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In this paper we describe an approach that aims to provide fundamental information towards a scientific, biomechanical basis for the use of natural coral scaffolds to initiate mesenchymal stem cells into osteogenic differentiation for transplant purposes. Biomaterial, such as corals, is an osteoconductive material that can be used to home human derived stem cells for clinical regenerative purposes. In bone transplantation, the use of biomaterials may be a solution to bypass two main critical obstacles, the shortage of donor sites for autografts and the risk of rejection with allograft procedures. Bone regeneration is often needed for multiple clinical purposes for instance, in aesthetic reconstruction and regenerative procedures. Coral graft Porites lutea has been used by our team for a decade in clinical applications on over a thousand patients with different bone pathologies including spinal stenosis and mandibular reconstruction. It is well accepted that human bone marrow (hBM) is an exceptional source of mesenchymal stem cells (MSCs), which may differentiate into different cell phenotypes such as osteoblasts, chondrocytes, adipocytes, myocytes, cardiomyocytes and neurons. Isolated MSCs from human bone marrow were induced into osteoblasts using an osteogenic medium enriched with two specific growth factors, FGF9 and vitamin D2. Part of the cultured MSCs were directly transferred and seeded onto coral scaffolds ( Porites Lutea) and induced to differentiate into osteoblasts and part were cultured in flasks for osteocell culture. The data support the concept that hBM is a reliable source of MSCs which may be easily differentiated into osteoblasts and seeded into coral as an optimal device for clinical application. Within this project we have also discussed the biological nature of MSCs, their potential application for clinical transplantation and the prospect of their use in gene therapy. [ABSTRACT FROM AUTHOR]

Background: There is mounting evidence to suggest the involvement of the immune system by means of activation by metal ions released via biocorrosion, in the pathophysiologic mechanisms of aseptic loosening of orthopedic implants. However, the detailed mechanisms of how metal ions become antigenic and are presented to T-lymphocytes, in addition to how the local inflammatory response is driven, remain to be investigated. Methods: Human T-lymphocytes were cultured in the presence of a variety of metal ions before investigating functional and phenotypic changes using flow cytometric analysis. Additionally, human monocyte-derived dendritic cells (mDC) loaded with metal ions were used as antigen-presenting cells and incubated with naive T-lymphocytes with the aim of generating titanium-specific T-lymphocytes. Results: Using an autologous in vitro model, with mDC treated with Titanium (IV), we were able to induce Titanium (IV)- specific T-lymphocytes. These T-lymphocytes responded in a dose-related manner to Titanium (IV), while they did not crossreact with Titanium (III) or other metal ions, indicating that the new antigenic peptide complexes formed by Titanium (IV) are highly specific. Conclusion: This study showed that mDC exposed to Titanium (IV) are able to induce the generation of Titanium (IV)-specific T-lymphocytes, demonstrating the strong and specific antigenicity of Titanium (IV) ions released by biocorrosion. [ABSTRACT FROM AUTHOR]

There have been many attempts to acquire and culture human keratinocytes for clinical purposes including from keratotome slices in media with fetal calf serum (FCS) or pituitary extract (PE), from skin specimens in media with feeder layers, from suction blister epidermal roofs' in serum-free culture and from human umbilical cord blood (hUCB) mesenchymal stem cells (MSCs) in media with skin feeder layers. Conversely this study was designed to investigate whether keratinocytes could be obtained directly from hUCB MSCs in vitro. It is widely established that mesenchymal stem cells from human umbilical cord blood have multipotent capacity and the ability to differentiate into disparate cell lineages hUCB MSCs were directly induced to differentiate into keratinocytes by using a specific medium composed of primary culture medium (PCM) and serum free medium (SFM) in a ratio 1:9 for a period of 7 days and tested by immunostain p63 and K1-K10. Cells thus cultured were positive in both tests, confirming the possibility to directly obtain keratinocytes from MSCs hUCB in vitro. [ABSTRACT FROM AUTHOR]

