Aging does not change the compressive stiffness of mandibular condylar cartilage in horses.

OBJECTIVE: Aging can cause an increase in the stiffness of hyaline cartilage as a consequence of increased protein crosslinks. By induction of crosslinking, a reduction in the diffusion of solutions into the hyaline cartilage has been observed. However, there is a lack of knowledge about the effects of aging on the biophysical and biochemical properties of the temporomandibular joint (TMJ) cartilage. Hence, the aim of this study was to examine the biophysical properties (thickness, stiffness, and diffusion) of the TMJ condylar cartilage of horses of different ages and their correlation with biochemical parameters. MATERIALS AND METHODS: We measured the compressive stiffness of the condyles, after which the diffusion of two contrast agents into cartilage was measured using Contrast Enhanced Computed Tomography technique. Furthermore, the content of water, collagen, GAG, and pentosidine was analyzed. RESULTS: Contrary to our expectations, the stiffness of the cartilage did not change with age (modulus remained around 0.7 MPa). The diffusion of the negatively charged contrast agent (Hexabrix) also did not alter. However, the diffusion of the uncharged contrast agent (Visipaque) decreased with aging. The flux was negatively correlated with the amount of collagen and crosslink level which increased with aging. Pentosidine, collagen, and GAG were positively correlated with age whereas thickness and water content showed negative correlations. CONCLUSION: Our data demonstrated that aging was not necessarily reflected in the biophysical properties of TMJ condylar cartilage. The combination of the changes happening due to aging resulted in different diffusive properties, depending on the nature of the solution.

Increased epidermal thickness and abnormal epidermal differentiation in keloid scars.

BACKGROUND: The pathogenesis underlying keloid formation is still poorly understood. Research has focused mostly on dermal abnormalities, while the epidermis has not yet been studied. OBJECTIVES: To identify differences within the epidermis of mature keloid scars compared with normal skin and mature normotrophic and hypertrophic scars. METHODS: Rete ridge formation and epidermal thickness were evaluated in tissue sections. Epidermal proliferation was assessed using immunohistochemistry (Ki67, keratins 6, 16 and 17) and with an in vitro proliferation assay. Epidermal differentiation was evaluated using immunohistochemistry (keratin 10, involucrin, loricrin, filaggrin, SPRR2, SKALP), reverse-transcriptase polymerase chain reaction (involucrin) and transmission electron microscopy (stratum corneum). RESULTS: All scars showed flattening of the epidermis. A trend of increasing epidermal thickness correlating to increasing scar abnormality was observed when comparing normal skin, normotrophic scars, hypertrophic scars and keloids. No difference in epidermal proliferation was observed. Only the early differentiation marker involucrin showed abnormal expression in scars. Involucrin was restricted to the granular layer in healthy skin, but showed panepidermal expression in keloids. Normotrophic scars expressed involucrin in the granular and upper spinous layers, while hypertrophic scars resembled normotrophic scars or keloids. Abnormal differentiation was associated with ultrastructural disorganization of the stratum corneum in keloids compared with normal skin. CONCLUSIONS: Keloids showed increased epidermal thickness compared with normal skin and normotrophic and hypertrophic scars. This was not due to hyperproliferation, but possibly caused by abnormal early terminal differentiation, which affects stratum corneum formation. Our findings indicate that the epidermis is associated with keloid pathogenesis and identify involucrin as a potential diagnostic marker for abnormal scarring.

The intricate anatomy of the periodontal ligament and its development: Lessons for periodontal regeneration.

The periodontal ligament (PDL) connects the tooth root and alveolar bone. It is an aligned fibrous network that is interposed between, and anchored to, both mineralized surfaces. Periodontal disease is common and reduces the ability of the PDL to act as a shock absorber, a barrier for pathogens and a sensor of mastication. Although disease progression can be stopped, current therapies do not primarily focus on tissue regeneration. Functional regeneration of PDL may be achieved using innovative techniques, such as tissue engineering. However, the complex fibrillar architecture of the PDL, essential to withstand high forces, makes PDL tissue engineering very challenging. This challenge may be met by studying PDL anatomy and development. Understanding PDL anatomy, development and maintenance provides clues regarding the specific events that need to be mimicked for the formation of this intricate tissue. Owing to the specific composition of the PDL, which develops by self-organization, a different approach than the typical combination of biomaterials, growth factors and regenerative cells is necessary for functional PDL engineering. Most specifically, the architecture of the new PDL to be formed does not need to be dictated by textured biomaterials but can emerge from the local mechanical loading conditions. Elastic hydrogels are optimal to fill the space properly between tooth and bone, may house cells and growth factors to enhance regeneration and allow self-optimization by the alignment to local stresses. We suggest that cells and materials should be placed in a proper mechanical environment to initiate a process of self-organization resulting in a functional architecture of the PDL.

