Liigeskudede molekulaarsed markerid põlveliigese varase osteoartroosi korral: rahvastikupõhine longitudinaalne uuring keskealistel isikutel

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Osteoartroosi (OA) globaalne levik loob vajaduse detailsema teabe järgi haiguse varastest faasidest. Kaua on OA peetud vananeva liigeskõhre „kulumiseks“. Tänapäeval käsitletakse OA metaboolselt aktiivse protsessina, mis võib alata kõikidest liigeskudedest: kõhrest, luust või pehmetest kudedest. OA preradioloogilise faasi hindamiseks on uute diagnostiliste ja prognostiliste vahenditena rakendust leidmas seerumist ja uriinist määratavad liigeskudede päritoluga molekulaarsed markerid. Siiani puuduvad süsteemsed uurimused liigeskudede sünteesi ja lammutamist peegeldavate biomarkerite väärtuse kohta OA varases faasis. Selle uurimuse eesmärkideks oli hinnata: (i) põlveliigese röntgenoloogilise OA levimust keskealistel põlvekaebustega isikutel ning progresseerumist 6 a. jooksul, (ii) liigeskudede biomarkerite diagnostilist ja ennustavat väärtust OA progresseerumise korral. Enam kui pooltel põlvevaevustega keskealistest inimestest esinesid OA röntgenoloogilised tunnused. Kuue jälgimisaasta jooksul süvenes haigus enamasti progresseeruva osteofütoosina. Põlveliigese OA röntgenoloogiline kulg oli heterogeenne ja mittepidev, hõlmates vahelduvalt haiguse stabiliseerumise ja süvenemise perioode. Käesoleva uurimusega õnnestus esmakordselt näidata kõhre-, luu- ja pehmete kudede aine¬-vahetuse samaaegset aktiveerumist OA varases faasis. Kõigil 3 uuritud kõhrekoe markeril (COMP, CTx-II, PIIANP) oli diagnostiline väärtus progresseeruva osteofütoosi suhtes, ning kahel neist (COMP ja CTx-II) oli ka ennustav roll OA väljendunud progressiooni suhtes. Kolmel uuritud luumarkeril (PINP, OC, MidOC) oli diagnostiline väärtus progresseeruva osteofütoosi suhtes, ja ühel neist (PINP) ka ennustavat roll, kui tegu OA laialdasema progressiooniga liigeses. Selgus, et vähemalt OA algfaasis võib luukoe ainevahetus intensiivistuda enamgi võrreldes kõhrekoega. Uus luukoe biomarker – osteokaltsiini keskfragment (MidOC) osutus tugevaimaks riski ennustajaks progresseeruva osteofütoosi suhtes. Biomarkerite väärtuste muutused demonstreerisid liigeskudedes toimuvate ainevahetuslike nihete kindlat mustrit varase OA progresseeruvatel juhtudel. Käesolev uurimus selgitas liigeskudede mitmete biomarkerite diagnostilist ja prognostilist väärtust OA varases faasis.The globally increasing prevalence of osteoarthritis (OA) calls for more detailed knowledge of the early phases of the disease. OA has long been considered to be a “wear and tear” disease of ageing articular cartilage. Nowadays, OA is viewed as a metabolically active process that may arise from any joint tissue: cartilage, bone or soft tissues. It progresses through molecular and radiographic stages. The investigation of pre-radio¬graphic phases is dependent on serum and/or urinary biomarkers originating from articular tissues as new diagnostic and prognostic tools for early OA management. So far, the systemic evaluation of biomarkers reflecting the synthesis and degradation of cartilage and bone has not been simultaneously addressed. The main objectives of the study were to determine: (i) the prevalence and six-year progression of radiographic knee OA in middle-aged subjects with chronic knee joint complaints, and (ii) the diagnostic and prognostic value of biomarkers for progressive radiographic knee OA. More than half of the middle-aged subjects with chronic knee complaints had early radiographic signs of knee OA. In the majority of cases, the six-year OA progression was based on osteophytosis. The radiographic course of knee OA was heterogeneous and non-continuous, with intermittent periods of progression and stabilization. The changes in biomarker values revealed a certain pattern of metabolic shifts in joint tissues in progressive knee OA. All three studied cartilage markers (COMP, CTx-II and PIIANP) had diagnostic value for progressive osteophytosis, and two of them (COMP and CTx-II) also had prognostic value for progressive osteophytosis and joint space narrowing. At the same time, three of the studied bone markers (PINP, OC and MidOC) had diagnostic value for progressive osteophytosis, and one of them, PINP, demonstrated a predictive value for extensive OA progression. Unexpectedly, the activation of bone metabolism seemed to dominate over that of cartilage. The novel bone marker urinary mid-fragments of osteocalcin (MidOC) turned out to be the strongest risk predictor for progressive osteophytosis. The present study proposed a possible sequence of metabolic events in joint tissues during early OA. We discovered the diagnostic and prognostic potential of several biomarkers for progressive OA

