Bone loss is a major and growing health problem. When bone gets thinner and more fragile, it increases the risk of fractures. Fractures increase morbidity and mortality as well as being a great economic burden for society. To improve treatment of bone loss, it is necessary to improve the understanding of bone remodeling, the process responsible for maintaining bone strength. In bone remodeling, bone-degrading cells called osteoclasts resorb old or damaged bone, which is replaced with new bone mass formed by osteoblasts. Resorption and formation have traditionally been considered separate processes, but recent studies found that they are coupled by a reversal-resorption phase. This project focuses on cortical bone because cortical remodeling occurs in canal structures called Haversian systems, giving the opportunity to investigate remodeling in isolated systems with only the involved cells present.

In cortical remodeling, osteoclasts create a canal through the bone matrix. This canal is subsequently partly filled with new bone mass. Osteoclasts open new canals (type 1) and remodel existing canals (type 2). During the reversal-resorption phase, reversal cells colonize the canal and eventually differentiate into mature bone-forming osteoblasts. A threshold density of reversal cells is necessary for initiation of bone formation. Deterioration of the coupling prolong or arrest the reversal-resorption phase, leading to extended bone resorption and no initiation of formation. This is thought to be the cause of cortical bone loss and subsequent bone fragility. The hypothesis of this project was that prolonging or arrest of the reversal-resorption phase is caused by lack of reversal cells and/or reduced differentiation. If so, the most suitable treatment would be an anabolic drug such as parathyroid hormone (PTH) rather than the traditional anti-resorptives. The overall aim of this thesis was to assess the origin and maturation of reversal cells during the reversal-resorption phase and the effect of iPTH treatment on cortical bone remodeling.

The aim of study 1 was to investigate recruitment, proliferation, and differentiation of reversal cells and the influence of canal type on these factors. Cortical pores (2D cross-sections of canals) in human femoral neck biopsies from adolescents with no diseases known to affect remodeling were classified according to pore type, and eroded, osteoid, and quiescent surfaces were identified. Immunostainings and in situ hybridization made it possible to investigate the expression of immature osteoprogenitors, mature osteoblasts, and proliferation. The cell densities and cells per circumference were determined on each surface type. As expected, reversal cells were identical to osteoprogenitors and eventually differentiated into osteoblasts. Cell density and cells per circumference increased gradually during the reversal-resorption phase as seen before. Osteoprogenitors proliferated to a bigger extend than osteoblasts. This has previously been observed in trabecular bone.

The aim of study 2 was to summarize the current research on the effect of different types of PTH-treatment on trabecular and cortical bone remodeling on a cellular level. Papers investigating remodeling markers and histomorphometric changes during resorption, reversal-resorption and formation were included in the review. PTH-treatment increased clinical markers of both resorption and formation. Histomorphometric studies showed signs of increased formation but no to little evidence of increased resorption. This indicates that the effect on formation overcomes that of resorption. The anabolic effects were more prominent in trabecular than cortical bone but may be temporary, although more data on long-term treatment are needed. Different types of iPTH and cyclic PTH had similar effects.

The aim of study 3 was to investigate the effects of short- and long-term treatment with PTH on cortical bone remodeling. Otherwise healthy patients with hypoparathyroidism received daily subcutaneous injections with either PTH (1-84) or placebo. After 6 months, the patients in the PTH group either continued the treatment or shifted to placebo. The study lasted for another 24 months. Biopsies were taken from the iliac crest at 6 and 30 months. Cortical microstructure was investigated. Pores were classified according to type, remodeling stage, and position, and their number and area were determined. iPTH treatment did not affect cortical microstructure but did increase resorption and formation equally. The effect lasted for as long as the treatment but stopped when treatment was discontinued.

Overall, this project found that recruitment, proliferation, and differentiation of osteoprogenitors are crucial for initiation of bone formation, and that PTH promotes this transition from resorption to formation. This work provides the basis for future studies on osteoprogenitor behavior in elderly and osteoporotic people.

Aldersrelateret knogletab er et stort og voksende sundhedsproblem. Når knogle gradvist bliver tyndere og mere porøs, øger det risikoen for frakturer. Frakturer øger sygeligheden og dødeligheden hos ældre og udgør desuden en stor økonomisk byrde for samfundet. For at forbedre behandlingen af knogletab er det nødvendigt at forbedre forståelsen af knogleremodellering, den proces, der er ansvarlig for knoglestyrken. I knogleremodellering resorberer knoglenedbrydende celler kaldet osteoklaster gammel eller skadet knogle, som efterfølgende erstattes af ny knoglemasse lavet af osteoblaster. Resorption og formation har traditionelt været regnet for to separate processer, men nye studier har vist, at de er koblet sammen af en reversal-resorption fase. Dette projekt fokuserer på kortikal knogle, fordi kortikal remodellering foregår i Haversianske systemer. Dette giver mulighed for at undersøge remodellering i isolerede systemer, hvor kun de involverede celler er til stede.

