Porous 3D Scaffolds Enhance MSC Vitality and Reduce Osteoclast Activity.

In the context of an aging population, unhealthy Western lifestyle, and the lack of an optimal surgical treatment, deep osteochondral defects pose a great challenge for the public health system. Biodegradable, biomimetic scaffolds seem to be a promising solution. In this study we investigated the biocompatibility of porous poly-((D,L)-lactide-ε-caprolactone)dimethacrylate (LCM) scaffolds in contrast to compact LCM scaffolds and blank cell culture plastic. Thus, morphology, cytotoxicity and metabolic activity of human mesenchymal stromal cells (MSC) seeded directly on the materials were analyzed after three and six days of culturing. Further, osteoclastogenesis and osteoclastic activity were assessed using reverse-transcriptase real-time PCR of osteoclast-specific genes, EIA and morphologic aspects after four, eight, and twelve days. LCM scaffolds did not display cytotoxic effects on MSC. After three days, metabolic activity of MSC was enhanced on 3D porous scaffolds (PS) compared to 2D compact scaffolds (CS). Osteoclast activity seemed to be reduced at PS compared to cell culture plastic at all time points, while no differences in osteoclastogenesis were detectable between the materials. These results indicate a good cytocompatibility of LCM scaffolds. Interestingly, porous 3D structure induced higher metabolic activity of MSC as well as reduced osteoclast activity.

Effects of BDNF and PEC Nanoparticles on Osteocytes.

Bone substitute materials loaded with mediators that stimulate fracture healing are demanded in the clinical treatment in trauma surgery and orthopedics. Brain-derived neurotrophic factor (BDNF) enhances the proliferation and differentiation of mesenchymal stem cells into osteoblast. To load the implants with BDNF, a drug delivery system that allows the release of BDNF under spatiotemporal control would improve functionality. Polyelectrolyte complex nanoparticles (PECNP) have been reported as a suitable drug delivery system. The suitability of PECNP in contact with osteocytes as the main cell type of bone is not known so far. Thus, we aimed to verify that BDNF and PECNP loaded with BDNF (PECNP+BDNF) as well as pure PECNP have no negative effects on osteocytes in vitro. Therefore, the murine osteocyte cell line MLO-Y4 was treated with BDNF and PECNP+BDNF. The effects on proliferation were analyzed by the BrdU test (n = 5). The results demonstrated a significant increase in proliferation 24 h after BDNF application, whereas PECNP+BDNF did not lead to significant changes. Thus, we conclude that BDNF is an appropriate mediator to stimulate osteocytes. Since the addition of PECNP did not affect the viability of osteocytes, we conclude that PECNP are a suitable drug delivery system for bone implants.

Importance of Mechanoreceptors and Other Neural Structures Within the Anterior Intermeniscal Ligament in the Etiology of Anterior Knee Pain After Tibial Nailing.

BACKGROUND: Anterior knee pain is the most common complication after intramedullary tibial nailing. The cause is often multifactorial and varies among individuals. Violation of the anterior intermeniscal ligament (AIL) during intramedullary tibial nailing might be a possible source of postsurgical anterior knee pain. Although there is a certain ambiguity regarding the importance and function of the AIL, neural structures in the AIL tissue might play a significant role with respect to functional purposes and pain perception. METHODS: We subjected 6 AIL specimens to histologic examination to identify the neural structures that are a mandatory requirement as a source of anterior knee pain. Specifically, we performed three-dimensional immunohistochemical investigation of subtyping, orientation, and detailed characterization of neural structures within the AIL tissue. RESULTS: Histologic and three-dimensional immunohistochemical examinations confirmed the presence of neural structures in all 6 AIL specimens. We identified myelinated and unmyelinated nerve fibers, as well as all types of mechanoreceptors. CONCLUSIONS: Free nerve endings are a mandatory requirement for pain perception as a result of AIL violation during tibial nailing. Our verification of all different types of mechanoreceptors in the AIL tissue makes a role of the ligament in knee joint function and proprioception highly probable. Further investigations are necessary to clarify possible correlations between neural supply and function of the AIL. Violation of the ligament during operative procedures should be avoided, although the significance of the AIL is still debated.

Application of donepezil increased collagen 1 expression in mesenchymal stroma cells of an ovine osteoporosis model.

