Experimental Induction of Physeal Injuries by Fracture, Drill, and Ablation Techniques: Analyses of Immunohistochemical Findings.

BACKGROUND: Although physeal fractures and physeal bars can result in significant clinical consequences to growth and development of the injured physis, little orthopaedic research has focused upon this topic. Our objective was to extend a previously developed rat model to examine the immunohistochemical features following surgical application of techniques disrupting the physis. METHODS: Physes were surgically disrupted using fracture (control), epiphyseal scrape (ES), or epiphyseal drill (ED). After 1, 3, 6, 10, or 21 days, animals were euthanized, sites processed for histology and immunohistochemical localization of vascular endothelial growth factor (VEGF), Factor VIII, Sox-9, PTHrP (parathyroid hormone-related protein) and PTHrP-R (parathyroid hormone-related protein receptor) in resting, proliferative, and hypertrophic physeal zones. Incidence of physeal bars, vertical septa and islands within the metaphysis was quantified. Semiquantitative analysis of immunohistochemistry was performed. RESULTS: Physeal bars, vertical septa, and displaced cartilage islands were present each of the surgical treatments. Fisher's exact test showed a statistically significant increase in the presence of physeal bars (P=0.002) and vertical septa (P=0.012) in the ED group at 10 and 21 days. Analysis of VEGF showed significant differences among the surgical treatments involving the resting zone, and the proliferative zone for days 1, 6, and 21 (P≤0.02) with greater mean scores present in the fracture (control) group, followed by the ED group; the lowest scores were present in the ES group. PTHrP-R immunolocalization showed significant differences among treatments in the hypertrophic zone at days 6 and 21 (P=0.022 and 0.044, respectively). CONCLUSIONS: On the basis of the type of surgical treatment, results show significant differences in the presence of VEGF (reflecting the vascular bed) in the resting and proliferating zones at days 1, 6, and 21. VEGF localization was less abundant in the ED group (which had more physeal bars), suggesting that lack of vascular ingrowth plays a role in physeal bar formation. CLINICAL RELEVANCE: Basic science data presented here provide insight into the importance of the various regions of the physis and its repair and continued growth after physeal fracture. We suggest that a better understanding of the cellular basis of physeal arrest following physeal fracture may have future relevance for the development of treatments to prevent or correct arrest.

Mucin 1, a signal transduction membrane-bound mucin, is present in human disc tissue and is downregulated in vitro by exposure to IL-1ß or TNF-α.

BACKGROUND: Back pain and disc degeneration have a growing socioeconomic healthcare impact. Mucin 1 (MUC1) is a transmembrane glycoprotein whose extracellular and intracellular domains participate in cellular signaling. Little is currently known about the presence or role of MUC1 in human disc degeneration. METHODS: In this IRB-approved research study, 29 human disc specimens were analyzed for MUC1 immunohistochemical localization and gene expression, and annulus fibrosus (annulus) cells were also isolated and cultured in 3D. Microarray analysis assessed expression levels of MUC1 in healthy and degenerated disc tissue and in cells exposed to proinflammatory cytokines (IL-1ß or TNF-α). RESULTS: MUC1 was shown to be present in annulus cells at the protein level using immunochemistry, and its expression was significantly upregulated in annulus tissue from more degenerated grade V discs compared to healthier grade I-II discs (p = 0.02). A significant positive correlation was present between the percentage of MUC1-positive cells and disc grade (p = 0.009). MUC1 expression in annulus cells cultured in 3D was also analyzed following exposure to IL-1ß or TNF-α; exposure produced significant MUC1 downregulation (p = 0.0006). CONCLUSIONS: Here we present the first data for the constitutive presence of MUC1 in the human disc, and its altered expression during disc degeneration. MUC1 may have an important role in disc aging and degeneration by acting as a regulator in the hypoxic environment, helping disc cells to survive under hypoxic conditions by stabilization and by activation of HIF-1α as previously recognized in pancreatic cancer cells.

Proinflammatory cytokines modulate the chemokine CCL2 (MCP-1) in human annulus cells in vitro: CCL2 expression and production.

