Bone Regeneration Based on Tissue Engineering Conceptions — A 21st Century Perspective

The role of Bone Tissue Engineering in the field of Regenerative Medicine has been the topic of substantial research over the past two decades. Technological advances have improved orthopaedic implants and surgical techniques for bone reconstruction. However, improvements in surgical techniques to reconstruct bone have been limited by the paucity of autologous materials available and donor site morbidity. Recent advances in the development of biomaterials have provided attractive alternatives to bone grafting expanding the surgical options for restoring the form and function of injured bone. Specifically, novel bioactive (second generation) biomaterials have been developed that are characterised by controlled action and reaction to the host tissue environment, whilst exhibiting controlled chemical breakdown and resorption with an ultimate replacement by regenerating tissue. Future generations of biomaterials (third generation) are designed to be not only osteoconductive but also osteoinductive, i.e. to stimulate regeneration of host tissues by combining tissue engineering and in situ tissue regeneration methods with a focus on novel applications. These techniques will lead to novel possibilities for tissue regeneration and repair. At present, tissue engineered constructs that may find future use as bone grafts for complex skeletal defects, whether from post-traumatic, degenerative, neoplastic or congenital/developmental “origin” require osseous reconstruction to ensure structural and functional integrity. Engineering functional bone using combinations of cells, scaffolds and bioactive factors is a promising strategy and a particular feature for future development in the area of hybrid materials which are able to exhibit suitable biomimetic and mechanical properties. This review will discuss the state of the art in this field and what we can expect from future generations of bone regeneration concepts.     

Effect of excessive methionine on the development of the cranial growth plate in newborn rats

ObjectiveMethionine is an essential amino acid and pivotal for normal growth and development. However, previous animal studies have shown that excessive maternal intake of methionine causes growth restrictions, organ damages, and abnormal growth of the mandible in newborn animals. However, the effect of excessive methionine on the development of the cranial growth plate is unknown. This study investigated histological alterations of the cranial growth plate induced by high methionine administration in newborn rats.DesignTwenty pregnant dams were divided into a control and an experimental group. The controls received a diet for rats and the experimental group was fed from the 18th gestational day with a special manufactured high methionine diet for rats. The high methionine diet was maintained until the end of the lactation phase (day 20). The offspring of both groups were killed at day 10 or 20 postnatally and their spheno-occipital synchondroses were collected for histological analysis.ResultsThe weight of the high-dose methionine treated experimental group was considerably reduced in comparison to the control group at day 10 and 20 postnatally. The cartilaginous area of the growth plate and the height of the proliferative zone were markedly reduced at postnatal day 10 in the experimental group.ConclusionsIn summary, the diet-induced hypermethioninemia in rat dams resulted in growth retardations and histomorphological changes of the spheno-occipital synchondrosis, an important craniofacial growth centre in newborns. This finding may elucidate facial dysmorphoses reported in patients suffering from hypermethioninemia.     

Influence of multisystemic affection on health-related quality of life in patients with myotonic dystrophy type 1

Aim

To assess health-related quality of life (HRQoL) in patients with DM1, to identify muscular, multisystemic, central and social factors that may affect QoL and to define a DM1 patient in risk of poor QoL.

Patients and method

This cross-sectional study comprised 120 DM1 consecutive patients. The following scales were used: Multidimensional Scale of Perceived Social Support (MSPSS), Muscular Impairment Rating Scale (MIRS), battery of neuropsychological tests, acceptance of illness scale (AIS), Hamilton rating scale for depression (Ham-D), Krupp's Fatigue Severity Scale (FSS), Daytime Sleepiness Scale (DSS) and SF-36 questionnaire.