OBJECTIVES: Donepezil inhibits the acetylcholine degradation molecule acetylcholinesterase (AChE). Clinical studies reported that Alzheimer's disease (AD) patients with hip fractures had improved bone quality and better fracture healing if they were treated with AD medication donepezil. We asked whether mesenchymal stroma cells (MSC) from an osteoporosis sheep model treated with donepezil increased their proliferation rate and mRNA expression. METHODS: Sheep were divided into 4 groups: a) untreated control group, b) sheep with bilateral ovariectomy (OVX), c) sheep with OVX and malnutrition, and d) sheep with OVX, malnutrition, and application of corticosteroid. After 8 months MSC were isolated of iliac crest biopsy, treated with donepezil, and AChE activity, proliferation rate, and mRNA expression were analyzed. RESULTS: Application of donepezil resulted in a significant decrease of AChE activity. Inhibition of AChE did not lead to a significant increase in proliferation. Expression of the osteogenic marker osteocalcin was not regulated by donepezil while the mRNA concentration of collagen was increased. CONCLUSION: AChE inhibition via donepezil resulted in an increased synthesis of osteoid which consists mainly of collagen. Thus, we suppose that increased acetylcholine levels through AChE inhibition do not support MSC proliferation but osteogenic activity probably combined with osteogenic differentiation.

Effects of new beta-type Ti-40Nb implant materials, brain-derived neurotrophic factor, acetylcholine and nicotine on human mesenchymal stem cells of osteoporotic and non osteoporotic donors.

INTRODUCTION: Treatment of osteoporotic fractures is still challenging and an urgent need exists for new materials, better adapted to osteoporotic bone by adjusted Young's modulus, appropriate surface modification and pharmaceuticals. MATERIALS AND METHODS: Titanium-40-niobium alloys, mechanically ground or additionally etched and titanium-6-aluminium-4-vanadium were analyzed in combination with brain-derived neurotrophic factor, acetylcholine and nicotine to determine their effects on human mesenchymal stem cells in vitro over 21 days using lactate dehydrogenase and alkaline phosphatase assays, live cell imaging and immunofluorescence microscopy. RESULTS: Cell number of human mesenchymal stem cells of osteoporotic donors was increased after 14 d in presence of ground titanium-40-niobium or titanium-6-aluminium-4-vanadium, together with brain-derived neurotrophic factor. Cell number of human mesenchymal stem cells of non osteoporotic donors increased after 21 d in presence of titanium-6-aluminium-4-vanadium without pharmaceuticals. No significant increase was measured for ground or etched titanium-40-niobium after 21 d. Osteoblast differentiation of osteoporotic donors was significantly higher than in non osteoporotic donors after 21 d in presence of etched, ground titanium-40-niobium or titanium-6-aluminium-4-vanadium accompanied by all pharmaceuticals tested. In presence of all alloys tested brain-derived neurotrophic factor, acetylcholine and nicotine increased differentiation of cells of osteoporotic donors and accelerated it in non osteoporotic donors. CONCLUSION: We conclude that ground titanium-40-niobium and brain-derived neurotrophic factor might be most suitable for subsequent in vivo testing.

Impact of acetylcholine and nicotine on human osteoclastogenesis in vitro.

Recent studies showed that the non-neuronal cholinergic system (NNCS) is taking part in bone metabolism. Most studies investigated its role in osteoblasts, but up to now, the involvement of the NNCS in human osteoclastogenesis remains relatively unclear. Thus, aim of the present study was to determine whether the application of acetylcholine (ACh, 10(−4) M), nicotine (10(−6) M), mineralized collagen membranes or brain derived neurotrophic factor (BDNF, 40 ng/mL) influences the mRNA regulation of molecular components of the NNCS and the neurotrophin family during osteoclastogenesis. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of young healthy donors (n = 8) and incubated with bone fragments and osteoclast differentiation media for 21 days. All the results are based on the measurement of RNA. Real-time RT-PCR analysis demonstrated a down-regulation of nicotinic acetylcholine receptor (nAChR) subunit α2 and muscarinic acetylcholine receptor (mAChR) M3by osteoclastogenesis while BDNF mRNA expression was not regulated. Application of ACh, nicotine, BDNF or collagen membranes did not affect osteoclastic differentiation.No regulation was detected for nAChR subunit α7, tropomyosin-related kinase receptor B (TrkB), and cholineacetyl transferase (ChAT). Taken together, we assume that the transcriptional level of osteoclastogenesis of healthy young humans is not regulated by BDNF, ACh, and nicotine. Thus, these drugs do not seem to worsen bone degradation and might therefore be suitable as modulators of bone substitution materials if having a positive effect on bone formation.

Regulation of acetylcholine receptors during differentiation of bone mesenchymal stem cells harvested from human reaming debris.