Bone-site-specific responses to zoledronic acid.

OBJECTIVES: Bisphosphonates are widely used to treat bone diseases such as osteoporosis. However, they may cause osteonecrosis of the jaw. Here, we investigated whether in vivo exposure to bisphosphonates has a different effect on long bone and jaw osteoclasts, and on the turnover of these different bones. MATERIALS AND METHODS: Zoledronic acid (0.5 mg kg-1 weekly) was administered intraperitoneally to 3-month-old female mice for up to 6 months. The effects on the number of osteoclasts, bone mineralization and bone formation were measured in the long bones and in the jaw. RESULTS: Long-term treatment with zoledronic acid reduced the number of jaw bone marrow cells, without affecting the number of long bone marrow cells. Zoledronic acid treatment did not affect the number of osteoclasts in vivo. Yet, the bisphosphonate increased bone volume and mineral density of both long bone and jaw. Interestingly, 6 months of treatment suppressed bone formation in the long bones without affecting the jaw. Unexpectedly, we showed that bisphosphonates can cause molar root resorption, mediated by active osteoclasts. CONCLUSIONS: Our findings provide more insight into bone-site-specific effects of bisphosphonates and into the aetiology of osteonecrosis of the jaw. We demonstrated that bisphosphonates can stimulate osteoclast activity at the molar roots.

The contribution of collagen fibers to the mechanical compressive properties of the temporomandibular joint disc.

OBJECTIVE: The Temporomandibular Joint (TMJ) disc is a fibrocartilaginous structure located between the mandibular condyle and the temporal bone, facilitating smooth movements of the jaw. The load-bearing properties of its anisotropic collagenous network have been well characterized under tensile loading conditions. However, recently it has also been speculated that the collagen fibers may contribute dominantly in reinforcing the disc under compression. Therefore, in this study, the structural-functional role of collagen fibers in mechanical compressive properties of TMJ disc was investigated. DESIGN: Intact porcine TMJ discs were enzymatically digested with collagenase to disrupt the collagenous network of the cartilage. The digested and non-digested articular discs were analyzed mechanically, biochemically and histologically in five various regions. These tests included: (1) cyclic compression tests, (2) biochemical quantification of collagen and glycosaminoglycan (GAG) content and (3) visualization of collagen fibers' alignment by polarized light microscopy (PLM). RESULTS: The instantaneous compressive moduli of the articular discs were reduced by as much as 50-90% depending on the region after the collagenase treatment. The energy dissipation properties of the digested discs showed a similar tendency. Biochemical analysis of the digested samples demonstrated an average of 14% and 35% loss in collagen and GAG, respectively. Despite the low reduction of collagen content the PLM images showed considerable perturbation of the collagenous network of the TMJ disc. CONCLUSIONS: The results indicated that even mild disruption of collagen fibers can lead to substantial mechanical softening of TMJ disc undermining its reinforcement and mechanical stability under compression.

Clonidine increases bone resorption in humans.

SUMMARY: Inhibition of sympathetic signaling to bone reduces bone resorption in rodents. In contrast, we show that pharmacological reduction of the sympathetic tone increases bone resorption in humans in vivo. This effect does not appear to be mediated via a direct pharmacological effect on the osteoclast. INTRODUCTION: Inhibition of sympathetic signaling to bone reduces bone resorption in rodents. It is uncertain whether a similar role for the sympathetic nervous system exists in humans. The sympathetic tone can be reduced by clonidine, which acts via alpha-2-adrenergic receptors in the brainstem. Our objective was to determine the effect of clonidine on bone turnover in humans. METHODS: The acute effect of a single oral dose of 0.3 mg clonidine on serum bone turnover markers (C-terminal cross-linking telopeptides of collagen type I (CTx), a marker for bone resorption, and procollagen type 1 N propeptide (P1NP), a marker for bone formation) was determined in a randomized crossover design in 12 healthy volunteers, aged 18-70 years. In addition, we assessed the effect of clonidine on the number of tartrate-resistant acid phosphatase-positive multinucleated cells (TRAcP(+) MNCs) and bone resorption. RESULTS: CTx concentrations increased after clonidine treatment compared to the control condition (p = 0.035). P1NP concentrations were not affected by clonidine (p = 0.520). In vitro, clonidine had no effect on the number of TRAcP(+) MNCs (p = 0.513) or on bone resorption (p = 0.996). CONCLUSIONS: We demonstrated that clonidine increases bone resorption in humans in vivo. This effect does not appear to be mediated via a direct effect on the osteoclast.

