFN-γ and low IL-10 levels in GDSC, low IFN-γ and high IL-10 levels in HDSC) provide an explanation why phagocytosis of collagen by macrophages is only seen in GDSC. In summary, we show for the first time that the IFN-γ dependent co-expression of Endo180 and MT1-MMP on macrophages coincides with collagen phagocytosis, thus providing evidence that the mechanism of collagen phagocytosis operating in the foreign body reaction by macrophages is comparable with the mechanism of intracellular collagen degradation by fibroblasts seen under physiological conditions.

The effect of ethylene oxide, glow discharge and electron beam on the surface characteristics of poly(L-lactide-co-caprolactone) and the corresponding cellular response of adipose stem cells.

Bioabsorbable polymers are increasingly being used in tissue engineering strategies. Despite the knowledge that some sterilization techniques may affect the physical properties of these polymers, this aspect is often overlooked. We speculate that the type of sterilization method used may influence cellular responses by altering the surface characteristics. We cultured adipose stem cells on bioabsorbable poly(l-lactide-co-caprolactone) (PLCL) sheets, sterilized using either ethylene oxide (EO), argon glow discharge (aGD) or electron beam (e-beam). Significantly higher values for surface roughness in the order EO>aGD>e-beam and significant differences in contact angles (EO>e-beam>aGD) and surface energies (aGD>e-beam>EO) were observed. Increased cell attachment and proliferation rates were observed with lower contact angles. The alkaline phosphatase activity was significantly higher for the ethylene oxide sterilized PLCL sheet. In conclusion, the type of sterilization for bioabsorbable polymers should be considered in the design of new scaffolds, since it might affect, or can be used to enhance, the outcome of the tissue engineered construct.

Pentosidine accumulates in the aging vitreous body: a gender effect.

The human vitreous body undergoes structural changes with aging. This can be followed by a posterior vitreous detachment, which can result in ocular pathology. As in many collagenous tissues, age-related changes in the vitreous could be caused by the formation of advanced glycation end products (AGEs). The goal of this study was to find out whether the AGE pentosidine accumulates in the human vitreous with aging. With this data we were able to estimate the half-life of vitreous collagen. Furthermore, we analyzed whether there was a gender difference in pentosidine accumulation, as this was seen in other tissues as well. Using high performance liquid chromatography, pentosidine contents were determined in whole vitreous bodies and in separate parts of vitreous bodies, which were all obtained from human donor eyes. Our results show that pentosidine accumulates in the human vitreous. From the rate of accumulation we could roughly estimate that vitreous collagen has as a similar or shorter half-life compared to skin collagen. This supports the concept of collagen turnover in the vitreous. In general, the female vitreous experiences a faster pentosidine accumulation than the male vitreous, and most of the pentosidine accumulation in the former occurs after 50 years of age.

Beta1 integrins regulate chondrogenesis and rock signaling in adipose stem cells.

Beta1 integrins play a controversial role during chondrogenesis. Since the maturation of chondrocytes relies on a signaling switch from cell-cell to cell-matrix interactions, we hypothesized that beta1 integrins play a different role at the earlier (mainly cell-cell interaction) from the later stage (mainly cell-matrix interaction) of chondrogenesis. Our data showed: in plain medium, sox9, collagen X, and collagen II gene expressions of ASCs were induced by beta1-integrin blockage at day 14. In chondrogenic medium, however, sox 9, sox6, and collagen II gene expression were induced at day 4 but inhibited at day 14. In addition, both beta1-integrin blockage and TGF-beta1 down-regulated Rock-1 and -2 gene expression and produced the round cells. We concluded that beta1 integrins play a more important role at the later stages than earlier stages of chondrogenesis, and that the onset of chondrogenesis promoted by beta1-integrin blockage might be through inhibiting Rock signaling.

Differences in matrix composition between calvaria and long bone in mice suggest differences in biomechanical properties and resorption: Special emphasis on collagen.