Acetylcholine (ACh) is an important signaling molecule in non-neuronal systems where it is involved in regulation of viability, proliferation, differentiation and migration of mesenchymal stem cells (MSC) that are capable to differentiate into osteoblasts, chondrocytes and adipocytes. Patients with the systemic disease osteoporosis show altered MSC properties, reduced bone formation and mineral density followed by increased bone fragility and high fracture incidence. Here we asked whether nicotinic and muscarinic acetylcholine receptors (AChR) are expressed in osteoblasts, adipocytes and chondrocytes differentiated from bone MSC extracted from human reaming debris (RDMSC) that was harvested during surgery of long bone diaphyseal fractures. Using RT-PCR, AChR were detected in RDMSC, osteoblasts, chondrocytes and adipocytes of male and female bone-healthy and of female osteoporotic donors. An up-regulation in multiplicity and occurrence of AChR subtypes was found in female compared to male donors and in osteoblast of male donors compared to adipocytes. Real-time RT-PCR analysis resulted in a significant increase in relative expression of nAChR α9 in chondrocytes compared to adipocytes of healthy female donors. The nAChR subunit α10 was significantly up-regulated in osteoblasts of healthy compared to osteoporotic donors as well as the mAChR M5 that is additionally decreased in osteoporotic osteoblasts compared to MSC and chondrocytes of osteoporotic donors. In summary, the gene expression of AChR during differentiation of RDMSC and its regulation in cells of osteoporotic donors lead to the assumption that AChR signaling is involved in bone formation and might be utilized to stimulate bone remodeling processes.

Enhanced osteogenesis on titanium implants by UVB photofunctionalization of hydrothermally grown TiO2 coatings.

Even though Ti-based implants are the most used materials for hard tissue replacement, they may present lack of osseointegration on the long term, due to their inertness. Hydrothermal treatment (HT) is a useful technique for the synthesis of firmly attached, highly crystalline coatings made of anatase titanium dioxide (TiO2), providing favorable nanoroughness and higher exposed surface area, as well as greater hydrophilicity, compared to the native amorphous oxide on pristine titanium. The hydrophilicity drops even more by photofunctionalization of the nanostructured TiO2-anatase coatings under UV light. Human mesenchymal stem cells exhibited a good response to the combination of the positive surface characteristics, especially in respect to the UVB pre-irradiation. The results showed that the cells were not harmed in terms of viability; even more, they were encouraged to differentiate in osteoblasts and to become osteogenically active, as confirmed by the calcium ion uptake and the formation of well-mineralized, bone-like nodule structures. In addition, the enrichment of hydroxyl groups on the HT-surfaces by UVB photofunctionalization accelerated the cell differentiation process and greatly improved the osteogenesis in comparison with the nonirradiated samples. The optimal surface characteristics of the HT-anatase coatings as well as the high potentiality of the photo-induced hydrophilicity, which was reached during a relatively short pre-irradiation time (5 h) with UVB light, can be correlated with better osseointegration ability in vivo; among the samples, the superior biological behavior of the roughest and most hydrophilic HT coating makes it a good candidate for further studies and applications.

Expression of choline and acetylcholine transporters in synovial tissue and cartilage of patients with rheumatoid arthritis and osteoarthritis.

Increasing evidence is showing that the non-neuronal cholinergic system plays an important role in the pathology of rheumatoid arthritis (RA). Choline transport into the cell is the rate-limiting step for the synthesis of acetylcholine (ACh), which can be released directly or in vesicles from the cell. However, in the human joint little is known about choline import or the release of ACh from the cell. Thus, we analyze the expression of members of the organic cation transporter (OCT), of the newly discovered choline transporter-like (CTL) family and of classical neuronal components such as the high-affinity choline transporter (CHT1) and the vesicular ACh transporter (VAChT) in the synovium and cartilage of the human hip joint from patients with osteoarthritis (OA) and RA. OCT1, OCT3 and OCTN1 and all members of the CTL family were expressed in synovial and cartilage samples. The expression of CTL1 and CTL2 was localized in synovial macrophages and fibroblasts. CHT1 mRNA expression was detectable only in the synovium, whereas VAChT was completely absent in all samples. Therefore, in the human joint, choline transport into the cell and the release of ACh seems to be mediated mainly by members of the OCT and CTL family. Expression of transporters appears not to be influenced by the pathological state, as no differences have been detected between joints from OA or RA patients. Importantly, however, all necessary components for choline import and the release of non-neuronal ACh are present in the human joint.

BDNF and its TrkB receptor in human fracture healing.

