Long-lived murine osteocytes are embodied by craniofacial skeleton in young and old animals whereas they decrease in number in postcranial skeletons at older ages.

BACKGROUND: Osteocytes are engaged in life-enduring processes such as bone remodelling, fracture healing or osseointegration of implants. Over age, ossification processes and regenerative capacity can greatly differ in mandible and femur. OBJECTIVE: Mesenchymal stem cells from cranial and postcranial bones are of different embryologic origin. This may be the reason why the regenerative capacity differs between cranial and postcranial bones in old patients. It was hypothesised that different ageing patterns, reflected by osteocyte density, lacunar density and osteoid formation, exist between murine mandibles and femurs. MATERIAL AND METHODS: Mandible and femur of young (4 months) and old (34-36 months old) male C57Bl/6 mice were histologically investigated to determine the number of lacunae occupied with osteocytes. Osteoid formation was revealed by Masson-Goldner staining, and the spatial distribution of BMP-2 synthesis was examined. RESULTS: Over lifetime, the number of lacunae occupied with osteocytes only showed a modest decrease in mandibular bone (old 85.63%/young 91.12%) while greatly diverging in the femur (old 55.99%/young 93.28%). In equal measure, old femur exhibited less osteoid formation and decreased BMP-2 expression. CONCLUSION: Tissue-specific conduct of bone ageing is moulded by osteocytic activities, which was found to vary between postcranial and craniofacial skeleton. The latter harbours long-lived osteocytes also in old animals which assures lifelong bone integrity. Preliminary concurring findings from a human cadaver, also presented in this contribution, provided a rationale for recommending the translatability to humans.

Systemic impact molds mesenchymal stromal/stem cell aging.

Aging is associated with an accruing emergence of non-functional tissues. Mesenchymal stem cells (MSC) bring forth progenitors with multi-lineage differentiation potential, yet, they also exhibit anti-inflammatory and tissue-protective properties. Due to aging, altered tissue microenvironments constrict controlled stem cell proliferation and progenitor differentiation, thus diminishing the fitness of MSC. Therefore, deepening our understanding of metabolic, molecular and environmental factors impacting on MSC during human aging as well as providing new vistas on their role in promoting healthy aging and preventing age-associated disease is pivot. It is anticipated that integrative quantification of systemic parameters dominantly impacting on MSC will also enable effective personalized prognosis and provision of effective early medical interventions. Working along this line, it can be envisaged that standards in medical therapies can be individually adjusted by accounting not solely for the patient's chronological age or other physical parameters rather than specific physiological parameters which are believed to functionally shape stem cell niches within the bone marrow.

Human bone marrow hosts polyfunctional memory CD4+ and CD8+ T cells with close contact to IL-15-producing cells.

Recently, a key role in memory T cell homing and survival has been attributed to the bone marrow (BM) in mice. In the human BM, the repertoire, function, and survival niches of CD4(+) and CD8(+) T cells have not yet been elucidated. In this study, we demonstrate that CD4(+) and CD8(+) effector memory T cells accumulate in the human BM and are in a heightened activation state as revealed by CD69 expression. BM-resident memory T cells produce more IFN-γ and are frequently polyfunctional. Immunofluorescence analysis revealed that CD4(+) and CD8(+) T cells are in the immediate vicinity of IL-15-producing BM cells, suggesting a close interaction between these two cell types and a regulatory role of IL-15 on T cells. Accordingly, IL-15 induced an identical pattern of CD69 expression in peripheral blood CD4(+) and CD8(+) T cell subsets. Moreover, the IL-15-inducible molecules Bcl-x(L), MIP-1α, MIP-1ß, and CCR5 were upregulated in the human BM. In summary, our results indicate that the human BM microenvironment, in particular IL-15-producing cells, is important for the maintenance of a polyfunctional memory CD4(+) and CD8(+) T cell pool.

Bone marrow T cells from the femur are similar to iliac crest derived cells in old age and represent a useful tool for studying the aged immune system.