I kortikal remodellering danner osteoklaster en kanal gennem knoglemassen. Kanalen bliver efterfølgende fyldt delvist op med ny knoglemasse. Osteoklaster åbner både nye kanaler (type 1) og genåbner eksisterende kanaler (type 2). I løbet af reversal-resorption fasen koloniserer reversalceller kanalen, og senere differentierer de til modne, knogledannende osteoblaster. En bestemt celledensitet er nødvendig for at knogleformationen kan starte. Svækkelse af koblingen forlænger eller standser reversal-resorption fasen, så knogleresorptionen varer længere og formationen ikke starter. Dette regnes for at være årsagen til kortikalt knogletab og dermed skrøbelige knogler. Hypotesen i dette projekt var at forlængelse og standsning af reversal-resorption fasen er forårsaget af mangel på reversalceller og/eller reduceret differentiering. Hvis hypotesen passer, ville den mest passende behandling være et anabolsk lægemiddel såsom parathyroidhormon (PTH), i højere grad end de traditionelle antiresorptive lægemidler. Det overordnede formål med denne afhandling var at undersøge oprindelsen og modningen af reversalceller i reversal-resorption fasen samt effekten af PTH-behandling på kortikal remodellering.

Formålet med studie 1 var at undersøge rekruttering, proliferation og differentiering af reversalceller samt hvilken betydning kanaltypen har på disse faktorer. Kortikale porer (2D tværsnit af kanaler) i lårbenshalsbiopsier fra unge mennesker uden kendt sygdom der påvirker remodellering blev klassificeret efter poretype, og eroderede, osteoide og hvilende overflader blev identificeret. Immunfarvninger og in situ hybridiseringer gjorde det muligt at undersøge forekomsten af umodne osteoprogenitorer, modne osteoblaster og deres proliferation. Celledensiteterne og antallet af celler per omkreds blev bestemt for hver overfladetype. Som forventet var reversalceller identiske med osteoprogenitorer og differentierede senere til osteoblaster. Celledensitet og celler per omkreds steg gradvist gennem reversal-resorptionfasen som set tidligere. Osteoprogenitorer prolifererede i højere grad end osteoblaster. Dette er før set i trabekulær knogle.

Formålet med studie 2 var at opsummere den nuværende forskning om effekten af forskellige typer PTH-behandling på trabekulær og kortikal remodellering på celleniveau. Artikler, der undersøgte remodelleringsmarkører og histomorfometriske forandringer i løbet af resorption, reversal-resorption og formation blev inkluderet i reviewet. PTH-behandling øgede kliniske markører for både resorption og formation. Histomorfometriske studier viste tegn på øget formation, men ingen til få beviser på øget resorption. Dette tyder på at effekten på formation overgår effekten på resorption. Den anabolske effekt var større i trabekulær end i kortikal knogle men er muligvis midlertidig, selvom der er brug for flere data for langtidsbehandling. Forskellige typer iPTH og cyklisk PTH havde samme effekt.

Formålet med studie 3 var at undersøge effekten af kort- og langtidsbehandling med PTH på kortikal knogleremodellering. Patienter med hypoparathyroidisme, som ikke havde andre sygdomme fik daglige injektioner med enten PTH (1-84) eller placebo. Efter 6 måneder fortsatte patienterne i PTH-gruppen enten med PTH eller skiftede til placebo. Studiet varede i 24 måneder mere. Biopsier blev udtaget fra hofteskålen ved 6 og 30 måneder. Kortikal mikrostruktur blev undersøgt. Porer blev klassificeret efter type, remodelleringsstadie og position, og deres antal og areal blev bestemt. PTH-behandling påvirkede ikke kortikal mikrostruktur, men øgede resorption og formation lige meget. Effekten varede lige så længe som behandlingen, men stoppede når behandlingen blev stoppet.

Sammenlagt fandt dette projekt at rekruttering, proliferation og differentiering af osteoprogenitorer er essentielle for start på knogleformation og at PTH stimulerer overgangen fra resorption til formation. Dette projekt kan danne basis for fremtidige studier af osteoprogenitorernes opførsel hos ældre og mennesker med knogleskørhed.