Bone biopsy and histomorphometry as a tool for diagnosis of osteoporosis and other metabolic bone diseases

Osteoporoza je sistemska bolest skeleta karakterizirana redukcijom koštane mase i poremećajem unutrašnje arhitekture koštanog tkiva, što dovodi do povećane fragilnosti kostiju i sklonosti prijelomima. Prema WHO, glavni dijagnostički kriterij za osteoporozu je BMD za lumbalni dio kralježnice i femur više od 2,5 SD ispod normalne vrijednosti koštane mase referentne za određenu starosnu dob. Statistički izvještaji govore da je danas u Hrvatskoj oko 250.000 žena oboljelo od osteoporoze. Prema službenim smjernicama za dijagnostiku, prevenciju i liječenje osteoporoze u RH, u dijagnostici osteoporoze, pored denzitometrije, rendgenograma torako-lumbalne kralježnice te laboratorijskih pokazatelja pregradnje kosti, preporučuje se i biopsija kosti s histomorfometrijom. Cilj je ovog rada uputiti na metodu koštane biopsije i histomorfometrije i njenu vrijednost u dijagnostici, ali i praćenju liječenja metaboličkih bolesti kostiju i osteoporoze.Osteoporosis is systemic skeletal disease characterised by reduction of bone mass as well as disruption of internal architecture of the osseous tissue which leads to increased bone fragility and tendency for fracture. According to WHO the primary diagnostic criteria for osteoporosis is BMD more than 2,5 SD below normal values of bone mass referral for certain age, measured on lumbar spine and femoral bone. Over 250.000 women are diagnosed with osteoporosis in Croatia. According to guidance for osteoporosis diagnosis, prevention and treatment, except for bone densitometry, X-rays of thoraco-lumbar spine and biochemical markers of bone remodeling, bone biopsy and histomorphometry are suggested. The aim of this paper is to describe bone biopsy and histomorphometry as a valuable method for diagnostics and follow up in metabolic bone diseases and osteoporosis

A Method for Histological, Enzyme Histochemical and Immunohistochemical Analysis of Periapical Diseases on Undecalcified Bone with Teeth

Svrha je rada bila ispitati i primijeniti postupak histološke, histokemijske i imunohistokemijske raščlambe serijskih rezova nedekalcificiranih psećih zuba s okolnim koštanim tkivom čeljusti. Na premolarima pasa mješanaca induciran je pulpitis i apikalni parodontitis bakterijskom florom iz usne šupljine. Trepanirana pulpna komorica 50 je dana izložena djelovanju oralne mikroflore. Pošto je životinja žrtvovana, nedekalcificirana je kost s dekoroniranim eksperimentalnim zubima uložena u metilmetakrilat i rezana wolfram karbidnim nožem na debljinu 5 - 7 μm. Rezovi su za histološku raščlambu bojeni toluidinskim plavilom, te metodom bojenja kisele fosfataze i tartarat - rezistentne kisele fosfataze. Za detekciju CD 45+ limfocita uporabljeno je imunohistokemijsko bojenje. Debljina rezova 5 - 7 μm omogućila je na histološkim i histokemijskim pripravcima citološku raščlambu promjena u pulpi i periapeksu svjetlosnim mikroskopom in situ. Imunohistokemijsko bojenje nije bilo zadovoljavajuće. Postupak ulaganja nedekalcificirane kosti i zuba u metilmetakrilat i njihovo rezanje wolfram-karbidnim nožem zadovoljavajuća je tehnika za histološke i neke histokemijske raščlambe za potrebe istraživanja u endodonciji. Nužno je poboljšati metodu imunohistokemijskoga bojenja.The objective was to examine and apply a method for serial sectioning of undecalcified canine teeth with surrounding jawbone and to analyse it histologically, enzyme histochemically and immunohistochemically. Pulpitis and apical periodontitis were induced in a mongrel dog’s premolar teeth by exposing the pulp to the oral environmental influence for 50 days. After animal sacrifice undecalcified bone with the decoronated experimental teeth were embedded in methylmethacrylate and sectioned with a tungsten carbide knife at 5-7 μm. Sections were stained with toluidin blue (TB) for histological analysis and by a method of staining non-specific acid phosphatase (ACP), and tartrat-resistant acid phosphatase (TRAP). Immunohistochemical staining was performed for detection of CD 45+ lymphocytes. Because the specimens were 5-7 μm thick it was possible to perform a detailed cytological analysis of changes in the pulp and periapex in situ with a light microscope on histological and enzyme histochemical specimens. Immunohistochemical staining was not satisfactory. The method of embedding undecalcified bone and teeth in methylmethacrylate and sectioning with a tungstencarbide knife is satisfactory for histological and some enzyme histochemical analysis in endodontic research. However, immunohistochemical staining needs improvement