BACKGROUND: CD4+ and CD8+ T cells reside in the human bone marrow (BM) and show a heightened activation state. However, only small sample sizes are available from sources such as the iliac crest. Larger samples can be obtained from the femur in the course of hip replacement surgery. It was therefore the goal of the present study to compare the phenotype and function of BM T cells from different sources from elderly persons and to investigate how femur derived bone marrow T cells can serve as a tool to gain a better understanding of the role of adaptive immune cells in the BM in old age. RESULTS: Bone marrow mononuclear cells (BMMC) were isolated from either the iliac crest or the femur shaft. As expected the yield of mononuclear cells was higher from femur than from iliac crest samples. There were no phenotypic differences between BMMC from the two sources. Compared to PBMC, both BM sample types contained fewer naïve and more antigen experienced CD4+ as well as CD8+ T cells, which, in contrast to peripheral cells, expressed CD69. Cytokine production was also similar in T cells from both BM types. Larger sample sizes allowed the generation of T cell lines from femur derived bone marrow using non-specific as well as specific stimulation. The phenotype of T cell lines generated by stimulation with OKT-3 and IL-2 for two weeks was very similar to the one of ex vivo BM derived T cells. Such lines can be used for studies on the interaction of different types of BM cells as shown by co-culture experiments with BM derived stromal cells. Using CMVNLV specific T cell lines we additionally demonstrated that BM samples from the femur are suitable for the generation of antigen specific T cell lines, which can be used in studies on the clonal composition of antigen specific BM T cells. CONCLUSION: In conclusion, our results demonstrate that BMMC from the femur shaft are a useful tool for studies on the role of T cells in the BM in old age.

Mesenchymal stem cells show radioresistance in vivo.

Irradiation impacts on the viability and differentiation capacity of tissue-borne mesenchymal stem cells (MSC), which play a pivotal role in bone regeneration. As a consequence of radiotherapy, bones may develop osteoradionecrosis. When irradiating human bone-derived MSC in vitro with increasing doses, the cells' self-renewal capabilities were greatly reduced. Mitotically stalled cells were still capable of differentiating into osteoblasts and pre-adipocytes. As a large animal model comparable to the clinical situation, pig mandibles were subjected to fractionized radiation of 2 χ 9 Gy within 1 week. This treatment mimics that of a standardized clinical treatment regimen of head and neck cancer patients irradiated 30 χ 2 Gy. In the pig model, fractures which had been irradiated, showed delayed osseous healing. When isolating MSC at different time points post-irradiation, no significant changes regarding proliferation capacity and osteogenic differentiation potential became apparent. Therefore, pig mandibles were irradiated with a single dose of either 9 or 18 Gy in vivo, and MSC were isolated immediately afterwards. No significant differences between the untreated and 9 Gy irradiated bone with respect to proliferation and osteogenic differentiation were unveiled. Yet, cells isolated from 18 Gy irradiated specimens exhibited a reduced osteogenic differentiation capacity, and during the first 2 weeks proliferation rates were greatly diminished. Thereafter, cells recovered and showed normal proliferation behaviour. These findings imply that MSC can effectively cope with irradiation up to high doses in vivo. This finding should thus be implemented in future therapeutic concepts to protect regenerating tissue from radiation consequences.

A Tool for Rapid Assessment of Functional Outcomes in Patients with Head and Neck Cancer.

Head and neck cancer (HNC) and its treatment can lead to various functional impairments. We developed and validated an instrument for rapid physician-rated assessment of basic functional outcomes in HNC patients. HNC-relevant functional domains were identified through a literature review and assigned to verbal ratings based on observable criteria. The instrument draft was subjected to systematic expert review to assess its face and content validity. Finally, the empirical validity, reliability, and responsiveness of the expert-adapted Functional Integrity in Head and Neck Cancer (HNC-FIT) scales were assessed in healthy controls and in HNC patients. A matrix of the 6 functional domains of oral food intake, respiration, speech, pain, mood, and neck and shoulder mobility was created, each with 5 verbal rating levels. Face and content validity levels of the HNC-FIT scales were judged to be adequate by 17 experts. In 37 control subjects, 24 patients with HNC before treatment, and in 60 HNC patients after treatment, the HNC-FIT ratings in the 3 groups behaved as expected and functional domains correlated closely with the outcome of corresponding scales of the EORTC-HN35-QoL questionnaire, indicating good construct and criterion validity. Interrater reliability (rICC) was ≥0.9 for all functional domains and retest reliability (rICC) was ≥0.93 for all domains except mood (rICC = 0.71). The treatment effect size (eta-square) as a measure of responsiveness was ≥0.15 (p < 0.01) for fall domains except for breathing and neck and shoulder mobility. The median HNC-FIT scale completion time was 1 min 17 s. The HNC-FIT scale is a rapid tool for physician-rated assessment of functional outcomes in HNC patients with good validity, reliability, and responsiveness.