Background: An enhanced fracture healing response has been reported in patients with traumatic brain injury (TBI). This has been attributed to circulating humoral factors that are thought to be proteins produced and released by the injured brain. However, these factors remain unknown. The aim of this study was to identify osteogenic factors in serum and cerebrospinal fluid (CSF) from TBI patients. This was carried out using in vitro proliferation assays with the human foetal osteoblastic 1.19 cell line (hFOB) combined with a novel proteomic approach. Methods: Serum was collected from brain-injured ( n = 12) and non-brain-injured ( n = 9) patients with a comorbid femur shaft fracture. Similarly, CSF was obtained from TBI ( n = 7) and non-TBI ( n = 9) patients. The osteoinductive potential of these samples was determined by measuring the in vitro proliferation rate of hFOB cells. Highly osteogenic serum and CSF samples of TBI patients were chosen for protein analysis and were compared to those of non-brain-injured patients. A new hFOB cell-based method was used to enrich the proteins in these samples, which had a functional affinity for these osteoprogenitor cells. These enriched protein fractions were mapped using two-dimensional gel electrophoresis and protein imaging methods displaying serum and CSF proteins of brain-injured and control subjects that had an affinity for human osteoprogenitor cells. Results: Serum and CSF derived from brain-injured patients demonstrated a greater osteoinductive potential ( P < 0.05) than their non-brain-injured counterparts. Clear-cut differences in the pattern of proteins in two-dimensional gels were detected between TBI and control patients. Fourteen proteins were exclusively present in the serum of TBI patients, while other proteins were either up- or downregulated in samples collected from TBI patients ( P < 0.05). Conclusion: Osteoinductive factors are present in the serum and CSF of brain-injured patients. These may include one or more of those proteins identified as having an affinity for osteoprogenitor cells that are either exclusively present or up- or downregulated in the serum and CSF of brain-injured patients. [ABSTRACT FROM AUTHOR]

There is increasing evidence that titanium ions are released from orthopedic implants by biocorrosion. The aim of this study was to investigate titanium uptake by human T-lymphocytes and its effects on phenotype and proliferation. Freshly isolated human nonadherent peripheral blood mononuclear cells (NA-PBMC), were exposed to TiCl4 [Ti(IV)]. Bioavailability and distribution of Ti(IV) in T-lymphocytes was determined by energy-filtered electron microscopy (EFTEM). The effects of Ti(IV) challenge on nonactivated and PHA-activated cells were assessed by flow cytometric analysis of surface markers, RANK-L production, and proliferation assays. EFTEM colocalized Ti(IV) with phosphorus in the nucleus, ribosomes, cytoplasmic membranes, and the surface membrane of T-lymphocytes. Ti(IV) increased significantly the expression of CD69, CCR4, and RANK-L in a concentration-dependent manner. Titanium enters T-lymphocytes through a currently unknown mechanism and binds to phosphorus-rich cell structures. Titanium influences phenotype and function of T-lymphocytes, resulting in activation of a CD69+ and CCR4+ T-lymphocyte population and secretion of RANK-L. These results strongly suggest the involvement of titanium ions challenged T-lymphocytes in the complex pathophysiological mechanisms of aseptic loosening of orthopedic implants. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:341-347, 2010 [ABSTRACT FROM AUTHOR]

Background: Traumatic brain injury is associated with an increased rate of heterotopic ossification within skeletal muscle, possibly as a result of humoral factors. In this study, we investigated whether cells from skeletal muscle adopt an osteoblastic phenotype in response to serum from patients with traumatic brain injury. Methods: Serum was collected from thirteen patients with severe traumatic brain injury, fourteen patients with a longbone fracture, and ten control subjects. Primary cultures of skeletal muscle cells isolated from patients undergoing orthopaedic surgery were performed and characterized with use of immunofluorescence microscopy, reverse transcriptionpolymerase chain reaction, and Western blot analysis. Proliferation and osteoblastic differentiation were assessed with use of commercial cell assays, Western blot analysis (for Osterix protein), and the Villanueva bone stain. Results: All serum-treated cell populations expressed the osteoblast marker Osterix after one week in culture. Cells treated with serum from all study groups in mineralization medium had increased alkaline phosphatase activity and mineralized nodules within the mesenchymal cell subpopulation after three weeks in culture. Serum from patients with traumatic brain injury induced a significant increase (p = 0.02) in the rate of proliferation of primary skeletal muscle cells (1.87 [95% confidence interval, 1.66 to 2.09]) compared with the rate induced by serum from patients with a fracture (1.42 [95% confidence interval, 1.21 to 1.58]) or by serum from controls (1.35 [95% confidence interval, 1.15 to 1.54]). Conclusions: Human serum supports the osteoblastic differentiation of cells derived from human skeletal muscle, and serum from patients with severe traumatic brain injury accelerates proliferation of these cells. These findings suggest the early presence of humoral factors following traumatic brain injury that stimulate the expansion of mesenchymal cells and osteoprogenitors within skeletal muscle. Clinical Relevance: A better understanding of the pathophysiological mechanisms that lead to the formation of heterotopic ossification in patients with a traumatic brain injury may be helpful in the prevention and treatment of this complication. [ABSTRACT FROM AUTHOR]