Chemokines are important secondary inflammatory mediators released in response to stimuli which act as second-order cytokines with specialized functions in inflammation. The role of many of these specialized mediators is as yet poorly understood in the human intervertebral disc. Here we investigated CCL2 (chemokine (C-C motif) ligand 2, also known as monocyte chemotactic protein-1 (MCP-1)) in a study of its immunolocalization in disc tissue, and then hypothesized that exposure of cultured human annulus cells to proinflammatory cytokines might alter CCL2 gene expression and CCL2 production. CLL2 was localized to many disc cells in both herniated and non-herniated tissue specimens. Molecular analyses showed that cells exposed to IL-1ß showed a 5.5 fold upregulation in CCL2 gene expression vs. controls, p=0.017. Cells exposed to TNF-α showed a 7.7 fold upregulation vs. controls, p=0.005. Cultured cells (grades II-V) showed increased MCP-1 production in IL1-ß-treated cells vs. controls (p=0.016), with no significant difference in production in TNF-α-treated cells. Local production of CCL2 in vivo and vitro suggests that annulus cells may be primary effector cells (as well as target cells), with the ability to mediate physiological immune-related processes during disc degeneration by both autocrine and paracrine signaling.

High-mobility group box-1 gene, a potent proinflammatory mediators, is upregulated in more degenerated human discs in vivo and its receptor upregulated by TNF-α exposure in vitro.

Mechanisms which control and enhance proinflammatory cytokine expression during human disc degeneration are still poorly understood. The high-mobility group box-1 gene (HMGB1) produces a protein which can itself act as a cytokine, or can function as a potent proinflammatory mediator. Little is known about expression of HMGB1 in the human disc. Since proinflammatory cytokines increase significantly during human disc degeneration, in this work we hypothesized that HMGB1 may show upregulation with advancing stages of degeneration, and upregulation in cells exposed to TNF-α. Immunohistochemistry was performed to confirm the presence of HMGB1 in the human disc, and human annulus cells were cultured and challenged with 10(3)pM TNF-α for 14days in 3D culture. Cells with positive HMGB1 immunolocalization were abundant in the outer annulus. Molecular analysis of cultured cells showed an 8-fold significant increase in HMGB1 expression in more degenerated Thompson grade V discs compared to healthier grade I/II discs (p=0.033). Human disc tissue was assessed in molecular studies. Herniated specimens showed a 6.3-fold significantly greater expression level than that seen in control specimens (p=0.001). In culture experiments, expression of the receptor to HMGB1, toll-like receptor 2, showed a 24-fold upregulation in vitro in cells exposed to TNF-α vs. controls (p=0.0003).

Disease-modifying effects of phosphocitrate and phosphocitrate-ß-ethyl ester on partial meniscectomy-induced osteoarthritis.

BACKGROUND: It is believed that phosphocitrate (PC) exerts its disease-modifying effects on osteoarthritis (OA) by inhibiting the formation of crystals. However, recent findings suggest that PC exerts its disease-modifying effect, at least in part, through a crystal-independent action. This study sought to examine the disease-modifying effects of PC and its analogue PC-ß-ethyl ester (PC-E) on partial meniscectomy-induced OA and the structure-activity relationship. METHODS: Calcification- and proliferation-inhibitory activities were examined in OA fibroblast-like synoviocytes (FLSs) culture. Disease-modifying effects were examined using Hartley guinea pigs undergoing partial meniscectomy. Cartilage degeneration was examined with Indian ink, safranin-O, and picrosirius red. Levels of matrix metalloproteinase-13 (MMP-13), ADAM metallopeptidase with thrombospondin type 1 motif 5 (ADAMTS5), chemokine (C-C motif) ligand 5 (CCL5), and cyclooxygenase-2 (Cox-2) were examined with immunostaining. The effects of PC-E and PC on gene expressions in OA FLSs were examined with microarray. Results are expressed as mean ± standard deviation and analyzed using Student's t test or Wilcoxon rank sum test. RESULTS: PC-E was slightly less powerful than PC as a calcification inhibitor but as powerful as PC in the inhibition of OA FLSs proliferation. PC significantly inhibited cartilage degeneration in the partial meniscectomied right knee. PC-E was less powerful than PC as a disease-modifying drug, especially in the inhibition of cartilage degeneration in the non-operated left knee. PC significantly reduced the levels of ADAMTS5, MMP-13 and CCL5, whereas PC-E reduced the levels of ADAMTS5 and CCL5. Microarray analyses revealed that PC-E failed to downregulate the expression of many PC-downregulated genes classified in angiogenesis and inflammatory response. CONCLUSIONS: PC is a disease-modifying drug for posttraumatic OA therapy. PC exerts its disease-modifying effect through two independent actions: inhibiting pathological calcification and modulating the expression of many genes implicated in OA. The ß-carboxyl group of PC plays an important role in the inhibition of cartilage degeneration, little role in the inhibition of FLSs proliferation, and a moderate role in the inhibition of FLSs-mediated calcification.