Facial trauma: how dangerous are skiing and snowboarding?

PURPOSE: The aim of this study was to investigate maxillofacial injuries sustained in both skiing and snowboarding accidents and correlate injury mechanisms and patterns evaluating a large population. MATERIALS AND METHODS: Between 1991 and 2003, all patients with maxillofacial injuries due to skiing and snowboarding accidents (1,393 cases) were reviewed and statistically analyzed according to age, gender, type of injury, cause of accident, location of trauma, and associated injuries. RESULTS: Skiing accidents resulted in a total of 1,250 injuries, and snowboarding resulted in 143. In this study 686 skiers presented with 1,452 facial bone fractures and 80 snowboarders sustained 160 fractures of the face. Skiers had dentoalveolar trauma in 810 cases and 1,295 soft tissue injuries, whereas snowboarders had 88 dental injuries and 187 soft tissue lesions. Mechanisms of injury included 542 cases due to skiing and 85 falls due to snowboarding (a 1.79-fold higher risk for snowboarders). The gender distribution showed a male-female ratio of 3:1 in skiers and 5.5:1 in snowboarders. In both groups male patients were more prone to have a facial bone fracture than female patients. Snowboarders aged between 10 and 29 years had a 2.14-fold higher risk of sustaining a maxillofacial injury than skiers. CONCLUSIONS: In both groups facial bone fractures occurred more often in male patients, and they were more likely to result from falls and collisions with other persons. Young snowboarders had a higher risk of maxillofacial injuries (especially soft tissue lesions) than skiers, whereas for children and old persons, skiing posed a much higher risk. Wearing a helmet while skiing and snowboarding should be mandatory to prevent serious trauma to the head.

Osteocyte numbers decrease only in postcranial but not in cranial bones in humans of advanced age.

BACKGROUND: Bone ageing is governed by the linked activities of short-lived osteoblasts and osteoclasts in conjunction with long-lived osteocytes present in osseous structure. Besides their maintenance function, osteogenic cells also gain specific positional information, which may potentially trigger ageing-associated cellular deviations in terminally differentiated osteocytes differently in cranial versus postcranial tissues. METHODS: We therefore investigated bone taken from deceased aged humans explanted at five distinct anatomical positions throughout the body and assessed physical and biological determinants applying radiologic and histologic measures. RESULTS: We were able to show that significantly more osteocytes reside in aged cortical bone at cranial positions than within axial or limb skeleton. These cellular states and conditions were not found in the corresponding trabecular bone, where osteocyte numbers remain also high at postcranial positions. Parallel comparative analyses of bone microstructure as analyzed by means of computer tomography showed no significant differences. CONCLUSIONS: Considering differences and commonalities regarding the bone samples, such as loading, mechanisms of ossification or the surrounding stromal cell compartment, our findings indicate that positional information laid down during ontogenetic processes is instructive during the entire life thus potentially also moulding spatial-specific mechanistic distinctions of bone ageing.

The role of oxygen termination of nanocrystalline diamond on immobilisation of BMP-2 and subsequent bone formation.

Medical implants are increasingly often inserted into bone of frail patients, who are advanced in years. Due to age, severe trauma or pathology-related bone changes, osseous healing at the implant site is frequently limited. We were able to demonstrate that coating of endosseous implants with nanocrystalline diamond (NCD) allows stable functionalization by means of physisorption with BMP-2. Strong physisorption was shown to be directly related to the unique properties of NCD, and BMP-2 in its active form interacted strongly when NCD was oxygen-terminated. The binding of the protein was monitored under physiological conditions by single molecule force spectroscopy, and the respective adsorption energies were further substantiated by force-field-calculations. Implant surfaces refined in such a manner yielded enhanced osseointegration in vivo, when inserted into sheep calvaria. Our results further suggest that this technical advancement can be readily applied in clinical therapies with regard to bone healing, since primary human mesenchymal stromal cells strongly activated the expression of osteogenic markers when being cultivated on NCD physisorbed with physiological amounts of BMP-2.