Age-Related and Gender-Related Differences between Human Vertebral and Iliac Crest Bone – A Histomorphometric Study on the Population of the Mediterranean Coast of Croatia

In this study, osseous tissue was examined in normal adult population that has inhabited areas by the Croatian Adriatic Sea. The most of such studies have shown that women are prone to lose bone connectedness, while men are predisposed to be a stronger constitution and they start with greater bone mass, though. Bone samples from two different anatomic sites were analyzed. The crista iliaca and the lumbar vertebra represent functionally different organs too. We wanted to consider weather the same age- and gender-related changes affect these two organs due to normal aging. Static histomorphometry was used to quantify involution changes in the trabecular bone. Results showed that involution process more severely affects women than men. Age-related structural changes were more prominent in lumbar vertebra than in iliac crest bone. Severe structural changes in lumbar vertebra could subsequently lead to a dysfunctional and deformed vertebral column. Therefore, iliac crest bone biopsies could hardly explain involution process that affects lumbar spine

Histomorphological Analysis of the Osteophytic Appositions in Patients with Lumbar Lateral Recess Syndrome

Patients with lumbar lateral recess syndrome (LRS) can be successfully cured by removing osseous excrescences that grow on the peripheral edge of articular surface of the facet joint. They cause narrowing of the lateral recess and compress a root of the spinal nerve. Their appearance is related to the instability of respective dynamic vertebral segment. The aim of this study was to analyze the osteophytic composition morphohistochemically and elucidate cellular processes that lead to this new formation appearance. It is necessary to find a possible causative-consequential relation between the osteophyte and instability. The ideal object to explore was the osteophyte in the lateral recess because it had to be removed during operative treatment. The group of 30 patients with clinical feature of LRS was chosen. Each patient had clinically verified LRS with consequential radiculopathy. Bony outgrowths were removed surgically and analyzed by histological and immunohistochemical methods: toluidine blue, Goldner trichrome, TRAP, indirect peroxidase with antibodies against BMP 3 and BMP 7. The outgrowths that caused lateral recess stenosis were composed of fibrous and hyaline cartilage and cancellous bone. The changes in cartilage and bone, and occurrence of intramembranous bone formation in sense of enlargement of trabeculae, leads to the conclusion that marginal osteophytic formations could be an adaptation to changed conditions in the dynamic vertebral segment and an attempt to stabilize this segment by enlargement of articular surface