Matrix metalloproteinase-12 immunolocalization in the degenerating human intervertebral disc and sand rat spine: Biologic implications.

Matrix metalloproteinase-12 (MMP-12; macrophage metalloelastase) degrades a number of extracellular matrix components which are present in the intervertebral disc, including type IV collagen, fibronectin, laminin, chondroitin sulfates, elastin and fibrinogen. MMP-12 has recently discovered relationships with cytokines and chemokines which also relate to disc cell biology. To date, no study has assessed immunolocalization of MMP-12 in degenerating human intervertebral disc tissue. Immunocytochemical localization was performed on 18 human disc specimens and on lumbar spines of the sand rat, a small animal model with well-recognized age-related disc degeneration. In the human disc, intracellular localization was present in both the annulus and nucleus portions of the disc. The sand rat degenerating disc also showed MMP-12 disc localization, with additional presence in chondrocytes of the vertebral endplate of older animals. This is the initial characterization of the presence of MMP-12 in the human and sand rat disc, and in chondrocytes of the vertebral endplate in older sand rats with degenerating discs. Findings are important because they document the presence of an additional MMP-12 in disc tissue, thus expanding our understanding of disc extracellular matrix remodeling, and because they provide novel information on the presence of MMP-12 in the cartilage endplate as it undergoes sclerosis during disc degeneration in the aging sand rat.

Production and expression of RANTES (CCL5) by human disc cells and modulation by IL-1-ß and TNF-α in 3D culture.

Chemokines act as important secondary inflammatory mediators which are released by cells in response to a variety of stimuli. Chemokines bind to cell surface receptors and act as second-order cytokines with specialized functions in inflammation. The role of RANTES (Regulated upon Activation, Normal T-cell Expressed, and Secreted) (also called CCL5 (chemokine (C-C motif) ligand 5)) has received little attention to date in disc tissue. Microarray analyses of lumbar disc annulus tissue revealed that RANTES expression was significantly upregulated in more degenerated Thompson grades IV and V discs compared to expression levels in grades I, II and III discs (p=0.032). Immunolocalization confirmed the presence of RANTES in the annulus and nucleus of the disc, and localized the RANTES receptors CCR1, CCR3 and CCR5 to cells in the disc. In vitro studies with IL-1-ß and TNF-α challenges, both proinflammatory cytokines resulted in elevated levels of RANTES in conditioned media (p<0.01); TNF-α exposure, however, produced significantly greater levels than did IL-1alpha (p<0.0001), suggesting a differential regulation by TNF-α. Local production of RANTES in vivo by annulus and nucleus cells, and in vitro induction of RANTES by proinflammatory cytokines suggest that disc cells are primary effector cells as well as target cells, and thus can mediate physiological immune-related processes during disc degeneration by both autocrine and paracrine signaling.

Growth and differentiation factor-5 (GDF-5) in the human intervertebral annulus cells and its modulation by IL-1ß and TNF-α in vitro.

Growth and differentiation factor-5 (GDF-5) is a member of the TGF-ß superfamily which regulates cell division and differentiation. GDF-5 attracted high interest because of its role in skeletal development, especially in cartilaginous sites. Little is known, however, about the role of GFD-5 in disc cell biology. The present work demonstrated the immunohistologic presence of GDF-5 in human outer and inner annulus tissue. Microarray analysis of annulus cells showed significant upregulation of GDF-5 expression in herniated vs. non-herniated lumbar discs (2.14-fold change, p=0.021). In vitro three-dimensional culture studies challenged human annulus cells with IL-1ß and TNF-α, two proinflammatory cytokines known to be elevated in the human degenerating disc. Exposure resulted in significant downregulation of GDF-5 during both TNF-α exposure (5.83-fold change, p=0.044) and IL-1ß exposure (3.38-fold change, p=0.015). In vitro findings suggest that the degenerating disc milieu, with high proinflammatory cytokine levels, may limit expression of GDF-5, resulting in limited regenerative capacity of the intact disc.