EpCAM expression in squamous cell carcinoma of the oral cavity: frequency and relationship to clinicopathologic features.

This retrospective study was designed to investigate the prognostic significance of EpCAM expression in human oral squamous cell carcinoma on a long-term follow-up. EpCAM expression was examined immunohistochemically on a tissue microarray (TMA) of paraffin embedded tissue specimens from 77 consecutive patients who underwent surgical treatment for squamous cell carcinoma of the oral cavity in the period between 1980 and 1997 at the Department of Craniomaxillofacial and Oral Surgery, Innsbruck Medical University. High EpCAM expression was found in 17 (22.1%) of the tumor samples. Using Kaplan-Meier analysis no correlation of EpCAM overexpression was observed with conventional clinicopathologic features in this patient cohort. Taken together, these data suggest that EpCAM might become an attractive treatment target in a subgroup of patients with OSCC.

Traumatic intracranial haemorrhage in conscious patients with facial fractures--a review of 1959 cases.

OBJECTIVE: Facial fracture patients who are conscious with a Glasgow Coma Scale (GCS) score of 15 in the absence of clinical neurological abnormalities are commonly not expected to have suffered severe intracranial pathology. However, high velocity impact may result in intracranial haemorrhage in different compartments. METHODS: Over a 7-year period, 1959 facial fracture patients with GCS scores of 15 and the absence of neurological abnormalities were analysed. In 54 patients (2.8%) computed tomography scans revealed the presence of accompanying intracranial haemorrhage (study group). These patients were compared with the 1905 patients without intracranial haemorrhage (control group). RESULTS: Univariate analysis identified accompanying vomiting/nausea and seizures, cervical spine injuries, cranial vault and basal skull fractures to be significantly associated with intracranial bleeding. In multivariate analysis the risk was increased nearly 25-fold if an episode of vomiting/nausea had occurred. Seizures increased the risk of bleeding more than 15-fold. The mean functional outcome of the study group according to the Glasgow Outcome Scale was 4.7+/-0.7. CONCLUSION: Intracranial haemorrhage cannot be excluded in patients with facial fractures despite a GCS score of 15 and normal findings following neurological examination. Predictors, such as vomiting/nausea or seizures, skull fractures and closed head injuries, enhance the likelihood of an intracranial haemorrhage and have to be considered.

Changes of the Functional Capacity of Mesenchymal Stem Cells due to Aging or Age-Associated Disease - Implications for Clinical Applications and Donor Recruitment.

SUMMARY: In contrast to stem cells of embryonic origin, autologous tissue-specific stem cells are easier to introduce into the clinical practice. In this context, molecular and cellular changes, which alter tissue-specific stem cell properties with age, are of particular interest since elderly patients represent the main target group for cell-based therapies. The clinical use of mesenchymal stem cells is an emerging field, especially because this stem cell type appears to be amenable for the treatment of a large number of diseases, such as non-healing bone defects and fractures, inflammatory relief during arthritis, and the repair of suspensory ligament tears. More than that, mesenchymal stem cells provoke effective immune suppression in the context of graft-versus-host disease. Here, we present a comprehensive overview of the recent findings with special attention to age-related changes of mesenchymal stem cell properties and the consequential impact on tissue regeneration and repair, together with the current perception concerning their therapeutic application potential as well as the challenges associated with their clinical use.

Impact of Nano-Crystalline Diamond Enhanced Hydrophilicity on Cell Proliferation on Machined and SLA Titanium Surfaces: An In-Vivo Study in Rodents.