The mammalian skeleton consists of bones that are formed in two different ways: long bones via endochondral ossification and flat bones via intramembranous ossification. These different formation modes may result in differences in the composition of the two bone types. Using the 2D-difference in gel electrophoresis technique and mass spectrometry, we analyzed the composition of murine mineral-associated proteins of calvaria and long bone. Considerable differences in protein composition were observed. Flat bones (calvariae) contained more soluble collagen (8x), pigment epithelium derived factor (3x) and osteoglycin (4x); whereas long bones expressed more chondrocalcin (3x), thrombospondin- 1 (4x), fetuin (4x), secreted phosphoprotein 24 (3x), and thrombin (7x). Although cystatin motifs containing proteins, such as secreted phosphoprotein 24 and fetuin are highly expressed in long bone, they did not inhibit the activity of the cysteine proteinases cathepsin B and K. The solubility of collagen differed which coincided with differences in collagen crosslinking, long bone containing 3x more (hydroxylysine)-pyridinoline. The degradation of long bone collagen by MMP2 (but not by cathepsin K) was impaired. These differences in collagen crosslinking may explain the differences in the proteolytic pathways osteoclasts use to degrade bone. Our data demonstrate considerable differences in protein composition of flat and long bones and strongly suggest functional differences in formation, resorption, and mechanical properties of these bone types.

Influence of collagen type II and nucleus pulposus cells on aggregation and differentiation of adipose tissue-derived stem cells.

Tissue microenvironment plays a critical role in guiding local stem cell differentiation. Within the intervertebral disc, collagen type II and nucleus pulposus (NP) cells are two major components. This study aimed to investigate how collagen type II and NP cells affect adipose tissue-derived stem cells (ASCs) in a 3D environment. ASCs were cultured in collagen type I or type II hydrogels alone, or co-cultured in transwells with micromass NP cells for 4 and 14 days. ASCs seeded in collagen type II gels acquired dentritic cell shapes, and orchestrated cell density-dependent gel contraction rates. Up-regulation of collagen type X, but not of other chondrogenic markers was observed at day 4, irrespective of the hydrogel type. Strikingly, in co-cultures with NP cells, more pronounced differentiation of ASCs along the cartilaginous lineage was observed (up-regulation of collagen IIA, IIB and aggrecan gene expression, as well as stronger alcian blue staining), when ASCs were embedded in collagen type II in comparison with type I hydrogels. Interestingly, strong cellular condensations/aggregations were observed in ASC-seeded type II, but not type I gels, and this aggregation was markedly delayed when the same gels were co-cultured with NP cells. The NP cell-mediated inhibition of ASC aggregation in collagen type II gels coincided with down-regulation of integrin subunit alpha2 gene expression. We conclude that soluble factors released by NP cells can direct chondrogenic differentiation of ASCs in collagen hydrogels, and that combination with a nucleus-mimicking collagen type II microenvironment enhances differentiation towards a more pronounced cartilage/NP lineage relative to collagen type I hydrogels.

Adipose stem cells for intervertebral disc regeneration: current status and concepts for the future.

New regenerative treatment strategies are being developed for intervertebral disc degeneration of which the implantation of various cell types is promising. All cell types used so far require in vitro expansion prior to clinical use, as these cells are only limited available. Adipose-tissue is an abundant, expendable and easily accessible source of mesenchymal stem cells. The use of these cells therefore eliminates the need for in vitro expansion and subsequently one-step regenerative treatment strategies can be developed. Our group envisioned, described and evaluated such a one-step procedure for spinal fusion in the goat model. In this review, we summarize the current status of cell-based treatments for intervertebral disc degeneration and identify the additional research needed before adipose-derived mesenchymal stem cells can be evaluated in a one-step procedure for regenerative treatment of the intervertebral disc. We address the selection of stem cells from the stromal vascular fraction, the specific triggers needed for cell differentiation and potential suitable scaffolds. Although many factors need to be studied in more detail, potential application of a one-step procedure for intervertebral disc regeneration seems realistic.

Differentiation of adipose stem cells by nucleus pulposus cells: configuration effect.

Degenerative disc disease (DDD) is a major cause of chronic low back pain. For mild/intermediate DDD, regeneration by injecting adipose stem cells (ASCs) into the nucleus pulposus (NP) may be considered. The goal of this study is to investigate whether NP cells can direct ASCs towards the NP phenotype. Interactions between NP cells and ASCs were studied in transwell co-cultures, employing both monolayer and micromass configurations. Micromass culturing significantly up-regulated aggrecan and collagen type II gene expression in NP cells. In ASCs, expression of these genes and of osteopontin, collagen type I and PPAR-gamma were not significantly affected. Strikingly, only when both cell types were micromass-cultured, ASCs could be chondrogenically differentiated, as shown by induction of collagen type II and aggrecan, and concomitant down-regulation of osteopontin, collagen type I and PPAR-gamma. We conclude that ASCs can be directed towards the NP cell-like phenotype by soluble factor(s) secreted by NP cells.