Markers of Bone Turnover In Preclinical Development of Drugs for Skeletal Diseases

Skeletal tissue is constantly remodeled in a process where osteoclasts resorb old bone and osteoblasts form new bone. Balance in bone remodeling is related to age, gender and genetic factors, but also many skeletal diseases, such as osteoporosis and cancer-induced bone metastasis, cause imbalance in bone turnover and lead to decreased bone mass and increased fracture risk. Biochemical markers of bone turnover are surrogates for bone metabolism and may be used as indicators of the balance between bone resorption and formation. They are released during the remodeling process and can be conveniently and reliably measured from blood or urine by immunoassays. Most commonly used bone formation markers include N-terminal propeptides of type I collagen (PINP) and osteocalcin, whereas tartrate-resistant acid phosphatase isoform 5b (TRACP 5b) and C-terminal cross-linked telopeptide of type I collagen (CTX) are common resorption markers. Of these, PINP has been, until recently, the only marker not commercially available for preclinical use. To date, widespread use of bone markers is still limited due to their unclear biological significance, variability, and insufficient evidence of their prognostic value to reflect long term changes. In this study, the feasibility of bone markers as predictors of drug efficacy in preclinical osteoporosis models was elucidated. A non-radioactive PINP immunoassay for preclinical use was characterized and validated. The levels of PINP, N-terminal mid-fragment of osteocalcin, TRACP 5b and CTX were studied in preclinical osteoporosis models and the results were compared with the results obtained by traditional analysis methods such as histology, densitometry and microscopy. Changes in all bone markers at early timepoints correlated strongly with the changes observed in bone mass and bone quality parameters at the end of the study. TRACP 5b correlated strongly with the osteoclast number and CTX correlated with the osteoclast activity in both in vitro and in vivo studies. The concept “resorption index” was applied to the relation of CTX/TRACP 5b to describe the mean osteoclast activity. The index showed more substantial changes than either of the markers alone in the preclinical osteoporosis models used in this study. PINP was strongly associated with bone formation whereas osteocalcin was associated with both bone formation and resorption. These results provide novel insight into the feasibility of PINP, osteocalcin, TRACP 5b and CTX as predictors of drug efficacy in preclinical osteoporosis models. The results support clinical findings which indicate that short-term changes of these markers reflect long-term responses in bone mass and quality. Furthermore, this information may be useful when considering cost-efficient and clinically predictive drug screening and development assays for mining new drug candidates for skeletal diseases.Luun biokemialliset merkkiaineet luustosairauksien prekliinisessä lääkekehityksessä Luun uudismuodostusta tapahtuu koko elämän ajan. Tässä prosessissa osteoklastit hajottavat vanhaa luuta ja osteoblastit muodostavat uutta luuta. Tasapainoon vaikuttaa ikä, sukupuoli ja perinnöllisyys, mutta myös monissa luustosairauksissa, kuten osteoporoosissa ja syövän luustometastaaseissa, tämä tasapaino on järkkynyt johtaen vähentyneeseen luun määrään ja lisääntyneeseen murtumaherkkyyteen. Luun biokemialliset merkkiaineet kertovat luun aineenvaihdunnasta eli hajotuksen ja muodostuksen välisestä tasapainosta. Merkkiaineita vapautuu luun uudismuodostuksessa ja niitä voidaan helposti ja luotettavasti mitata seerumista tai virtsasta immunomääritysmenetelmillä. Yleisesti käytettyjä luun muodostuksen merkkiaineita ovat tyypin I kollageenin aminoterminaalinen propeptidi (PINP) ja osteokalsiini, sekä luun hajotuksen merkkiaineita tartraatti-resistentti hapan fosfataasi alatyyppi 5b (TRACP 5b) ja tyypin I kollageenin karboksiterminaalinen telopeptidi (CTX). Näistä PINP on ainoa merkkiaine, jolle ei ole aiemmin ollut saatavilla prekliiniseen käyttöön soveltuvaa kaupallista immunomääritysmenetelmää. Tällä hetkellä biokemiallisten merkkiaineiden laajamittainen käyttö on vielä rajoittunutta, koska niihin liittyy paljon biologista ja analyyttistä variaatiota eikä niiden merkitsevyydestä ja käyttökelpoisuudesta pitkän aikavälin muutosta ennustavana tekijänä ole riittävästi näyttöä. Tämän tutkimuksen tavoitteena oli selvittää luuston biokemiallisten merkkiaineiden soveltuvuutta lääkemolekyylien tehokkuuden ennustajina prekliinisissä osteoporoositutkimusmalleissa. Tutkimuksessa karakterisoitiin ja validoitiin PINP:lle prekliiniseen käyttöön kehitetty immunomääritysmenetelmä. PINP:n, osteokalsiinin N-terminaalisen keskifragmentin, TRACP 5b:n ja CTX:n tasoja tutkittiin prekliinisissä osteoporoosimalleissa, ja saatuja tuloksia verrattiin perinteisillä menetelmillä kuten histologialla, tiheysmittauksilla ja mikroskopialla saatuihin tuloksiin. Kaikkien tutkittujen luuston merkkiaineiden alkuvaiheen muutosten havaittiin korreloivan kokeen lopussa nähtyihin luuston rakenteellisiin muutoksiin. TRACP 5b korreloi osteoklastien lukumäärään ja CTX osteoklastien aktiivisuuteen sekä in vitro että in vivo kokeissa. CTX/TRACP 5b suhteelle luotiin termi resorptio-indeksi, joka kuvaa osteoklastien keskimääräistä aktiivisuutta. Indeksi antoi tarkempaa tietoa kuin kumpikaan merkkiaine erikseen käytetyissä prekliinisissä osteoporoosimalleissa. PINP:n havaittiin korreloivan vahvasti luun muodostukseen, kun taas osteokalsiini kuvasi sekä luun muodostusta että hajotusta. Tämän tutkimuksen tulokset antavat uutta tietoa luun biokemiallisten merkkiaineiden soveltuvuudesta lääkemolekyylien tehokkuuden ennustajina prekliinisissä osteoporoosimalleissa. Havainnot tukevat kliinisiä tutkimuksia, joissa merkkiaineiden on havaittu korreloivan myöhemmin nähtävien luuston rakenteellisten muutosten kanssa. Tutkimuksessa saatu tieto auttaa suunniteltaessa kliinisen ennustettavuuden kannalta parempia määritysmenetelmiä, joiden avulla voidaan nopeammin ja tehokkaammin löytää uusia toimivia lääkemolekyylejä luustosairauksien hoitoon.Siirretty Doriast