By coating surfaces with nano-crystalline diamond (NCD) particles, hydrophilicity can be altered via sidechain modifications without affecting surface texture. The present study aimed to assess the impact of NCD hydrophilicity on machined and rough SLA titanium discs on soft tissue integration, using a rodent model simulating submerged healing. Four different titanium discs (machined titanium = M Titanium, NCD-coated hydrophilic machined titanium = M-O-NCD, sand blasted acid etched (SLA Titanium) titanium, and hydrophilic NCD-coated SLA titanium = SLA O-NCD) were inserted in subdermal pockets of 12 Wistar rats. After one and four weeks of healing, the animals were sacrificed. Biopsies were embedded in methyl methacrylate (MMA), and processed for histology. The number of cells located within a region of interest (ROI) of 10 µm around the discs were counted and compared statistically. Signs of inflammation were evaluated descriptively employing immunohistochemistry. At one week, M-O-NCD coated titanium discs showed significantly higher amounts of cells compared to M Titanium, SLA Titanium, and SLA-O-NCD (p < 0.001). At four weeks, significant higher cell counts were noted at SLA-O-NCD surfaces (p < 0.01). Immunohistochemistry revealed decreased inflammatory responses at hydrophilic surfaces. Within the limits of an animal study, M-O-NCD surfaces seem to stimulate cell proliferation in the initial healing phase, whereas SLA-O-NCD surfaces appeared advantageous afterwards.

Nanocrystalline diamond--an excellent platform for life science applications.

Nanocrystalline diamond (NCD) has recently been successfully utilized in a variety of life science applications. NCD films are favorable and salubrious substrates for cells during cultivation. Therefore NCD has also been employed in tissue engineering strategies. NCD as reported in this contribution was grown by means of a modified hot-filament chemical vapor deposition technique, which results in less than 3% sp2-hybridization and yields grain sizes of 5-20 nm. After production the NCD surface was rather hydrophobic, however it could be efficiently refined to exhibit more hydrophilic properties. Changing of the surface structure was found to be an efficient means to influence growth and differentiation capacity of a variety of cells. The particular needs for any given cell type has to be proven empirically. Yet flexible features of NCD appear to be superior to plastic surfaces which can be hardly changed in quality. Besides its molecular properties, crystal structural peculiarities of NCD appear to influence cell growth as well. In our attempt to facilitate, highly specialized applications in biomedicine, we recently discovered that growth factors can be tightly bound to NCD by mere physisorption. Hence, combination of surface functionalization together with further options to coat NCD with any kind of three-dimensional structure opens up new avenues for many more applications. In fact, high through-put protein profiling of early disease stages may become possible from serum samples, because proteins bound to NCD can now be efficiently analyzed by MALDI/TOF-MS. Given these results, it is to be presumed that the physical properties and effective electrochemical characteristics of NCD will allow tailoring devices suitable for many more diagnostic as well as therapeutic applications.

A domestic porcine model for studying the effects of radiation on head and neck cancers.

BACKGROUND: Radiation therapy (RT) of the head and neck region is often accompanied by serious side effects. Research in this area is needed to improve treatment outcomes and ameliorate therapy tolerance. Laboratory rodents are barely matching today's clinical standards in RT research. Yet domestic swine (Sus scrofa domestica) have previously proved suitable for various advanced tests in clinical research and training. We therefore investigated whether S. scrofa domestica is also appropriate for irradiation of the mandible. STUDY DESIGN: A common scheme for irradiation treatment of S. scrofa domestica mandibles in a split-mouth design was acquired by applying computed tomography (CT) scanning under sedation. Basing on close anatomic resemblance, a standard treatment plan comprising 2 opposed irradiation fields could be accomplished. RESULTS: RT was carried out in a clinical environment with 2 × 9 Gy. The resulting operating procedure facilitated complication-free sedation, transport, positioning, CT scanning, and effective irradiation. CONCLUSION: Based on common standards applied for RT in humans, domestic pigs can be employed to progress RT clinical research. Due to their human-like anatomy, physiology, size, and weight, the swine model is expedient for advancing experimental RT of the head and neck area.

Bone and aging: effects on the maxillofacial skeleton.

The originally balanced system of normal bone metabolism with an equilibrium of bone resorption and bone formation experiences involutionary changes along with increasing age. This article provides an overview on the effects of aging on bone, in particular on the bone of the maxillofacial skeleton. It explains differences between the postmenopausal type and the senile type of osteoporosis, deals with age-related morphologic changes of bone, mechanisms leading to age-related changes, depicts bones at high fracture risk in aging persons, analyses the reduced bone quantity and quality in cranial bone, and discusses the preservation of maxillofacial bone dimensions by implants. Therefore, research on wound healing, in particular bone healing, and on the regenerative potential of tissue of mesenchymal origin is of major interest and will eventually translate into improved care for patients during daily clinical routine.