The remodeling of the skeleton

Koštani sustav kralježnjaka vrši dvije glavne uloge: prva je biomehanička uloga koja uključuje podupiranje tijela i zaštitu unutarnjih organa i struktura te inzerciju mišićima u svrhu izvođenja pokreta, a druga uloga je metabolička koja uključuje održavanje mineralne homeostaze organizma. Zato, iako su smatrane „mrtvim” inertnim strukturama, kao što to studenti tijekom svog studija medicine doživljavaju, kosti su metabolički iznimno aktivni organi podvrgnuti kontinuiranim cjeloživotnim promjenama. Tijekom rasta i razvoja kosti se formiraju procesom oblikovanja (engl. bone modeling), što uključuje izgradnju i odlaganje koštanog matriksa na razvojno određenim i biomehanički induciranim mjestima skeleta. Čak i prije negoli se skelet u potpunosti razvije, kosti se počinju kontinuirano pregrađivati (engl. bone remodeling) u svrhu održavanja tkivnog integriteta i biomehaničke funkcije, prilagodbe koštane arhitekture prema mehaničkim zahtjevima i zbog popravaka mikrooštećenja, te ako je potrebno mobilizirati kalcij i fosfor. Proces pregradnje uključuje niz precizno reguliranih i tijesno spregnutih staničnih i molekularnih procesa koje omogućuje koordinirana aktivnost osteocita, osteoblasta i osteoklasta. Ova tri različita tipa stanica koštanog tkiva organizirana su u specifičnu morfološku i funkcionalnu višestaničnu jedinicu (engl. basic multicellular unit) koja obavlja i koordinira dva temeljna procesa u biologiji kosti: razgradnju i izgradnju kosti tijekom ciklusa nazvanog ciklus koštane pregradnje. Ovaj ciklus počinje fazom aktivacije koja uključuje mehanosenzaciju i mehanotransdukciju osteocita ili staničnim odgovorom na sistemski humoralni faktor kao što je paratireoidni hormon. Ovi startni signali potiču stvaranje specifične strukture nad koštanom površinom koju treba pregraditi i nazivamo je odjeljak koštane pregradnje (engl. bone remodeling compartment). Nadalje ovi startni signali aktiviraju osteoklastne prekursore na proliferaciju, diferencijaciju, pričvršćivanje i razgradnju određene koštane površine u sljedećoj fazi resorpcije. Nakon što se razgradi određena količina koštanog matriksa, počinje faza obrata koja uključuje oslobađanje brojnih, još uvijek nedovoljno poznatih činitelja koji povezuju razgradnju i osteoblastnu izgradnju s mineralizacijom kosti u sljedećoj fazi. Kada se stvori ista količina kosti koja se razgradila, slijedi završna faza ciklusa u kojoj se ciklus koštane pregradnje zaustavlja, a koštana površina ulazi u mirnu fazu.The bones of the vertebrate skeleton serve two main functions: the first is biomechanical function which includes support and protection of internal organs and structures, so as providing a muscle attachment for the locomotion and the second is essential metabolic function which includes mineral homeostasis maintaining. In spite skeleton is being presented as “dead”, inert structure, as often medical students learn in anatomy, it is metabolically, extremely active organ and undergoes continuous lifelong changes. Bones are initially formed by modeling processes, which include the production and deposition of bone tissue at developmental and biomechanical sites of the skeleton. Before being fully formed, bones undergo remodeling process which continues throughout life, in order to preserve the structural integrity and biomechanical function, to adjust bone architecture according to mechanical demands, to repair microdamages, and if it is needed, to mobilize stored calcium and phosphorus. The remodeling process involves a series of highly regulated and tightly coupled cellular and molecular processes, performed by coordinated activity of osteocytes, osteoblasts and osteoclasts. These different bone cells are organized into specific morphological and functional bone multicellular units which perform and coordinate two basic bone biology processes: bone resorption and bone formation, throughout a cycle of events called bone remodeling. The bone remodeling cycle starts with phase of activation, which includes mechanosensation and mechanotransduction of the osteocytes, or with cellular response to systemic humoral factors such as PTH. These are start signals which induce a formation of a specific canopy structure over the bone surfaces which need to be remodeled, called the remodeling compartment. Furthermore, these signals will activate osteoclastic precursor to proliferate, differentiate, attach and to resorb bone surface in the following resorption phase. After the certain quantity of bone was removed, the reversal phase occurres, which involves releasing of numerous, yet unknown factors that couple bone resorption and upcoming bone formation and mineralization, which are done by osteoblasts in the formation phase. After the same quantity of new bone was formed, the termination phase proceeds in which the cycle ends and bone surfaces become quiescent or “at rest”