Fracture healing is a physiological process of repair which proceeds in stages, each characterized by a different predominant tissue in the fracture gap. Matrix reorganization is regulated by cytokines and growth factors. Neurotrophins and their receptors might be of importance to osteoblasts and endothelial cells during fracture healing. The aim of this study was to examine the presence of brain-derived neurotrophic factor (BDNF) and its tropomyosin-related kinase B receptor (TrkB) during human fracture healing. BDNF and TrkB were investigated in samples from human fracture gaps and cultured cells using RT-PCR, Western blot, and immunohistochemistry. Endothelial cells and osteoblastic cell lines demonstrated a cytoplasmic staining pattern of BDNF and TrkB in vitro. At the mRNA level, BDNF and TrkB were expressed in the initial and osteoid formation phase of human fracture healing. In the granulation tissue of fracture gap, both proteins--BDNF and TrkB--are concentrated in endothelial and osteoblastic cells at the margins of woven bone suggesting their involvement in the formation of new vessels. There was no evidence of BDNF or TrkB during fracture healing in chondrocytes of human enchondral tissue. Furthermore, BDNF is absent in mature bone. Taken together, BDNF and TrkB are involved in vessel formation and osteogenic processes during human fracture healing. The detection of BDNF and its TrkB receptor during various stages of the bone formation process in human fracture gap tissue were shown for the first time. The current study reveals that both proteins are up-regulated in human osteoblasts and endothelial cells in fracture healing.

Podoplanin immunopositive lymphatic vessels at the implant interface in a rat model of osteoporotic fractures.

Insertion of bone substitution materials accelerates healing of osteoporotic fractures. Biodegradable materials are preferred for application in osteoporotic patients to avoid a second surgery for implant replacement. Degraded implant fragments are often absorbed by macrophages that are removed from the fracture side via passage through veins or lymphatic vessels. We investigated if lymphatic vessels occur in osteoporotic bone defects and whether they are regulated by the use of different materials. To address this issue osteoporosis was induced in rats using the classical method of bilateral ovariectomy and additional calcium and vitamin deficient diet. In addition, wedge-shaped defects of 3, 4, or 5 mm were generated in the distal metaphyseal area of femur via osteotomy. The 4 mm defects were subsequently used for implantation studies where bone substitution materials of calcium phosphate cement, composites of collagen and silica, and iron foams with interconnecting pores were inserted. Different materials were partly additionally functionalized by strontium or bisphosphonate whose positive effects in osteoporosis treatment are well known. The lymphatic vessels were identified by immunohistochemistry using an antibody against podoplanin. Podoplanin immunopositive lymphatic vessels were detected in the granulation tissue filling the fracture gap, surrounding the implant and growing into the iron foam through its interconnected pores. Significant more lymphatic capillaries were counted at the implant interface of composite, strontium and bisphosphonate functionalized iron foam. A significant increase was also observed in the number of lymphatics situated in the pores of strontium coated iron foam. In conclusion, our results indicate the occurrence of lymphatic vessels in osteoporotic bone. Our results show that lymphatic vessels are localized at the implant interface and in the fracture gap where they might be involved in the removal of lymphocytes, macrophages, debris and the implants degradation products. Therefore the lymphatic vessels are involved in implant integration and fracture healing.

Effects of multi-deficiencies-diet on bone parameters of peripheral bone in ovariectomized mature rat.

Many postmenopausal women have vitamin D and calcium deficiency. Therefore, vitamin D and calcium supplementation is recommended for all patients with osteopenia and osteoporosis. We used an experimental rat model to test the hypothesis that induction of osteoporosis is more efficiently achieved in peripheral bone through combining ovariectomy with a unique multi-deficiencies diet (vitamin D depletion and deficient calcium, vitamin K and phosphorus). 14-week-old Sprague-Dawley rats served as controls to examine the initial bone status. 11 rats were bilaterally ovariectomized (OVX) and fed with multi-deficiencies diet. Three months later the treated group and the Sham group (n = 8) were euthanized. Bone biomechanical competence of the diaphyseal bone was examined on both, tibia and femur. Image analysis was performed on tibia via µCT, and on femur via histological analysis. Lower torsional stiffness indicated inferior mechanical competence of the tibia in 3 month OVX+Diet. Proximal metaphyseal region of the tibia showed a diminished bone tissue portion to total tissue in the µCT despite the increased total area as evaluated in both µCT and histology. Cortical bone showed higher porosity and smaller cross sectional thickness of the tibial diaphysis in the OVX+Diet rats. A lower ALP positive area and elevated serum level of RANKL exhibited the unbalanced cellular interaction in bone remodeling in the OVX+Diet rat after 3 month of treatment. Interestingly, more adipose tissue area in bone marrow indicated an effect of bone loss similar to that observed in osteoporotic patients. Nonetheless, the presence of osteoid and elevated serum level of PTH, BGP and Opn suggest the development of osteomalacia rather than an osteoporosis. As the treatment and fracture management of both osteoporotic and osteomalacia patients are clinically overlapping, this study provides a preclinical animal model to be utilized in local supplementation of minerals, drugs and growth factors in future fracture healing studies.