Spontaneous age-related cervical disc degeneration in the sand rat.

BACKGROUND: Disc space narrowing, osteophytes, and disc degeneration are common and increase with aging. Few animal models are appropriate for the study of spontaneous age-related cervical disc degeneration. QUESTIONS/PURPOSES: We used the sand rat, a member of the gerbil family with well-recognized age-related lumbar disc degeneration, to determine whether spontaneous cervical disc degeneration differed from lumbar degeneration when evaluated by (1) radiologic and (2) histologic measures. Animals 2 to 25 months of age were used in these analyses. METHODS: Cervical and lumbar discs of 99 sand rats were analyzed with radiology, and cervical discs of 67 sand rats were studied with histology. Lateral digital radiographs of cervical and lumbar spines were scored for presence or absence of wedging, disc space narrowing, osteophytes, end plate calcification, and irregular disc margins at C2-C3 through C6-C7 and T12-L1 through L7-S1. Percentages for presence were calculated and statistically analyzed for younger (range, 2-11.9 months old) versus older (range, 12.0-25 months old) animals. RESULTS: Cervical discs in younger animals exhibited a greater proportion of irregular margins compared with lumbar sites (94% versus 83%; p = 0.02; 95% CI for difference, 2.7, 19.0%). In older animals, cervical discs showed a greater proportion of osteophytes than did lumbar discs (7% versus 0%; p < 0.0001). The incidence of disc space narrowing was greater in cervical versus lumbar sites (99% versus 90%; p = 0.0008). Cervical spine sites which contained osteophytes morphologically showed irregular disc margins and revealed an extrusion of herniated disc material in the osteophytes. CONCLUSIONS: Radiologic and morphologic studies confirmed age-related disc degeneration in the cervical spine of the sand rat. CLINICAL RELEVANCE: Clinical cervical aging studies have shown that 14% of asymptomatic subjects younger than 40 years have abnormal MRI scans with an increase to 50% by 50 years old. We studied an economic rodent model for cervical age-related spontaneous disc.

Leveraging natural capital accounting to support businesses with nature-related risk assessments and disclosures.

Nature loss threatens businesses, the global economy and financial stability. Understanding and addressing these risks for business will require credible measurement approaches and data. This paper explores how natural capital accounting (NCA) can support business data and information needs related to nature, including disclosures aligned with the Taskforce on Nature-related Financial Disclosures recommendations. As businesses seek to measure, manage and disclose their nature-related risks and opportunities, they will need well-organized, consistent and high-quality information regarding their dependencies and impacts on nature, which few businesses currently collect or track in-house. NCA may be useful for these purposes but has not been widely used or applied by businesses. National NCA guided by the U.N. System of Environmental-Economic Accounting may provide: (i) a useful framework for businesses in conceptualizing, organizing and managing nature-related data and statistics; and (ii) data and information that can directly support business disclosures, corporate NCA and other business applications. This paper explores these opportunities as well as synergies between national and corporate natural capital accounts. In addition, the paper discusses key barriers to advancing the wider use and benefits of NCA for business, including: awareness of NCA, data access, business capabilities related to NCA, spatial and temporal scales of data, audit and assurance considerations, potential risks, and costs and incentives. This article is part of the theme issue 'Bringing nature into decision-making'.

Human annulus cells regulate PAPP-A and IGFBP-4 expression, and thereby insulin-like growth factor bioavailability, in response to proinflammatory cytokine exposure in vitro.