The interaction between growth hormone and the thyroid axis in hypopituitary patients: in vivo and ex vivo studies

Alterations in the hypothalamo-pituitary-thyroid (HPT) axis have been reported following growth hormone (GH) replacement. The aim of this study was to examine potential mechanisms responsible for GH-induced changes in the HPT axis. Furthermore, we aimed to explore the relationship between changes in the serum concentration of thyroid hormones and peripheral biomarkers of thyroid hormone action, before and after GH replacement. Twenty men with severe GH deficiency participated in a prospective, observational study of physiological GH replacement. Research measurements were conducted immediately prior to commencing and 3-6 months after GH replacement. Following GH replacement, the ratio of freeT3:freeT4 in serum increased. In subcutaneous fat, Type 2 deiodinase enzyme activity declined. Type 1 and Type 3 deiodinase activity remained unchanged following GH substitution. Serum TSH, thyroglobulin and thyroid binding globulin levels were unchanged by GH therapy. Hepatic-derived serum markers of thyroid hormone action, including ferritin and caeruloplasmin, declined following GH replacement. In contrast, serum markers of bone turnover increased in parallel with changes in serum concentration of thyroid hormones. Resting energy expenditure and cardiac time intervals did not change throughout the study. Health-related quality of life (QOL) improved following GH replacement in the full study group. However, the improvement in QOL was poorer than expected in subjects who experienced a fall in serum free T4. This study confirms the significant impact of GH replacement on the thyroid axis. In vitro analysis of subcutaneous fat from hypopituitary subjects demonstrates that GH replacement is associated with significant changes in deiodinase isoenzyme activity. However, the observed variation in enzyme activity does not explain changes in the circulating concentration of thyroid hormones induced by GH replacement. Notwithstanding the underlying mechanism, changes in the HPT axis, induced by GH replacement, have complex clinical implications for patients with hypopituitarism