Implications of combined ovariectomy/multi-deficiency diet on rat bone with age-related variation in bone parameters and bone loss at multiple skeletal sites by DEXA.

BACKGROUND: Osteoporosis is a multi-factorial, chronic, skeletal disease highly prevalent in post-menopausal women and is influenced by hormonal and dietary factors. Because animal models are imperative for disease diagnostics, the present study establishes and evaluates enhanced osteoporosis obtained through combined ovariectomy and deficient diet by DEXA (dual-energy X-ray absorptiometry) for a prolonged time period. MATERIAL/METHODS: Sprague-Dawley rats were randomly divided into sham (laparotomized) and OVX-diet (ovariectomized and fed with deficient diet) groups. Different skeletal sites were scanned by DEXA at the following time points: M0 (baseline), M12 (12 months post-surgery), and M14 (14 months post-surgery). Parameters analyzed included BMD (bone mineral density), BMC (bone mineral content), bone area, and fat (%). Regression analysis was performed to determine the interrelationships between BMC, BMD, and bone area from M0 to M14. RESULTS: BMD and BMC were significantly lower in OVX-diet rats at M12 and M14 compared to sham rats. The Z-scores were below -5 in OVX-diet rats at M12, but still decreased at M14 in OVX-diet rats. Bone area and percent fat were significantly lower in OVX-diet rats at M14 compared to sham rats. The regression coefficients for BMD vs. bone area, BMC vs. bone area, and BMC vs. BMD of OVX-diet rats increased with time. This is explained by differential percent change in BMD, BMC, and bone area with respect to time and disease progression. CONCLUSIONS: Combined ovariectomy and deficient diet in rats caused significant reduction of BMD, BMC, and bone area, with nearly 40% bone loss after 14 months, indicating the development of severe osteoporosis. An increasing regression coefficient of BMD vs. bone area with disease progression emphasizes bone area as an important parameter, along with BMD and BMC, for prediction of fracture risk.

Polyspecific cation transporters mediate luminal release of acetylcholine from bronchial epithelium.

In airway epithelia, non-neuronal cholinergic regulations have been described; however, the route for acetylcholine (ACh) release has not been verified. To investigate whether organic cation transporters (OCTs) serve this function, we studied the expression of OCTs in airway epithelia and their capability to translocate ACh. Using immunohistochemistry in rats and humans, OCT1, OCT2, and OCT3 were localized to the luminal membrane of ciliated epithelial cells. In humans, OCT2 showed the strongest expression in the luminal membrane. We expressed the OCT isoforms in oocytes of Xenopus laevis and measured uptake and efflux of ACh. Tracer flux measurements showed that ACh is transported by OCT1 and OCT2 but not by OCT3. Two-electrode-voltage-clamp measurements revealed that OCT2 mediates electrogenic uptake and efflux of ACh. For ACh uptake by human OCT2, a K(M) value of approximately 0.15 mM was determined. At -50 mV, ACh efflux by human OCT2 was trans-inhibited by micromolar concentrations of the inhalational glucocorticoid budesonide, which is used in treatment of asthma (K(i) approximately 2.7 microM). The data show that OCT1 and OCT2 mediate luminal ACh release in human airways and suggest that ACh release is blocked after inhalation of budesonide.

Nicotinic acetylcholine receptor subtypes in nociceptive dorsal root ganglion neurons of the adult rat.

Stimulation of nicotinic acetylcholine receptors (nAChR) excites peripheral sensory nerve fibres, but also exert antinociceptive effects. The differences in these nAChR-mediated effects could be related to the expression of different nAChR subtypes located on nociceptive neurons. In the present study, we focused on the recently described alpha 10-nAChR subunit, and on alpha 4 and alpha 7 subunits, which are the most abundant subunits in the central nervous system. In nociceptive neurons from thoracic and lumbar dorsal root ganglia (DRG), nAChR subunits were found at transcriptional (RT-PCR), translational (immunohistochemistry) and functional levels. Cultured DRG neurons express mRNA for the subunits alpha 2-7 and alpha 10. The alpha-subunit proteins 4, 7 and 10 were colocalised in virtually all nociceptive neurons that were identified by immunoreactivity for the vanilloid receptor TRPV-1. These findings were corroborated by current recordings and calcium measurements, which revealed excitatory inward currents and calcium responses in capsaicin sensitive neurons.