Pregnancy-associated plasma protein-A (PAPP-A) is a metalloproteinase which cleaves IGF binding protein (BP)-4 in the extracellular matrix, making IGF available to nearby cells. We have shown that PAPP-A is present in the human intervertebral disc, and is significantly upregulated in more degenerated discs where increased proinflammatory cytokine levels are present. We hypothesized that increased proinflammatory cytokines present in the degenerating disc might be related to PAPP-A expression. Experiments exposed human annulus cells to IL-1-ß or TNF-α to test this hypothesis. Treated cells showed significantly increased PAPP-A in conditioned media versus controls (p < 0.001). PAPP-A production following exposure to IL-1ß was significantly greater in cells derived from more degenerated versus healthier discs (p = 0.05). PAPP-A gene expression (microarray analysis) was significantly upregulated in IL-1ß- or TNF-α-exposed cells (p = 0.01-0.004). Quantitative RT-PCR confirmed significant upregulation of IGFBP-4 in IL-1ß- or TNF-α-exposed cells. Data have potential relevance to future cell-based biologic therapies for disc degeneration.

Genome-wide analysis of pain-, nerve- and neurotrophin -related gene expression in the degenerating human annulus.

BACKGROUND: In spite of its high clinical relevance, the relationship between disc degeneration and low back pain is still not well understood. Recent studies have shown that genome-wide gene expression studies utilizing ontology searches provide an efficient and valuable methodology for identification of clinically relevant genes. Here we use this approach in analysis of pain-, nerve-, and neurotrophin-related gene expression patterns in specimens of human disc tissue. Control, non-herniated clinical, and herniated clinical specimens of human annulus tissue were studied following Institutional Review Board approval. RESULTS: Analyses were performed on more generated (Thompson grade IV and V) discs vs. less degenerated discs (grades I-III), on surgically operated discs vs. control discs, and on herniated vs. control discs. Analyses of more degenerated vs. less degenerated discs identified significant upregulation of well-recognized pain-related genes (bradykinin receptor B1, calcitonin gene-related peptide and catechol-0-methyltransferase). Nerve growth factor was significantly upregulated in surgical vs. control and in herniated vs. control discs. All three analyses also found significant changes in numerous proinflammatory cytokine- and chemokine-related genes. Nerve, neurotrophin and pain-ontology searches identified many matrix, signaling and functional genes which have known importance in the disc. Immunohistochemistry was utilized to confirm the presence of calcitonin gene-related peptide, catechol-0-methyltransferase and bradykinin receptor B1 at the protein level in the human annulus. CONCLUSIONS: Findings point to the utility of microarray analyses in identification of pain-, neurotrophin and nerve-related genes in the disc, and point to the importance of future work exploring functional interactions between nerve and disc cells in vitro and in vivo. Nerve, pain and neurotrophin ontology searches identified numerous changes in proinflammatory cytokines and chemokines which also have significant relevance to disc biology. Since the degenerating human disc is primarily an avascular tissue site into which disc cells have contributed high levels of proinflammatory cytokines, these substances are not cleared from the tissue and remain there over time. We hypothesize that as nerves grow into the human annulus, they encounter a proinflammatory cytokine-rich milieu which may sensitize nociceptors and exacerbate pain production.

Matrix metalloproteinase-26, a novel MMP, is constitutively expressed in the human intervertebral disc in vivo and in vitro.

Matrix metalloproteinase (MMP) regulation and expression is important in the aging/degenerating human intervertebral disc. MMP-26 (also known as matrilysin-2 or endometase) is a newly discovered MMP which degrades type IV collagen, fibronectin, fibrinogen, vitronectin, denatured collagen types I-IV, insulin-like growth factor binding protein 1, and activated pro-MMP-9. Our objective here was to determine if it is present in human disc tissue and cultured disc cells. Immunohistochemistry and microarray gene expression analyses were used to evaluate the presence of MMP-26 in human disc tissue from healthy and degenerated discs. Immunohistochemistry was also applied to human annulus cells cultured in a collagen sponge. Cellular and matrix localization of MMP-26 was identified in the outer and inner annulus and in the nucleus pulposus. Fewer cells showed localization in the inner vs. outer annulus, and localization was sparse in the nucleus. During in vitro culture of annulus cells, MMP-26 was also expressed. Molecular analyses showed significant downregulation of expression of MMP-26 (p=0.03), and significant 9.8-fold upregulation of TGF-beta (p=0.01) in more degenerated discs vs. healthier discs. Findings document the first identification of MMP-26 in the disc at the molecular and protein levels. Results point to the potentially important role of MMP-26 in matrix modulation during disc health and degeneration.