The remodeling of the skeleton

Koštani sustav kralježnjaka vrši dvije glavne uloge: prva je biomehanička uloga koja uključuje podupiranje tijela i zaštitu unutarnjih organa i struktura te inzerciju mišićima u svrhu izvođenja pokreta, a druga uloga je metabolička koja uključuje održavanje mineralne homeostaze organizma. Zato, iako su smatrane „mrtvim” inertnim strukturama, kao što to studenti tijekom svog studija medicine doživljavaju, kosti su metabolički iznimno aktivni organi podvrgnuti kontinuiranim cjeloživotnim promjenama. Tijekom rasta i razvoja kosti se formiraju procesom oblikovanja (engl. bone modeling), što uključuje izgradnju i odlaganje koštanog matriksa na razvojno određenim i biomehanički induciranim mjestima skeleta. Čak i prije negoli se skelet u potpunosti razvije, kosti se počinju kontinuirano pregrađivati (engl. bone remodeling) u svrhu održavanja tkivnog integriteta i biomehaničke funkcije, prilagodbe koštane arhitekture prema mehaničkim zahtjevima i zbog popravaka mikrooštećenja, te ako je potrebno mobilizirati kalcij i fosfor. Proces pregradnje uključuje niz precizno reguliranih i tijesno spregnutih staničnih i molekularnih procesa koje omogućuje koordinirana aktivnost osteocita, osteoblasta i osteoklasta. Ova tri različita tipa stanica koštanog tkiva organizirana su u specifičnu morfološku i funkcionalnu višestaničnu jedinicu (engl. basic multicellular unit) koja obavlja i koordinira dva temeljna procesa u biologiji kosti: razgradnju i izgradnju kosti tijekom ciklusa nazvanog ciklus koštane pregradnje. Ovaj ciklus počinje fazom aktivacije koja uključuje mehanosenzaciju i mehanotransdukciju osteocita ili staničnim odgovorom na sistemski humoralni faktor kao što je paratireoidni hormon. Ovi startni signali potiču stvaranje specifične strukture nad koštanom površinom koju treba pregraditi i nazivamo je odjeljak koštane pregradnje (engl. bone remodeling compartment). Nadalje ovi startni signali aktiviraju osteoklastne prekursore na proliferaciju, diferencijaciju, pričvršćivanje i razgradnju određene koštane površine u sljedećoj fazi resorpcije. Nakon što se razgradi određena količina koštanog matriksa, počinje faza obrata koja uključuje oslobađanje brojnih, još uvijek nedovoljno poznatih činitelja koji povezuju razgradnju i osteoblastnu izgradnju s mineralizacijom kosti u sljedećoj fazi. Kada se stvori ista količina kosti koja se razgradila, slijedi završna faza ciklusa u kojoj se ciklus koštane pregradnje zaustavlja, a koštana površina ulazi u mirnu fazu.The bones of the vertebrate skeleton serve two main functions: the first is biomechanical function which includes support and protection of internal organs and structures, so as providing a muscle attachment for the locomotion and the second is essential metabolic function which includes mineral homeostasis maintaining. In spite skeleton is being presented as “dead”, inert structure, as often medical students learn in anatomy, it is metabolically, extremely active organ and undergoes continuous lifelong changes. Bones are initially formed by modeling processes, which include the production and deposition of bone tissue at developmental and biomechanical sites of the skeleton. Before being fully formed, bones undergo remodeling process which continues throughout life, in order to preserve the structural integrity and biomechanical function, to adjust bone architecture according to mechanical demands, to repair microdamages, and if it is needed, to mobilize stored calcium and phosphorus. The remodeling process involves a series of highly regulated and tightly coupled cellular and molecular processes, performed by coordinated activity of osteocytes, osteoblasts and osteoclasts. These different bone cells are organized into specific morphological and functional bone multicellular units which perform and coordinate two basic bone biology processes: bone resorption and bone formation, throughout a cycle of events called bone remodeling. The bone remodeling cycle starts with phase of activation, which includes mechanosensation and mechanotransduction of the osteocytes, or with cellular response to systemic humoral factors such as PTH. These are start signals which induce a formation of a specific canopy structure over the bone surfaces which need to be remodeled, called the remodeling compartment. Furthermore, these signals will activate osteoclastic precursor to proliferate, differentiate, attach and to resorb bone surface in the following resorption phase. After the certain quantity of bone was removed, the reversal phase occurres, which involves releasing of numerous, yet unknown factors that couple bone resorption and upcoming bone formation and mineralization, which are done by osteoblasts in the formation phase. After the same quantity of new bone was formed, the termination phase proceeds in which the cycle ends and bone surfaces become quiescent or “at rest”