Myosin Heavy Chain Expression Can Vary over the Length of Jaw and Leg Muscles.

Muscle fiber type classification can be determined by its myosin heavy chain (MyHC) composition based on a few consecutive sections. It is generally assumed that the MyHC expression of a muscle fiber is the same over its length since neural stimulation and systemic influences are supposed to be the same over its length. We analyzed this in detail in three muscle types: the temporalis (closer) and digastricus (opener; both first brachial arch), and the medial gastrocnemius (somite). Sections of the muscles were incubated with monoclonal antibodies against various MyHC isoforms, and the distribution of these isoforms within individual fibers was followed over a distance of approximately 1 mm. The staining intensity of a fiber was measured and compared with the other fibers in the section. In the temporalis, digastricus, and gastrocnemius, 46, 11, and 15%, respectively, of their MyHC-I fibers showed a variation in the staining intensity over the length of their fibers, as well as 47, 87, and 22%, respectively, of their MyHC-IIA fibers. Most variable fibers were found amongst those with an overall relative intermediate staining intensity, which are presumably hybrid fibers. We conclude that different parts of a muscle fiber can have different fiber type compositions and, thus, contractile properties. Some muscle parts might reach their maximum contraction peak sooner or later than a muscle part a few microns further away. Next to stimulation by the nerve and systemic influences, local influences might also have an impact on the MyHC expression of the fiber.

Tumor necrosis factor-α antagonist infliximab inhibits osteoclast formation of peripheral blood mononuclear cells but does not affect periodontal ligament fibroblast-mediated osteoclast formation.

BACKGROUND AND OBJECTIVE: The inflammatory cytokine tumor necrosis factor-alpha (TNF-α) is elevated in inflamed periodontal tissues and may contribute to periodontitis progression. TNF-α stimulates formation and activity of osteoclasts, the cells that are recruited in periodontitis, that cause alveolar bone degradation and subsequent tooth loss. We previously showed that TNF-α is elevated in co-cultures of periodontal ligament fibroblast (PDLF) and peripheral blood mononuclear cells (PBMC). Hence, TNF-α could be a determining factor in osteoclast formation in these cultures, as osteoclasts are formed despite the fact that prototypical osteoclast differentiation factor receptor activator of nuclear factor kappa-B ligand is outnumbered at least 100-fold by its inhibitor osteoprotegerin in these cultures. MATERIAL AND METHODS: To assess the role of TNF-α in periodontitis-associated osteoclast formation in vitro, osteoclast formation was analyzed in the presence of the anti-TNF-α therapeutic agent infliximab in two culture systems: (i) PBMC in co-culture with PDLFs from controls and patients with periodontitis, or (ii) with PBMC only. PDLFs from control and patients with periodontitis were exposed to infliximab, PBMCs were added and the formation of osteoclast-like cells was assessed. RESULTS: TNF-α was highest levels in supernatants at 7 d in co-cultures and declined at 14 and 21 d. TNF-α was undetectable in cultures that received infliximab. The formation and activity of osteoclasts in co-cultures was not affected by infliximab. In contrast, infliximab in cultures of only PBMC significantly reduced the formation of osteoclasts. This reduction was accompanied by a decreased number and size of cell clusters, a step that precedes the formation of osteoclasts. TNF-α was again undetectable in the supernatant of infliximab-treated cultures, but was detectable at similar levels in cell lysates of control and infliximab-treated PBMC cultures. CONCLUSION: Our study shows that the contribution of TNF-α to osteoclast formation is cell system dependent. It contributes to PBMC-induced osteoclast formation, possibly by establishing stronger cell-cell interactions that precede osteoclast formation.