Variations in aggrecan localization and gene expression patterns characterize increasing stages of human intervertebral disk degeneration.

During disk degeneration, annulus dehydration and matrix fraying culminate in the formation of tears through which nucleus and annulus disk material may rupture, causing radicular pain. Annular tears are present in more than half of the patients in early adulthood and are almost always present in the elderly. Aggrecan, which provides the disk with a shock absorber function under loading, is a key disk extracellular matrix (ECM) component. The objective of the present study was to assess the immunolocalization of aggrecan in the annulus, and to assess molecular gene expression patterns in the annulus ECM utilizing microarray analysis. Immunohistochemistry was performed on 45 specimens using an anti-human aggrecan antibody. Affymetrix microarray gene expression studies used the extracellular matrix ontology approach to evaluate an additional 6 grade I-II, 9 grade III, and 4 grade IV disks. Grade III/IV disks were compared to healthier grade I/II disks. Healthy and less degenerated disks showed a general uniform aggrecan immunolocalization; more degenerated disks contained regions with little or no identifiable aggrecan localization. In degenerated disks, molecular studies showed a significant downregulation of aggrecan, ADAMTS-like 3, and ADAMTS10. Collagen types III and VIII, fibronectin, decorin, connective tissue growth factor, TIMP-3, latent TGF-ß binding protein 2 and TGF-ß1 were significantly upregulated with fold changes ranging from 2.4 to 9.8. Findings here help us better understand changes in the immunohistochemical distribution of a key proteoglycan during disk aging. Such information may have application as we work towards biologic therapies to improve the aging/degenerating disk matrix.

Senescent vs. non-senescent cells in the human annulus in vivo: cell harvest with laser capture microdissection and gene expression studies with microarray analysis.

BACKGROUND: Senescent cells are well-recognized in the aging/degenerating human disc. Senescent cells are viable, cannot divide, remain metabolically active and accumulate within the disc over time. Molecular analysis of senescent cells in tissue offers a special challenge since there are no cell surface markers for senescence which would let one use fluorescence-activated cell sorting as a method for separating out senescent cells. METHODS: We employed a novel laser capture microdissection (LCM) design to selectively harvest senescent and non-senescent annulus cells in paraffin-embedded tissue, and compared their gene expression with microarray analysis. LCM was used to separately harvest senescent and non-senescent cells from 11 human annulus specimens. RESULTS: Microarray analysis revealed significant differences in expression levels in senescent cells vs non-senescent cells: 292 genes were upregulated, and 321 downregulated. Genes with established relationships to senescence were found to be significantly upregulated in senescent cells vs. non-senescent cells: p38 (MPAK14), RB-Associated KRAB zinc finger, Discoidin, CUB and LCCL domain, growth arrest and DNA-damage inducible beta, p28ING5, sphingosine-1-phosphate receptor 2 and somatostatin receptor 3; cyclin-dependent kinase 8 showed significant downregulation in senescent cells. Nitric oxidase synthase 1, and heat shock 70 kDa protein 6, both of which were significantly down-regulated in senescent cells, also showed significant changes. Additional genes related to cytokines, cell proliferation, and other processes were also identified. CONCLUSIONS: Our LCM-microarray analyses identified a set of genes associated with senescence which were significantly upregulated in senescent vs non-senescent cells in the human annulus. These genes include p38 MAP kinase, discoidin, inhibitor of growth family member 5, and growth arrest and DNA-damage-inducible beta. Other genes, including genes associated with cell proliferation, extracellular matrix formation, cell signaling and other cell functions also showed significant modulation in senescent vs non-senescent cells. The aging/degenerating disc undergoes a well-recognized loss of cells; understanding senescent cells is important since their presence further reduces the disc's ability to generate new cells to replace those lost to necrosis or apoptosis.

Testing ecosystem accounting in the United States: A case study for the Southeast.