Most metals in contact with biological systems undergo corrosion by an electrochemical process. This study investigated whether human osteoclasts (OC) are able to grow on stainless steel (SS) and directly corrode the metal alloy leading to the formation of corresponding metal ions, which may cause inflammatory reactions and activate the immune system. Scanning electron microscopy analysis demonstrated long-term viable OC cultures and evident resorption features on the surface of SS discs on which OC were cultured for 21 days. The findings were confirmed by atomic emission spectrometry investigations showing significantly increased levels of chromium, nickel, and manganese in the supernatant of OC cultures. Furthermore, significant levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α, which are considered to be major mediators of osteolysis, were revealed in the same cultures by cytometric bead array analysis. Within the present study, it was shown that human osteoclast precursors are able to grow and differentiate towards mature OC on SS. The mature cells are able to directly corrode the metal surface and release corresponding metal ions, which induce the secretion of pro-inflammatory cytokines that are known to enhance osteoclast differentiation, activation, and survival. Enhanced corrosion and the subsequently released metal ions may therefore result in enhanced osteolytic lesions in the peri-prosthetic bone, contributing to the aseptic loosening of the implant. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 841-846, 2009 [ABSTRACT FROM AUTHOR]

Background: Patients with a traumatic brain injury (TBI) and bone fractures often show an enhanced fracture healing, as well as an increased incidence of heterotopic ossifications (HO). It has been suggested that unknown osteoinductive factors may be released by the injured brain into the systemic blood circulation and act peripherally on the affected tissues. The aim of this study was to investigate whether serum from TBI patients is osteoinductive. Methods: Sixty-one consecutive patients were classified into four groups: TBI and long-bone fracture (group I, n = 12), isolated severe TBI (group II, n = 21), isolated long-bone fracture (group III, n = 19) and controls (group IV, n = 9). Blood samples were collected at 6, 24, 72 and 168 h post-injury. The osteogenic potential was determined by measuring the in vitro proliferation rate of the human fetal osteoblastic cell line hFOB1.19, and primary human osteoblasts. Additionally, serum induced osteoblastic differentiation was assessed by measuring the mRNA expression of specific osteoblastic markers, including alkaline phosphatase, runt-related transcription factor 2, cathepsin K and serine protease 7. Results: The sera of group I induced a higher mean proliferation rate of primary human osteoblasts at all time points of sampling than group III ( P < 0.05). Group I had a higher mean proliferation rate of hFOB1.19 cells than all other groups at 6, 24 and 72 h post-injury ( P < 0.05). The expression of alkaline phosphatase, cathepsin K and runt-related transcription factor 2 mRNA was increased in group I compared with group III and serine protease 7 was exclusively expressed in group I. Conclusion: The study results strongly support a humoral mechanism in enhanced fracture healing and the induction of HO after TBI. Increased proliferation of osteoblastic cells and an accelerated differentiation of osteoprogenitor cells may be responsible for increased osteogenesis in TBI. [ABSTRACT FROM AUTHOR]

Background: Scientific evidence is mounting for an association between traumatic brain injury and enhanced osteogenesis. The aim of this study was to correlate the in vitro osteoinductive potential of serum with the features of fracture-healing and the extent of brain damage in patients with severe traumatic brain injury and bone fracture. Methods: Patients with a long-bone fracture and a traumatic brain injury (seventeen patients) or without a brain injury (twenty-four patients) were recruited. The Glasgow Coma Scale score was determined on admission. Radiographs of the fracture were made before surgery, at six weeks, and at three, six, and twelve months after surgery. The time to union was estimated clinically and radiographically, and the callus ratio to shaft diameter was calculated. Serum samples were collected at six, twenty-four, seventy-two, and 168 hours after injury, and their osteogenic potential was determined by measurement of the in vitro proliferation rate of the human fetal osteoblastic cell line hFOB1.19. Results: Patients with a traumatic brain injury had a twofold shorter time to union (p = 0.01), a 37% to 50% increased callus ratio (p < 0.01), and their sera induced a higher proliferation rate in hFOB cells (p < 0.05). A linear relationship was revealed between hFOB cell proliferation rates and the amount of callus formed (p <0.05). The Glasgow Coma Scale score was correlated with the callus ratio on both radiographic projections (p < 0.05), time to union (p = 0.04), and the proliferation rate of hFOB cells at six hours after injury (p = 0.03). Conclusions: Patients with a severe brain injury release unknown humoral factors into the blood circulation that enhance and accelerate fracture-healing. [ABSTRACT FROM AUTHOR]