Role of periodontal ligament fibroblasts in osteoclastogenesis: a review.

During the last decade it has become clear that periodontal ligament fibroblasts may contribute to the in vitro differentiation of osteoclasts. We surveyed the current findings regarding their osteoclastogenesis potential. Periodontal ligament fibroblasts have the capacity to select and attract osteoclast precursors and subsequently to retract and enable migration of osteoclast precursors to the bone surface. There, fusion of precursors takes place, giving rise to osteoclasts. The RANKL-RANK-osteoprotegerin (OPG) axis is considered crucial in this process. Periodontal ligament fibroblasts produce primarily OPG, an osteoclastogenesis-inhibitory molecule. However, they may be influenced in vivo by direct or indirect interactions with bacteria or by mechanical loading. Incubation of periodontal ligament fibroblasts with bacteria or bacterial components causes an increased expression of RANKL and other osteoclastogenesis-stimulating molecules, such as tumor necrosis factor-α and macrophage-colony stimulating factor. Similar results are observed after the application of mechanical loading to these fibroblasts. Periodontal ligament fibroblasts may be considered to play an important role in the remodelling of alveolar bone. In vitro experiments have demonstrated that periodontal ligament fibroblasts adapt to bacterial and mechanical stimuli by synthesizing higher levels of osteoclastogenesis-stimulating molecules. Therefore, they probably contribute to the enhanced osteoclast formation observed during periodontitis and to orthodontic tooth movement.

Submicron-scale surface architecture of tricalcium phosphate directs osteogenesis in vitro and in vivo.

A current challenge of synthetic bone graft substitute design is to induce bone formation at a similar rate to its biological resorption, matching bone's intrinsic osteoinductivity and capacity for remodelling. We hypothesise that both osteoinduction and resorption can be achieved by altering surface microstructure of beta-tricalcium phosphate (TCP). To test this, two TCP ceramics are engineered with equivalent chemistry and macrostructure but with either submicron- or micron-scale surface architecture. In vitro, submicron-scale surface architecture differentiates larger, more active osteoclasts--a cell type shown to be important for both TCP resorption and osteogenesis--and enhances their secretion of osteogenic factors to induce osteoblast differentiation of human mesenchymal stem cells. In an intramuscular model, submicrostructured TCP forms 20 % bone in the free space, is resorbed by 24 %, and is densely populated by multinucleated osteoclast-like cells after 12 weeks; however, TCP with micron-scale surface architecture forms no bone, is essentially not resorbed, and contains scarce osteoclast-like cells. Thus, a novel submicron-structured TCP induces substantial bone formation and is resorbed at an equivalent rate, potentially through the control of osteoclast-like cells.

Prostaglandin E2 inhibits in-vitro mineral deposition by human periodontal ligament cells via modulating the expression of TWIST1 and RUNX2.

BACKGROUND AND OBJECTIVE: Prostaglandin E2 (PGE2) has been shown to be able to influence both bone formation and resorption. The purpose of this study was to investigate the effect of PGE2 on the osteogenic differentiation of human periodontal ligament (HPDL) cells. MATERIAL AND METHODS: HPDL cells were cultured with 0.001-1 µm PGE2 in osteogenic medium. In-vitro mineral deposition was determined by Alizarin Red S staining, and gene expression was determined by real-time PCR. RESULTS: PGE2 inhibited in-vitro mineral deposition by HPDL cells in a dose-dependent manner. PCR analyses showed that PGE2 upregulated the expression of Runt-related transcription factor 2 (RUNX2), but had no effect on osteocalcin expression. Upregulation of TWIST-related protein1 (TWIST1), a functional antagonist of RUNX2, was also observed. In addition, increased levels of RUNX2 and TWIST1 proteins, induced by PGE2, were detected by western blot analysis. Using a chemical activator of E prostanoid (EP) receptors as well as small interfering RNA against an EP receptor, it was shown that PGE2 regulated RUNX2 and TWIST1 via the EP2 receptor. The role of protein kinase A in the inductive effect of PGE2 was also demonstrated. CONCLUSION: The results of this study revealed that PGE2 modulates the osteogenic differentiation of HPDL cells via regulating the expression of RUNX2 and TWIST1. The results suggest a possible role for PGE2 in regulating the homeostasis of periodontal ligament tissue.