Strong binding of bioactive BMP-2 to nanocrystalline diamond by physisorption.

Nano-crystalline diamond (NCD)-coated surfaces were efficiently functionalized with bone morphogenetic protein-2 (BMP-2) by means of physisorption. Due to their randomly oriented texture, NCD-coated surfaces appear to bind complex molecules firmly. Applying various highly sensitive analytical methods, the interaction was found extremely stable. The strength of the experimentally measured adherence between BMP-2 and NCD was further corroborated by theoretical calculations. Oxygen treatment rendered NCD hydrophilic by the appearance of surface oxygen containing groups. This particular NCD surface exhibited even higher binding energies towards BMP-2 than the hydrophobic surface, and this surface was also favoured by cultured cells. Most importantly in this context, bound BMP-2 was found fully active. When cultured on BMP-2-treated NCD, osteosarcoma cells strongly up-regulated alkaline phosphatase, a specific marker for osteogenic differentiation. Hence, this simple method will allow generating highly versatile surfaces with complex biomimetic coatings, essentials for novel medical devices and implants as well as for innovative scaffolds in tissue engineering.

A central role for the nuclear factor-kappaB pathway in anti-inflammatory and proinflammatory actions of mechanical strain.

Mechanical signals play an integral role in bone homeostasis. These signals are observed at the interface of bone and teeth, where osteoblast-like periodontal ligament (PDL) cells constantly take part in bone formation and resorption in response to applied mechanical forces. Earlier, we reported that signals generated by tensile strain of low magnitude (TENS-L) are antiinflammatory, whereas tensile strain of high magnitude (TENS-H) is proinflammatory and catabolic. In this study, we examined the mechanisms of intracellular actions of the antiinflammatory and proinflammatory signals generated by TENS of various magnitudes. We show that both low and high magnitudes of mechanical strain exploit nuclear factor (NF)-kappaB as a common pathway for transcriptional inhibition/activation of proinflammatory genes and catabolic processes. TENS-L is a potent inhibitor of interleukin (IL)-1 beta-induced I-kappaBbeta degradation and prevents dissociation of NF-kB from cytoplasmic complexes and thus its nuclear translocation. This leads to sustained suppression of IL-1beta-induced NF-kappaB transcriptional regulation of proinflammatory genes. In contrast, TENS-H is a proinflammatory signal that induces I-kappaBbeta degradation, nuclear translocation of NF-kappaB, and transcriptional activation of proinflammatory genes. These findings are the first to describe the largely unknown intracellular mechanism of action of applied tensile forces in osteoblast-like cells and have critical implications in bone remodeling.

Anti-inflammatory effects of continuous passive motion on meniscal fibrocartilage.

Motion-based therapies have been applied to promote healing of arthritic joints. The goal of the current study was to determine the early molecular events that are responsible for the beneficial actions of motion-based therapies on meniscal fibrocartilage. Rabbit knees with Antigen-Induced-Arthritis (AIA) were exposed to continuous passive motion (CPM) for 24 or 48 h and compared to immobilized knees. The menisci were harvested and glycosaminoglycans (GAG), interleukin-1beta (IL-1beta), matrix metalloproteinase-1 (MMP-1), cyclooxygenase-2 (COX-2), and interleukin-10 (IL-10) were determined by histochemical analysis. Within 24 h, immobilized knees exhibited marked GAG degradation. The expression of proinflammatory mediators MMP-1, COX-2, and IL-1beta was notably increased within 24 h and continued to increase during the next 24 h in immobilized knees. Knees subjected to CPM revealed a rapid and sustained decrease in GAG degradation and the expression of all proinflammatory mediators during the entire period of CPM treatment. More importantly, CPM induced synthesis of the anti-inflammatory cytokine IL-10. The results demonstrate that mechanical signals generated by CPM exert potent anti-inflammatory signals on meniscal fibrochondrocytes. Furthermore, these studies explain the molecular basis of the beneficial effects of CPM observed on articular cartilage and suggest that CPM suppresses the inflammatory process of arthritis more efficiently than immobilization.