Results

HRQoL was impaired in DM1 patients in both physical and mental domains (PCS was 41.8 ± 23.5, MCS 47.0 ± 24.3 and total SF-36 score 45.6 ± 24.0). The most significant factors correlating with better SF-36 total score were younger age (β = −0.45, p < 0.001), shorter duration of disease (β = −0.27, p = 0.001), higher education (β = 0.20, p = 0.009), less severe muscular weakness (β = −0.52, p < 0.001), normal swallowing (β = 0.22, p = 0.005), absence of fainting (β = 0.31, p = 0.002), absence of snoring (β = 0.21, p = 0.036), better acceptance of disease (β = −0.17, p = 0.036), lower depressiveness (β = −0.46, p = 0.001), lower fatigue (β = −0.32, p = 0.001), absence of cataract (β = −0.21, p = 0.034), absence of kyphosis (β = 0.31, p = 0.004) and absence of constipation (β = 0.24, p = 0.016). Second linear regression analysis revealed that depressed (β = −0.38, p < 0.001) and elder patients (β = −0.27, p = 0.007) and as well as those with poor acceptance of illness (β = −0.21, p = 0.006) were in especially higher risk of having poor HRQoL (R² = 0.68).

Conclusion

We identified different central, social, muscular, cardiorespiratory and other factors correlating with HRQoL. It is of great importance that most of these factors are amenable to treatment.     

The predictive value of the presence of different antibodies and thymus pathology to the clinical outcome in patients with generalized myasthenia gravis

Objectives

To analyze the predictive value of anti-acetylcholine receptor antibodies (anti-AChR Ab) and anti-muscle specific kinase antibodies (anti-MuSK Ab), as well as the thymus pathology to the clinical outcome in patients with generalized myasthenia gravis (MG).

Methods

We analyzed 138 patients with generalized MG, who were thymectomized and assayed for anti-AChR Ab and anti-MuSK Ab.

Results

Anti-AChR Ab were detected in 84% of patients, while anti-MuSK Ab were present in 36% of the AChR Ab negative patients. Severe forms of the disease were more frequent in MuSK Ab positive, compared to the AChR Ab positive and complete seronegative patients. Thymic lymphoid follicular hyperplasia (LFH) was present in 60%, thymoma in 23%, atrophic thymus in 9% and the normal thymus in 8% of patients. LFH was more frequent among women, while thymoma and atrophic thymus were more frequent in men. The younger patients mainly had LFH and normal thymus, while thymoma and atrophic thymus were more frequent in older patients. The mildest clinical presentation was present in patients with normal thymus, while severe forms of the disease were registered in the patients with thymoma. The AChR Ab positive patients had more often LFH and thymoma, while within MuSK Ab positive patients atrophic thymus was most common.

Conclusion

The best disease outcome was observed in patients with normal thymus or LFH with anti-AChR Ab or without both types of antibodies.     

Cardiologic predictors of sudden death in patients with myotonic dystrophy type 1

The aim of this study was to analyze survival, causes of death and cardiologic predictors of sudden death in a large cohort of patients with myotonic dystrophy type 1 (DM1). The study was comprised of 171 adult DM1 patients hospitalized at the Neurology Clinic in a 20-year period. Severe electrocardiographic (ECG) abnormality included at least one of the following: rhythm other than sinus, PR interval of ?240 ms, QRS complex duration of 120 ms or more, and second-degree or third-degree atrioventricular (AV) block. Survival data were analyzed by the Kaplan–Meier test, log–rank test and Cox regression analysis. During the mean follow-up period of 9.4 ± 5.4 years, a pacemaker was implanted in 5.8% of DM1 patients and 14% of patients died. The mean age at death was 55.6 ± 12.5 years. The most common causes of death in our cohort were sudden death (41.7%) and respiratory failure (29.2%). The presence of palpitations (hazard ratio [HR] = 4.7, p < 0.05) and increased systolic blood pressure (HR = 9.8, p < 0.05) were significant predictors of sudden death. Among ECG parameters, severe ECG abnormality (HR = 4.7, p < 0.05), right bundle branch block (RBBB; HR = 3.9, p < 0.05) and bifascicular block (HR = 5.8, p < 0.05) were significant predictors of sudden death.     