Demineralized bone matrix and platelet-rich plasma do not improve healing of osteochondral defects of the talus: an experimental goat study.

OBJECTIVE: The purpose of this study was to evaluate the effectiveness of demineralized bone matrix (DBM) with and without platelet-rich plasma (PRP) in the treatment of osteochondral defects (OCDs) of the talus. We hypothesized that treatment with DBM would result in more bone formation than no treatment in control OCDs, and that PRP would further enhance the regenerative capacity of DBM. METHOD: A standardized 6-mm OCD was created in each talus of 16 adult goats. According to a randomization scheme, one OCD of each goat was treated with allogeneic DBM hydrated with normal saline (n = 8) or hydrated with autologous PRP (n = 8). The contralateral OCD (n = 16) served as control. After 24 weeks, the animals were euthanized and the tali excised. Various outcome parameters were analyzed with use of macroscopic evaluation, micro-computed tomography (µCT), histology, histomorphometry, and fluorescence microscopy. RESULTS: None of the analyses revealed statistically significant differences between the groups for any of the parameters analyzed in any volume of interest. For example, the mean bone volume fraction (BV/TV) of the defect, as measured by µCT, was 0.56 (95% confidence interval [CI], 0.44-0.68) for DBM hydrated with normal saline and 0.52 (95% CI, 0.40-0.65) for DBM hydrated with PRP, compared to 0.53 (95% CI, 0.45-0.61) and 0.54 (95% CI, 0.44-0.64) for the internal controls, respectively (P > 0.05). CONCLUSION: In contrast to our hypotheses, no beneficial treatment effect of DBM with or without PRP was found for OCDs of the caprine talus.

[Dissertations 25 years after date 34. breakdown of collagen by fibroblasts and osteoclasts]. / Proefschriften 25 jaar na dato 34. Collageenafbraak door fibroblasten en osteoclasten.

The remodeling of soft and hard connective tissue is ssential for the proper functioning of an organism and also for the proper functioning of a tooth. An element of this remodeling is the disintegration mediated by fibroblasts or osteoclasts. The precise means by which the remodeling process takes place was and continues to be in part unknown. This doctoral research was able to show that collagen fibres are absorbed by fibroblasts from the surrounding tissue and consequently broken down in the lysosomal apparatus. Osteoclasts, the only cell type capable of breaking down mineralized bone, also appear to be capable of absorbing collagens. It was demonstrated that intracellular bone collagens in these cells can be found in patients who sufferfrom the rare disease pycnodysostosis. It is postulated that, in the osteoclasts of these patients, the activity of one or more enzymes which break down protein were reduced or absent. During the last 25 years, significant advances in the understanding of the processes which underlie the bone remodeling.

Periodontal ligament and gingival fibroblasts from periodontitis patients are more active in interaction with Porphyromonas gingivalis.

BACKGROUND AND OBJECTIVE: Inflammatory responses of host cells to oral pathogenic bacteria, such as Porphyromonas gingivalis, are crucial in the development of periodontitis. Host cells, such as periodontal ligament and gingival fibroblasts, from periodontitis patients may respond to P. gingivalis in a different manner compared with cells from healthy persons. The aim of this study was to investigate inflammatory responses to viable P. gingivalis by periodontal ligament and gingival fibroblasts from periodontitis patients and healthy control subjects. MATERIAL AND METHODS: Primary periodontal ligament and gingival fibroblasts from periodontitis patients (n=14) and healthy control subjects (n=8) were challenged in vitro with viable P. gingivalis. Gene expression of Toll-like receptors (TLRs) 1, 2, 4, 6, 7 and 9, CD14, nuclear factor-κB1 and its putative inhibitor NF-κB inhibitor-like protein1, and of interleukin-1ß, interleukin-6, interleukin-8, tumour necrosis factor-α, monocyte chemotactic protein-1 and regulated upon activation, normal T-cel expressed, and secreted, were assessed by real-time PCR. RESULTS: Periodontal ligament fibroblasts from periodontitis patients had a higher mRNA expression of TLR1, TLR4, TLR7 and CD14, and a lower expression of NFKBIL1, both before and after P. gingivalis challenge. In contrast, gingival fibroblasts from periodontitis patients had stronger induction of TLR1, TLR2 and TLR7 by P. gingivalis. Cytokine responses were not different between patients and control subjects. Interestingly, periodontal ligament, but not gingival, fibroblasts from P. gingivalis culture-positive persons responded more strongly to P. gingivalis than periodontal ligament fibroblasts from P. gingivalis-negative persons. CONCLUSION: Periodontal ligament and gingival fibroblasts respond to P. gingivalis in a different manner and may play different roles in periodontitis. Both subsets of fibroblasts from patients appear more active in interaction with P. gingivalis. Moreover, periodontal ligament fibroblasts from P. gingivalis-positive donors are more responsive to an in vitro P. gingivalis challenge.