The remodeling of the skeleton

Koštani sustav kralježnjaka vrši dvije glavne uloge: prva je biomehanička uloga koja uključuje podupiranje tijela i zaštitu unutarnjih organa i struktura te inzerciju mišićima u svrhu izvođenja pokreta, a druga uloga je metabolička koja uključuje održavanje mineralne homeostaze organizma. Zato, iako su smatrane „mrtvim” inertnim strukturama, kao što to studenti tijekom svog studija medicine doživljavaju, kosti su metabolički iznimno aktivni organi podvrgnuti kontinuiranim cjeloživotnim promjenama. Tijekom rasta i razvoja kosti se formiraju procesom oblikovanja (engl. bone modeling), što uključuje izgradnju i odlaganje koštanog matriksa na razvojno određenim i biomehanički induciranim mjestima skeleta. Čak i prije negoli se skelet u potpunosti razvije, kosti se počinju kontinuirano pregrađivati (engl. bone remodeling) u svrhu održavanja tkivnog integriteta i biomehaničke funkcije, prilagodbe koštane arhitekture prema mehaničkim zahtjevima i zbog popravaka mikrooštećenja, te ako je potrebno mobilizirati kalcij i fosfor. Proces pregradnje uključuje niz precizno reguliranih i tijesno spregnutih staničnih i molekularnih procesa koje omogućuje koordinirana aktivnost osteocita, osteoblasta i osteoklasta. Ova tri različita tipa stanica koštanog tkiva organizirana su u specifičnu morfološku i funkcionalnu višestaničnu jedinicu (engl. basic multicellular unit) koja obavlja i koordinira dva temeljna procesa u biologiji kosti: razgradnju i izgradnju kosti tijekom ciklusa nazvanog ciklus koštane pregradnje. Ovaj ciklus počinje fazom aktivacije koja uključuje mehanosenzaciju i mehanotransdukciju osteocita ili staničnim odgovorom na sistemski humoralni faktor kao što je paratireoidni hormon. Ovi startni signali potiču stvaranje specifične strukture nad koštanom površinom koju treba pregraditi i nazivamo je odjeljak koštane pregradnje (engl. bone remodeling compartment). Nadalje ovi startni signali aktiviraju osteoklastne prekursore na proliferaciju, diferencijaciju, pričvršćivanje i razgradnju određene koštane površine u sljedećoj fazi resorpcije. Nakon što se razgradi određena količina koštanog matriksa, počinje faza obrata koja uključuje oslobađanje brojnih, još uvijek nedovoljno poznatih činitelja koji povezuju razgradnju i osteoblastnu izgradnju s mineralizacijom kosti u sljedećoj fazi. Kada se stvori ista količina kosti koja se razgradila, slijedi završna faza ciklusa u kojoj se ciklus koštane pregradnje zaustavlja, a koštana površina ulazi u mirnu fazu.The bones of the vertebrate skeleton serve two main functions: the first is biomechanical function which includes support and protection of internal organs and structures, so as providing a muscle attachment for the locomotion and the second is essential metabolic function which includes mineral homeostasis maintaining. In spite skeleton is being presented as “dead”, inert structure, as often medical students learn in anatomy, it is metabolically, extremely active organ and undergoes continuous lifelong changes. Bones are initially formed by modeling processes, which include the production and deposition of bone tissue at developmental and biomechanical sites of the skeleton. Before being fully formed, bones undergo remodeling process which continues throughout life, in order to preserve the structural integrity and biomechanical function, to adjust bone architecture according to mechanical demands, to repair microdamages, and if it is needed, to mobilize stored calcium and phosphorus. The remodeling process involves a series of highly regulated and tightly coupled cellular and molecular processes, performed by coordinated activity of osteocytes, osteoblasts and osteoclasts. These different bone cells are organized into specific morphological and functional bone multicellular units which perform and coordinate two basic bone biology processes: bone resorption and bone formation, throughout a cycle of events called bone remodeling. The bone remodeling cycle starts with phase of activation, which includes mechanosensation and mechanotransduction of the osteocytes, or with cellular response to systemic humoral factors such as PTH. These are start signals which induce a formation of a specific canopy structure over the bone surfaces which need to be remodeled, called the remodeling compartment. Furthermore, these signals will activate osteoclastic precursor to proliferate, differentiate, attach and to resorb bone surface in the following resorption phase. After the certain quantity of bone was removed, the reversal phase occurres, which involves releasing of numerous, yet unknown factors that couple bone resorption and upcoming bone formation and mineralization, which are done by osteoblasts in the formation phase. After the same quantity of new bone was formed, the termination phase proceeds in which the cycle ends and bone surfaces become quiescent or “at rest”