Therapeutic alliance and behavior inhibition

Abstract

The study examined whether the quality of the therapeutic alliance is associated with a tendency toward behavior inhibition. Inhibition was measured by the frequency with which the clients used the word “not” and by their verbal productivity. In treatment sessions with low-quality therapeutic alliance, clients tended to use the word “not” more often and speak fewer words. Results suggest that it could be useful for therapists to use indexes of behavior inhibition as problem markers in the therapeutic alliance.     

Pannexin‐1 and P2X7‐Receptor Are Required for Apoptotic Osteocytes in Fatigued Bone to Trigger RANKL Production in Neighboring Bystander Osteocytes

Osteocyte apoptosis is required to induce intracortical bone remodeling after microdamage in animal models, but how apoptotic osteocytes signal neighboring “bystander” cells to initiate the remodeling process is unknown. Apoptosis has been shown to open pannexin‐1 (Panx1) channels to release adenosine diphosphate (ATP) as a “find‐me” signal for phagocytic cells. To address whether apoptotic osteocytes use this signaling mechanism, we adapted the rat ulnar fatigue‐loading model to reproducibly introduce microdamage into mouse cortical bone and measured subsequent changes in osteocyte apoptosis, receptor activator of NF‐κB ligand (RANKL) expression and osteoclastic bone resorption in wild‐type (WT; C57Bl/6) mice and in mice genetically deficient in Panx1 (Panx1KO). Mouse ulnar loading produced linear microcracks comparable in number and location to the rat model. WT mice showed increased osteocyte apoptosis and RANKL expression at microdamage sites at 3 days after loading and increased intracortical remodeling and endocortical tunneling at day 14. With fatigue, Panx1KO mice exhibited levels of microdamage and osteocyte apoptosis identical to WT mice. However, they did not upregulate RANKL in bystander osteocytes or initiate resorption. Panx1 interacts with P2X₇R in ATP release; thus, we examined P2X₇R‐deficient mice and WT mice treated with P2X₇R antagonist Brilliant Blue G (BBG) to test the possible role of ATP as a find‐me signal. P2X₇RKO mice failed to upregulate RANKL in osteocytes or induce resorption despite normally elevated osteocyte apoptosis after fatigue loading. Similarly, treatment of fatigued C57Bl/6 mice with BBG mimicked behavior of both Panx1KO and P2X₇RKO mice; BBG had no effect on osteocyte apoptosis in fatigued bone but completely prevented increases in bystander osteocyte RANKL expression and attenuated activation of resorption by more than 50%. These results indicate that activation of Panx1 and P2X₇R are required for apoptotic osteocytes in fatigued bone to trigger RANKL production in neighboring bystander osteocytes and implicate ATP as an essential signal mediating this process. © 2016 American Society for Bone and Mineral Research.     