Gingival and periodontal ligament fibroblasts differ in their inflammatory response to viable Porphyromonas gingivalis.

BACKGROUND AND OBJECTIVE: Porphyromonas gingivalis is an oral pathogen strongly associated with destruction of the tooth-supporting tissues in human periodontitis. Gingival fibroblasts (GF) and periodontal ligament fibroblasts (PDLF) are functionally different cell types in the periodontium that can participate in the host immune response in periodontitis. This study aimed to investigate the effects of viable P. gingivalis on the expression of genes associated with inflammation and bone degradation by these fibroblast subsets. MATERIAL AND METHODS: Primary human GF and PDLF from six healthy donors were challenged in vitro with viable P. gingivalis W83 for 6 h. Gene expression of inflammatory cytokines in GF and PDLF was analyzed using real-time PCR, and protein expression was analyzed using ELISA. RESULTS: Viable P. gingivalis induced a strong in vitro inflammatory response in both GF and PDLF. We found increased gene expression of interleukin (IL)-1beta, IL-6, IL-8, tumor necrosis factor-alpha, monocyte chemotactic protein-1 and regulated upon activation, normal T-cell expressed and secreted (RANTES). Macrophage colony-stimulating factor was induced and the expression of osteoprotegerin was decreased in GF, but not in PDLF. In nonchallenged cells, a higher level of expression of IL-6 was observed in GF than in PDLF. Between individual donors there was large heterogeneity in responsiveness to P. gingivalis. Also, in each individual, either GF or PDLF was more responsive to P. gingivalis. CONCLUSION: Considerable heterogeneity in responsiveness to P. gingivalis exists both between GF and PDLF and between individuals, which may be crucial determinants for the susceptibility to develop periodontitis.

The anion exchanger Ae2 is required for enamel maturation in mouse teeth.

One of the mechanisms by which epithelial cells regulate intracellular pH is exchanging bicarbonate for Cl(-). We tested the hypothesis that in ameloblasts the anion exchanger-2 (Ae2) is involved in pH regulation during maturation stage amelogenesis. Quantitative X-ray microprobe mineral content analysis, scanning electron microscopy, histology, micro-computed tomography and Ae2 immuno-localisation analyses were applied to Ae2-deficient and wild-type mouse mandibles. Immuno-localisation of Ae2 in wild-type mouse incisors showed a very strong expression of Ae2 in the basolateral membranes of the maturation stage ameloblasts. Strikingly, zones of contiguous ameloblasts were found within the maturation stage in which Ae2 expression was extremely low as opposed to neighbouring cells. Maturation stage ameloblasts of the Ae2(a,b)(-/-) mice failed to stain for Ae2 and showed progressive disorganisation as enamel development advanced. Maturation stage enamel of the Ae2(a,b)(-/-) mice contained substantially less mineral and more protein than wild-type enamel as determined by quantitative X-ray microanalysis. Incisor enamel was more severely affected than molar enamel. Scanning electron microscopy revealed that the rod-inter-rod structures of the Ae2(a,b)(-/-) mice incisor enamel were absent. Mineral content of dentine and bone of Ae2(a,b)(-/-) mice was not significantly different from wild-type mice. The enamel from knockout mouse teeth wore down much faster than that from wild-type litter mates. Basolateral bicarbonate secretion via the anionic exchanger Ae2 is essential for mineral growth in the maturation stage enamel. The observed zonal expression of Ae2 in the maturation stage ameloblasts is in line with a model for cyclic proton secretion during maturation stage amelogenesis.

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

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

Expression of FcgammaRs and mCD14 on polymorphonuclear neutrophils and monocytes may determine periodontal infection.