Fibroblast-like synoviocyte-chondrocyte interaction in cartilage degradation.

OBJECTIVE: In vitro models for joint diseases often focus on a single cell type, such as chondrocytes in osteoarthritis (OA) or fibroblast-like synoviocytes (synoviocytes) in rheumatoid arthritis (RA). However, these joint diseases affect the whole joint and interaction between chondrocytes and synoviocytes may play an important role in disease pathology. The current study was designed to study the use of the alginate recovered chondrocyte method as a model for cartilage degradation and to study interaction between chondrocytes and synoviocytes. METHODS: Bovine chondrocytes were cultured in alginate beads for 1 week, subsequently chondrons were retrieved and seeded into transwells. Every two days cartilage-slices were analysed for proteoglycan content (colorimetric, Blyscan GAG kit), collagen content (HPLC) and collagen HP and LP crosslinking (HPLC). For degradation experiments, monocultures of cartilage-slices labelled with (35)S and cocultures with synoviocytes were stimulated with IL-1beta or TNF-alpha. After 7 days, (35)S release was measured taken as a measure of cartilage degradation. RESULTS: After biochemical analysis, three week old cartilage-like slices were chosen to perform cartilage-degradation experiments. Synoviocytes were able to induce cartilage degradation only in the presence of living chondrocytes. In addition, the cytokines interleukin 1 (IL-1beta) and tumor necrosis factor (TNF-alpha) were only able to induce cartilage degradation by chondrocytes, not by synoviocytes. CONCLUSION: These data indicate that the alginate recovered chondrocyte method provides a novel model for cartilage degradation in which the interaction between synoviocytes and chondrocytes can be studied.

Interleukin 4 and prolonged hypoxia induce a higher gene expression of lysyl hydroxylase 2 and an altered cross-link pattern: important pathogenetic steps in early and late stage of systemic scleroderma?

The major pathological processes of systemic scleroderma (SSc) comprise inflammation and microvascular damage in the early or acute progressive stage as well as tissue fibrosis and hypoxia in the chronic end stage. Fibrosis seems to be a general phenomenon characterized by an increase of hydroxylysine aldehyde derived collagen cross-links which has been shown in vitro for systemic scleroderma fibroblasts. In the present study, we analyzed the cross-link pattern and the gene expression of lysyl hydroxylase 2 (LH2) in the skin of SSc. Furthermore, we determined the modulatory impact of inflammatory cytokines (interleukin 4, TNF- alphaand interleukin 1alpha/beta) and prolonged hypoxia on the cross-link profile and the gene expression of LH2, respectively. The concentration of hydroxylysine aldehyde derived cross-links was significantly increased in SSc, while the level of lysine aldehyde derived cross-links was not changed. Accordingly, a marked increase of the transcriptional level of LH2 was found. In long term dermal fibroblast cultures, only interleukin 4 induced an increase of hydroxylysine aldehyde derived cross-links accompanied by a higher gene expression of LH2. Furthermore, prolonged hypoxia induced a marked increase of the mRNA level of LH2 in relation to collagen I. The skin of SSc is characterized by an increase of the transcriptional activity of LH2 leading to an altered cross-link pattern. The changes in the quality of the collagenous matrix can also be obtained in cell culture by the exposure of fibroblasts to interleukin 4 or prolonged hypoxia emphasizing the role of this mediator in the acute and the low oxygen tension in the chronic phase of the disease.

Adaptation of subchondral bone in osteoarthritis.

Osteoarthritis is a chronic joint disease with pathological changes in the articulating cartilage and all other tissues that occupy the joint. Radin and coworkers have suggested the involvement of subchondral bone in the disease process. However, evidence for an essential role in the etiology has never been proven. Recent studies showing reduced chemical and mechanical properties of subchondral bone in various stages of the disease have invigorated interest in the role of subchondral bone in the development and progression of the disease. The current study showed that the concept of bone adaptation might explain subchondral stiffening, a process where subchondral bone becomes typically sclerotic in osteoarthritis. In addition, we report reduced mechanical matrix tissue properties as well as an increase in denatured collagen content. In conclusion, although osteoarthritic bone tissue contains increased denatured collagen and has reduced matrix mechanical properties, the widely accepted concept of subchondral stiffening is compatible with the process of normal bone adaptation.