Assessing the sensitivity of diffusion MRI to detect neuronal activity directly

Functional MRI (fMRI) is widely used to study brain function in the neurosciences. Unfortunately, conventional fMRI only indirectly assesses neuronal activity via hemodynamic coupling. Diffusion fMRI was proposed as a more direct and accurate fMRI method to detect neuronal activity, yet confirmative findings have proven difficult to obtain. Given that the underlying relation between tissue water diffusion changes and neuronal activity remains unclear, the rationale for using diffusion MRI to monitor neuronal activity has yet to be clearly established. Here, we studied the correlation between water diffusion and neuronal activity in vitro by simultaneous calcium fluorescence imaging and diffusion MR acquisition. We used organotypic cortical cultures from rat brains as a biological model system, in which spontaneous neuronal activity robustly emerges free of hemodynamic and other artifacts. Simultaneous fluorescent calcium images of neuronal activity are then directly correlated with diffusion MR signals now free of confounds typically encountered in vivo. Although a simultaneous increase of diffusion-weighted MR signals was observed together with the prolonged depolarization of neurons induced by pharmacological manipulations (in which cell swelling was demonstrated to play an important role), no evidence was found that diffusion MR signals directly correlate with normal spontaneous neuronal activity. These results suggest that, whereas current diffusion MR methods could monitor pathological conditions such as hyperexcitability, e.g., those seen in epilepsy, they do not appear to be sensitive or specific enough to detect or follow normal neuronal activity.Developing a direct MRI method to detect neuronal activity in vivo and noninvasively is a major focus in neuroscience. Progress in this area is required to improve our understanding of normal brain function, and in a clinical setting, to develop new tools for studying normal and abnormal development to diagnose diseases and disorders of the brain. Functional MRI (fMRI) has been widely used in the cognitive neurosciences since its invention in the 1990s (1–3). The most widely used fMRI method, blood-oxygenation-level-dependent (BOLD) MRI, detects hemodynamic changes in the brain, which only indirectly reflects neuronal activity. Moreover, its hemodynamic origin limits both its spatial and temporal resolution and its interpretation as a direct proxy for neuronal activity (4, 5).More recently, several MRI methods were proposed to provide more direct measures of neuronal excitation (6). In particular, diffusion MRI, a method to measure the apparent diffusivity of water within tissues (7–9), has been suggested as a direct functional imaging method to detect neuronal activity (10–13). Early in vivo experiments in both humans and animals reported small but significant increases in highly diffusion-weighted MRI signals, which were ascribed to changes directly induced by the underlying neuronal activity rather than indirect hemodynamic changes (10–13). In vitro experiments on brain slices (14, 15) and spinal cord (16) reported similar reductions in water diffusivity under conditions of extreme hyperexcitability using strong pharmacologic stimulants.However, functional diffusion MRI (fDMRI) has not been widely used or adopted since its introduction almost two decades ago. Two major reasons for this may be a dearth of experiments that convincingly establish its neurophysiological basis and the poor reproducibility of the originally reported changes in diffusion MRI signals by different laboratories. The inability to detect the predicted changes using fDMRI and the possible confounds of hemodynamic contributions in fDMRI measurements in vivo do not argue for a robust connection between changes in diffusion MRI and underlying neuronal activity (17–20). Thus, “ground-truth” experiments, potentially establishing a connection between the changes in diffusion MRI and underlying neuronal activity, are needed, particularly to shed light on the possible biophysical basis of the fDMRI signal.Recently, we developed a novel test bed that could be used to assess non–hemodynamic-based functional MRI methods, in which MR signal acquisition and intracellular calcium fluorescence imaging to monitor neuronal activity can be performed simultaneously on organotypic cortical cultures from rat brains (21). The organotypic cortex culture is a well-characterized biological model of neuronal activity free of hemodynamic, respiratory, and other physiological confounds. Not only is the in vivo cortical cytoarchitecture preserved (including cortical layers and cortical cell types), but neuronal activity in the culture also displays bursts of spontaneous neuronal avalanches grouped into so-called up-states and separated by periods of low activity (22–25), resembling resting neuronal activity in vivo (26–28). Specifically, fluorometric calcium (Ca²⁺) imaging is used to detect intracellular Ca²⁺ concentration changes that closely follow action potentials in neurons under normal conditions and provide a direct method for detecting neuronal spiking activity in a neuronal network (29, 30). This test bed thus allows one to study precisely and accurately temporal correlations between the candidate functional MR signals, which are free of the usual in vivo confounds, and the underlying neuronal spiking activity by using an independent intracellular Ca²⁺ imaging experiment (21).In the current study, diffusion MR signals are obtained simultaneously with intracellular calcium fluorescence imaging of the organotypic cortex culture. The direct effects of neuronal activity on the diffusion MR signals are studied by time-series analysis of the simultaneous calcium and MR signals during normal neuronal activity and in different pathological states, which include induced hyperexcitability by kainic acid (kainate) and potassium, disinhibition by picrotoxin (PTX), suppression of excitability by tetrodotoxin (TTX), and cell volume modulation caused by osmotic pressure challenges. On the basis of these findings, it is possible to assess the prospect of detecting normal and abnormal neuronal activity using fDMRI and to better understand the relationship between fDMRI changes and biophysical mechanisms associated with neuronal excitation.