Ecosystem accounts, as formalized by the System of Environmental-Economic Accounting Experimental Ecosystem Accounts (SEEA EEA), have been compiled in a number of countries, yet there have been few attempts to develop them for the U.S. We explore the potential for U.S. ecosystem accounting by compiling ecosystem extent, condition, and ecosystem services supply and use accounts for a ten-state region in the Southeast. The pilot accounts address air quality, water quality, biodiversity, carbon storage, recreation, and pollination for selected years from 2001 to 2015. Results illustrate how information from ecosystem accounts can contribute to policy and decision-making. Using an example from Atlanta, we also show how ecosystem accounts can be considered alongside other SEEA accounts to give a more complete picture of a local area's environmental-economic trends. The process by which we determined where to place metrics within the accounting framework, which was strongly informed by the National Ecosystem Services Classification System (NESCS), can provide guidance for future ecosystem accounts in the U.S. and other countries. Finally, we identify knowledge gaps that limit the inclusion of certain ecosystem services in the accounts and suggest future research that can close these gaps and improve future U.S. ecosystem accounts.

A new small animal model for the study of spine fusion in the sand rat: pilot studies.

Spine fusion is used to treat traumatic or degenerative lumbar instability in the cervical or lumbar spine. Although degenerative radiological changes in discs adjacent to a fusion have been well-recognized, histopathological changes in adjacent discs have not been studied and are poorly understood. An economical small animal model for lumbar fusion would be a useful research tool. Study objectives were to: (1) develop a model of non-instrumented spine fusion in the sand rat, a rodent with spontaneous, age-related disc degeneration; (2) use radiological-histological analyses to study fusion and disc degeneration in cranial and caudal discs adjacent to the fusion. Studies were approved by our Institutional Animal Care and Use Committee. A small segment of outer annulus tissue was surgically removed from lumbar discs, radiographs obtained and the animal allowed to recover and age. At surgical harvest, radiographs of 28 spine fusion specimens were scored and statistically analysed for adjacent disc space narrowing, wedging, endplate sclerosis and irregular disc margins. At harvest, the incidence of these radiological indices of disc degeneration were significantly greater than at time of surgery. Pilot studies presented here indicate that this model provides a novel addition to basic science approaches studying the clinically important topic of spinal fusion and adjacent segment changes. The resulting fusion site can be assessed statistically with radiological scoring to determine development/progression of disc degeneration in adjacent segments, and correlative histological features can be examined. The sand rat is well-suited for use in spine fusion studies because of reliable/reproducible progression of disc degeneration and the favourable economics of small rodent studies.

Acute effects of different orthoses on lower extremity kinetics and kinematics during running; a musculoskeletal simulation analysis.

PURPOSE: The current investigation aimed to examine the effects of different orthotic conditions on the biomechanical mechanisms linked to the aetiology of chronic pathologies using musculoskeletal simulation. METHODS: 16 male and 20 females ran over an embedded force plate at 4.0 m/s, in five different conditions (medial, lateral, no-orthoses, semi-custom and off the shelf). Kinematics of the lower extremities were collected using an eight-camera motion capture system and lower extremity joint loading also explored using a musculoskeletal simulation approach. Differences between orthoses conditions were examined using 2 × 2 mixed ANOVA. RESULTS: External instantaneous load rate was significantly reduced in the off the shelf orthoses (male = 1290.60 and female = 1567.10 N/kg/s), compared to the medial (male = 1480.45 and female = 1767.05 N/kg/s) and semi-custom (male = 1552.99 and female = 1704.37 N/kg/s) conditions. In addition, peak patellofemoral stress was significantly lower in the off the shelf orthoses (male = 68.55 and female = 94.91 KPa/kg) compared to the lateral condition (male = 70.49 and female = 103.22 KPa/kg). Finally, peak eversion angles were significantly attenuated in the medial orthoses (male = -6.61 and female = -7.72 deg) compared to the lateral (male = -9.61 and female = -10.32 deg), no-orthoses (male = -8.22 and female = -10.10 deg), semi-custom (male = -8.25 and female = -9.49 deg) and off the shelf (male = -7.54 and female = -8.85 deg) conditions. CONCLUSIONS: The current investigation shows that different orthotic devices/ configurations may provide distinct benefits in terms of their effectiveness in attenuating the biomechanical parameters linked to the aetiology of chronic running injuries.