TRACE METALS IN BLOOD AND URINE AS POTENTIAL MARKERS OF BONE BREAKDOWN IN PATIENTS WITH BONE METASTASES

In the western world cancer is the second most important cause of death, after heart disease, accounting for 20-25% of all mortalities, and is today probably one of the most feared diseases. Approximately one third of all cancer patients will develop skeletal metastases (secondary bone cancer). Early diagnosis and effective monitoring during treatment is therefore essential in terms of making any impact on survival rates and developing new cancer therapies. Unfortunately, the current methods for diagnosing and measuring bone metastases, such as bone scans and urinary hydroxyproline determinations, lack sensitivity and specificity. The urinary pyridinium crosslinks, pyridinoline (PYD) and deoxypyridinoline (DPYD), have recently been identified as sensitive and specific markers of bone breakdown. However the analysis of the pyridinium crosslinks using high performance liquid chromatography (HPLC) has proved far from ideal for routine clinical assessment. The results from studies to critically evaluate this method are presented and particular problems encountered when the crosslinks are extracted from the urine samples are discussed. The tedious, time consuming and cumbersome sample preparation procedure are also shown to adversely effect the robustness and reproducibility of the method. The recent introduction of an immunoassay method potentially overcomes many of the inherent problems with the HPLC analysis. This enzyme linked immunoasorbant assay (ELISA) is evaluated and found to compare favourably with the HPLC method, offering several distinct advantages. The method is quick, simple, robust, demonstrates good accuracy and precision, is less prone to interferences and can be easily introduced into clinical laboratories on a routine basis. In addition it also minimises sample preparation time. However, there is still a requirement for alternative and better biochemical markers to measure bone breakdown. It is well known that bone is an active, living tissue and that bone metabolism and remodelling are tightly coupled processes, where the rate of bone formation equals the rate of bone resorption in healthy bone. When an imbalance occurs, this leads to unhealthy bone and ultimately a clinical disease of the skeleton. Some trace metals, e.g lead, accumulate in the bone and since the development of bone metastases results in extensive bone breakdown, the subsequent release of these metals into the blood and urine may potentially serve as markers. In this work inductively coupled plasma-mass spectrometry (ICP-MS) has been used and methods developed to determine such metals in clinical matrices. The development of a simple dilution method is described for use in preliminary trials to measure the blood lead levels and other trace metal profiles, in patients with bone metastases. The blood lead results attained agree closely with a certified reference material, and the method is shown to remain under analytical control. The trial results are presented and discussed with reference to further and more detailed investigations. The selection criteria for other suitable elements such as AI, Ba, Cd, Ce, Pb, Sr, and Zr in blood and urine, along with an assessment of the analytical and clinical praticalities of the methodology which must be considered for subsequent full clinical trials is also discussed following a critical evaluation. Finally the results obtained in a extended clinical trial are presented. The crosslink levels (serving as the reference marker), measured by ELISA, were compared with the trace metal levels (Cd, Pb and Sr) in blood and urine samples, measured by ICP-MS, in order to assess their diagnostic potential and effectiveness in monitoring treatment. The blood lead levels were found to offer the greatest potential, correlating well with the DPYD values in the majority of cases. The blood strontium levels also showed some promise. However the blood cadmium and the urinary trace metal levels proved less suitable. The results attained in this feasibility study support a more detailed clinical investigation, on a much larger scale, and over a longer period of time. The need to incorporate a full statistical evaluation of all factors that can influence the final results is highlighted.Freedom Fields Hospital, Plymout