Variance in expression of receptors for immunoglobulin G (FcgammaRs), complement (CR3) and lipopolysaccharide (mCD14) on polymorphonuclear neutrophils (PMNs) and monocytes might affect susceptibility for infection with certain pathogens in periodontitis, a chronic infectious disease of tooth-supportive tissues. Levels of FcgammaRI, IIa, III, CR3 and mCD14 on PMNs and monocytes were measured in 19 periodontitis patients and 18 healthy controls. Subgingival infection with Aggregatibacter actinomycetemcomitans (Aa) and Porphyromonas gingivalis (Pg) was determined. Activation of PMNs and monocytes in response to stimulation with Aa and Pg was assessed by means of change in mCD14 expression. Periodontitis is associated with an enrichment of the FcgammaRIII(+) monocytes (P = 0.015) with concomitant low mCD14 (P = 0.001). Unadjusted data showed that the subjects culture-positive for Aa (Aa(+)) had significantly lower expression of monocytic FcgammaRI (P = 0.005) and FcgammaRIIa (P = 0.015) than Pg(+) subjects. The FcgammaRI was still lower on monocytes from Aa(+) subjects after adjusting for the background factors (P = 0.037). PMNs from Aa(+) subjects responded in a hyper-reactive manner, in particular when stimulated with Aa (P = 0.011). Lower FcgammaRs expression by monocytes is related to a higher susceptibility of a subject to become infected with Aa. The higher proportion of FcgammaRIII(+) monocytes may be involved in the chronicity of this condition. Hyper-reactive PMNs in Aa(+) subjects may contribute to accelerated breakdown of tooth-supportive tissues.

Tissue identification with micro-magnetic resonance imaging in a caprine spinal fusion model.

Nonunion is a major complication of spinal interbody fusion. Currently X-ray and computed tomography (CT) are used for evaluating the spinal fusion process. However, both imaging modalities have limitations in judgment of the early stages of this fusion process, as they only visualize mineralized bone. Magnetic resonance imaging (MRI) could be of great value as it is able to discriminate between different types of tissue. A feasibility study was performed in nine animals from a goat spinal fusion study, to evaluate the detection capacity of different tissues with micro-MRI. In this study bioresorbable polylactic acid cages were used. Six- and 12-months follow-up specimens were scanned in a 6.3 T micro-MRI scanner. After scanning, the specimens were processed for histology. Different types of tissue as well as the degradable cage material were identified in the fusion zone and designated as regions of interest (ROIs). Subsequently, the location of these ROIs was determined on the corresponding micro-MRI image, and average signal intensities of every individual ROI were measured. An excellent match was seen between the histological sections and micro-MRI images. The micro-MRI images showed quantifiable differences in signal intensity between bone with adipose marrow, bone with hematopoietic marrow, fibrocartilage, fibrous tissue, and degradable implant material. In time the signal intensity of bone with adipose marrow, bone with hematopoietic red marrow, and of fibrous tissue remained relatively constant. On the other hand, the signal intensity of the degradable implant material and the fibrocartilage changed significantly in time, indicating change of structure and composition. In conclusion, in our model using bioresorbable cages the MRI provides us with detailed information about the early fusion process and may therefore, allow early diagnosis of non-union.

Fluid shear stress inhibits TNFalpha-induced osteocyte apoptosis.

Bone tissue can adapt to orthodontic load. Mechanosensing in bone is primarily a task for the osteocytes, which translate the canalicular flow resulting from bone loading into osteoclast and osteoblast recruiting signals. Apoptotic osteocytes attract osteoclasts, and inhibition of osteocyte apoptosis can therefore affect bone remodeling. Since TNF-alpha is a pro-inflammatory cytokine with apoptotic potency, and elevated levels are found in the gingival sulcus during orthodontic tooth movement, we investigated if mechanical loading by pulsating fluid flow affects TNF-alpha-induced apoptosis in chicken osteocytes, osteoblasts, and periosteal fibroblasts. During fluid stasis, TNF-alpha increased apoptosis by more than two-fold in both osteocytes and osteoblasts, but not in periosteal fibroblasts. One-hour pulsating fluid flow (0.70 +/- 0.30 Pa, 5 Hz) inhibited (-25%) TNF-alpha-induced apoptosis in osteocytes, but not in osteoblasts or periosteal fibroblasts, suggesting a key regulatory role for osteocyte apoptosis in bone remodeling after the application of an orthodontic load.