Three-dimensional culture of human meniscal cells: extracellular matrix and proteoglycan production.

BACKGROUND: The meniscus is a complex tissue whose cell biology has only recently begun to be explored. Published models rely upon initial culture in the presence of added growth factors. The aim of this study was to test a three-dimensional (3D) collagen sponge microenvironment (without added growth factors) for its ability to provide a microenvironment supportive for meniscal cell extracellular matrix (ECM) production, and to test the responsiveness of cells cultured in this manner to transforming growth factor-beta (TGF-beta). METHODS: Experimental studies were approved prospectively by the authors' Human Subjects Institutional Review Board. Human meniscal cells were isolated from surgical specimens, established in monolayer culture, seeded into a 3D scaffold, and cell morphology and extracellular matrix components (ECM) evaluated either under control condition or with addition of TGF-beta. Outcome variables were evaluation of cultured cell morphology, quantitative measurement of total sulfated proteoglycan production, and immunohistochemical study of the ECM components chondroitin sulfate, keratan sulfate, and types I and II collagen. RESULT AND CONCLUSION: Meniscal cells attached well within the 3D microenvironment and expanded with culture time. The 3D microenvironment was permissive for production of chondroitin sulfate, types I and II collagen, and to a lesser degree keratan sulfate. This microenvironment was also permissive for growth factor responsiveness, as indicated by a significant increase in proteoglycan production when cells were exposed to TGF-beta (2.48 microg/ml +/- 1.00, mean +/- S.D., vs control levels of 1.58 +/- 0.79, p < 0.0001). Knowledge of how culture microenvironments influence meniscal cell ECM production is important; the collagen sponge culture methodology provides a useful in vitro tool for study of meniscal cell biology.

Gene expression of types I, II, and VI collagen, aggrecan, and chondroitin-6-sulfotransferase in the human annulus: in situ hybridization findings.

BACKGROUND CONTEXT: Within each lamellar bundle in the annulus, disc cells produce a complex and sophisticated architectural organization which acts to meet the unique biomechanical needs of the disc. How cells coordinate expression of genes throughout the disc is an important but as yet poorly understood process. For the annulus, such coordination probably involves cell-cell communication as well as growth factor and mechanoreceptor signaling to appropriately maintain the disc extracellular matrix (ECM) for the prevention of annular tears. PURPOSE: To determine the percentage and patterns of gene expression for types I, II, and VI collagen, aggrecan, and chondroitin-6-sulfotransferase in the human annulus. STUDY DESIGN/SETTING: Human annulus specimens were obtained from surgical subjects and a control donor in a study approved by the authors' Human Subjects Institutional Review Board. PATIENT SAMPLE: Four Thompson grade II, three grade III, and four grade IV annulus specimens were evaluated with in situ hybridization to determine gene expression. OUTCOME MEASURES: The percentages of cells in the human annulus expressing type I, II, and VI collagen, aggrecan, and chondroitin-6-sulfotransferase. METHODS: In situ hybridization, a technique with high temporal and spatial resolution, was used to detect gene expression of types I, II, and VI collagen, aggrecan, and chondroitin-6 sulfotransferase in cells in adjacent sections of annulus from discs with Thompson grades of II, III, and IV. RESULTS: Overall, 30.8% of cells expressed aggrecan, 38.4% type I collagen, 45.6% type II collagen, 48.1% type VI collagen, and 57.7% chondroitin-6-sulfotransferase. An important finding was that adjacent cells could be expressing, or not expressing, the gene of interest. These data could not have been gained from other global molecular techniques such as microarray analysis or reverse transcription polymerase chain reaction (RT-PCR). Information on gene expression by individual disc cells is important to better understand disc matrix homeostasis, the pathogenesis of disc degeneration, and to formulate potential biologic therapies for disc degeneration. CONCLUSIONS: This in situ hybridization study revealed the important finding that adjacent cells differ in their gene expression patterns for specific genes. Factors that could contribute to this difference in adjacent cell gene expression include cellular heterogeneity within the annulus, the presence of senescent cells with altered gene expression, and/or loss of coordinated disc cell function as a result of disruption of cell-cell communication.