Cartilage Protection and Analgesic Activity of a Botanical Composition Comprised of Morus alba

Although there have been augmented advances in drug discovery, current OA management is inadequate due to the lack of successful therapies proven to be effective in modifying disease progression. For some, the risk outweighs the benefit. As a result, there is a desperate need for safe and efficacious natural alternatives. Here we evaluated a composition from Morus alba, Scutellaria baicalensis, and Acacia catechu in maintaining joint structural integrity and alleviating OA associated symptoms in monoiodoacetate- (MIA-) induced rat OA disease model. Study lasted for 6 weeks. 59.6%, 64.6%, 70.7%, 69.9%, and 70.3% reductions in pain sensitivity were observed for rats treated with the composition from week 1 to week 5, respectively. Statistically significant improvements in articular cartilage matrix integrity (maintained at 57.1% versus MIA + vehicle treated rats) were shown from the modified total Mankin score for animals treated with the composition. The composition showed a statistically significant reduction in uCTX-II level (54.1% reductions). The merit of combining these botanicals was also demonstrated in their synergistic analgesic activity. Therefore, the standardized blend of Morus alba, Scutellaria baicalensis, and Acacia catechu could potentially be considered as an alternative remedy from natural sources for the management of OA and/or its associated symptoms

The effects of plyometrics or resistance-training on markers of bone turnover and hormones in men

The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file.Title from PDF of title page (University of Missouri--Columbia, viewed on December 31, 2009).Thesis advisor: Dr. Pamela S. Hinton.Includes bibliographical references.M.A. University of Missouri--Columbia 2009.Weight-bearing exercise positively affects bone mineral density (BMD) and bone strength, presumably by altering the balance between bone formation and resorption. The purpose of the present study was to determine the acute response of markers of bone turnover and hormones to a single-bout of resistance-training or plyometrics. Twelve recreationally active males, aged 24-63 years, participated in this partially randomized cross-over study, which included five trials: PLY (fed/fasted), RT (fed/fasted), and a no-exercise, fed control trial (CON) (n=6). Blood was drawn immediately prior to exercise (PRE), immediately following exercise (POST), and 15, 30, 60, 120 min, and 24 hr following PRE. Total testosterone (T), intact parathyroid hormone (PTH), and cortisol (COR) concentrations in serum were determined. Serum bone-specific alkaline phosphatase (BAP) (bone formation marker) and tartrate-resistant acid phosphatase, isoform 5b (TRAP5b) (bone resorption marker) were measured. The results of the present study suggest favorable changes in bone formation and bone resorption as assessed by bone turnover markers (BAP and TRAP5b) following a single-bout of RT or PLY. The decrease in PTH following plyometrics and resistance-training positively correlated with a decrease in TRAP5b, suggesting that PTH may mediate the exercise-induced changes in osteoclast activity

Coronectomy of deeply impacted lower third molar : incidence of outcomes and complications after one year follow-up

Objectives: The purpose of present study was to assess the surgical management of impacted third molar with proximity to the inferior alveolar nerve and complications associated with coronectomy in a series of patients undergoing third molar surgery. Material and Methods: The position of the mandibular canal in relation to the mandibular third molar region and mandibular foramen in the front part of the mandible (i.e., third molar in close proximity to the inferior alveolar nerve [IAN] or not) was identified on panoramic radiographs of patients scheduled for third molar extraction. Results: Close proximity to the IAN was observed in 64 patients (35 females, 29 males) with an impacted mandibular third molar. Coronectomy was performed in these patients. The most common complication was tooth migration away from the mandibular canal (n = 14), followed by root exposure (n = 5). Re-operation to remove the root was performed in cases with periapical infection and root exposure. Conclusions: The results indicate that coronectomy can be considered a reasonable and safe treatment alternative for patients who demonstrate elevated risk for injury to the inferior alveolar nerve with removal of the third molars. Coronectomy did not increase the incidence of damage to the inferior alveolar nerve and would be safer than complete extraction in situations in which the root of the mandibular third molar overlaps or is in close proximity to the mandibular canal

EXPLORATION OF STRONTIUM UPTAKE BY BONE AND SOFT TISSUES USING STABLE ISOTOPE TECHNIQUES

Ph.DDOCTOR OF PHILOSOPH