The effect of age, anthropometric parameters, vertebral bone densitometry and ash density on iliac crest bone volume and microstructure

Background and Purpose: The purpose was to compare iliac crest bone static bone histomorphometry and vertebral bone densitometry as two methods frequently used in clinical practice. Patients and Methods: Cylindrical bone samples of the iliac crest bone (N=100) for bone histomorphometry and the whole bodies of the third lumbar vertebra (L3) (N=100) for bone densitometry and atomic absorption spectrometry were collected. Multiple regression analyses were carried out and results were considered significant when p<0.05 and 0.01. Results: Age was inversely proportional to all histomorphometric parameters except for Tb.Sp. Age predicted BV/TV with the largest share of contribution of 68%. Gender showed the highest share of contribution for the Tb.Th (32%), while BMD showed it for the Ct.Th (10%). Conclusions: After age and gender, BMD is the third strongest variable to predict iliac crest bone histomorphometric parameters, and thus we can conclude that iliac crest bone histomorphometry and vertebral bone densitometry are correlated, which is important for monitoring osteoporosis in good clinical practic

Expression of the BMP -2, -4 and -7 and their Antagonists Gremlin, Chordin, Noggin and Follistatin during Ectopic Osteogenesis

Molecular network of the osteogenic BMPs and extracellular inhibitors maintains homeostasis of the skeletal tissues. It is important to determine relationship between BMP-2, -4 and -7 and their inhibitors: gremlin, follistatin, chordin and noggin, during normal osteogenesis. To determine their expression pattern we conducted investigation by inducing ecto- pic bone formation in rats. The results shown that levels of the BMP-2 and BMP-4 expression in chondrocytes are similar to noggin and follistatin. The latter BMPs and inhibitors have increased levels of the expression at day 14 th of the osteo- genesis, which suggests their important roles in early phases of the chondrogenesis. Gremlin and chordin have shown increased levels of expression in late phase of chondrogenesis, which suggests their important role in regulation of the osteogenesis initiation. In this study, BMPs and inhibitors have the highest levels of the expression at 21 st day in the osteocytes, which suggests their strong interactions in osteogenesis

Immunohistochemical study of the BMPs and their extracellular antagonists in osteoarthritic human knee joint

The osteophytes are bone spurs overgrowing the edge of the articular cartilage during the course of osteoarthritis (OA). The cellular mechanism of their development and growth resembles the intramembranous and endochondral bone development during embrional and postnatal normal bone development, growth, modeling, remodeling and repair. The role of BMPs in bone development and metabolism is well documented and the members of the BMPs molecular network were recognized as important factors which could modulate new bone development and growth of osteophytes. The purpose of this study is to analyze the cooexpression of the most potent osteoinductive members of the BMP family (BMP-2, -4 and -7) and their extracellular antagonists gremlin, noggin, chordin, follistatin in order to establish their role during degenerative process of the synovial joints and growth of osteophyte. In this study, the BMP-2, BMP-4, BMP-7, gremlin, noggin, chordin, follistatin expressions were analyzed in joint tissues from OA patients and from healthy individuals by immunohistochemistry and Western blot. The immunohistochemistry showed different localization pattern of BMPs and BMP extracellular antagonists expressions in OA vs. normal joint tissues. In osteophyte, BMP-2 was not detected, while BMP-4 and BMP-7 were positive in hypertrophyc chondrocytes and osteocytes at the sites of endochondral bone development. BMP-7 was strongly positive, while BMP-4 was negative in synovial membrane of OA joints. Gremlin, chordin and noggin were found in chondrocytes, osteoblasts of the osteophytic bone and synovial epithelium, while follistatin was found in chondrocytes, blood vessels and synovial epithelium. Our result demonstrate the significant differences in BMPs and their antagonists expression in normal tissues compare to joint tissue affected by OA and revealed how molecular balance of the local growth factors such as BMPs and their antagonists could be disturbed during degenerative processes. Also, these findings suggest the potent role of these molecular factors in pathogenesis of the OA and/or during growth and development of the human knee joint osteophytes