Vascular casting is a widely used method for the representation of body vascularization. Many different injection materials have been described throughout the time to enhance the arterial vascular supply within a specifically defined anatomical location. The use of industrial polyurethane has been recently evaluated and applied to animal and human anatomy. The aim of this study was to confirm the safe and reliable use of industrial polyurethane in knee specimen in order to obtain a three-dimensional vascular tree of the distal femur. 10 fresh-frozen knees (mid-thigh to mid tibia) were used to assess the vascularity around the femoral condyles. Industrial polyurethane foam (Soudal™ foam) was diluted with acetone in order to obtain a runny fluid, easy to inject. After injection, the knees were bathed in a 10% NaOH solution, heated at 30°. The corrosion process took from 20 to 24 h and allowed all the soft tissue surrounding the knee to be subsided, leaving only the bone with polyurethane vascular architecture. After soft tissue corrosion, the vascular network around the knees was easily identified underlying the relation of the vessels to the bone. Even small arterioles (diameter< 1 mm) were distinguished with a good resistance to breakage. Corrosion casting remains an easy and reliable alternative to dissection for the understanding of tissue perfusion as the handling of the polyurethane is easy and has low costs. The described author's method can be used osteo-articular specimen as well as in other organs. The protocol of injection and corrosion needs however to be adapted to the different specimen and anatomical location. Polyurethane associated to acetone can safely be used as injection material in order to demonstrate the vascularity of a specimen and remains easy to use. [ABSTRACT FROM AUTHOR]

Background and aim. Postmenopausal osteoporosis, caused by estrogen deficiency, is characterized by the structural deterioration of bone accompanied by an increase in bone marrow adipocytes. Transgenic animal models have shown that there is a reciprocal relationship between osteoblastogenesis and adipogenesis in vivo. The present study investigated whether the estrogen and the canonical Wnt signaling pathways are linked together and regulate the phenotype and function, differentiation and proliferation of human osteoblasts using an in vitro estrogen-deficiency model. Methods. Human osteoblasts (hFOB 1.19) and fulvestrant, an estrogen receptor blocker, were used to mimic estrogen deficiency in vitro. Osteogenic and adipogenic differentiation was measured by using specific stains and microscopy, as well as by measuring the expression of bone cell-specific markers with reverse transcription polymerase chain reaction. Expression of estrogen receptor-α (ERα) and β-catenin was detected in Western blots and by immunoprecipitation. Results. The cells expressed the 46-kDa and the 77-kDa ERα isoforms and β-catenin. Fulvestrant reduced expression of ERα and β-catenin. β-Catenin was co-immunoprecipitated with ERα, indicating that these two proteins form a new signaling complex and transcription factor. In addition, it induced intracellular lipid droplet accumulation and downregulation of bone cell markers, indicating adipocyte differentiation. [ABSTRACT FROM AUTHOR]

Human papillomavirus-like particles (HPV-VLP) are a candidate vaccine for prevention of HPV infection, and also are a candidate for an immunogenic delivery system for incorporated antigen. VLP activatein vitrogenerated dendritic cells (DC) but not Langerhans cells (LC); however, the mechanism of this activation is unknown. We have shown that uptake and activation of DC by VLP involves proteoglycan receptors and can be inhibited by heparin. Heparin has been shown to activate DC by signalling through Toll-like receptor 4 (TLR4) and nuclear factor (NF)-κB. The pathway of DC activation by VLP was further investigated in the present study. Exposure to VLP induced costimulatory molecule expression, RelB translocation and IL-10 production by DC but not by LC. The lack of LC activation was reversible when TGF-β was removed from the LC medium. VLP-induced induction of costimulatory molecule expression, RelB activation and cytokine secretion by DC was blocked by inhibition of NF-κB activation, heparin or TLR4 mAb. The data provide evidence that HPV-VLP signal DC through a pathway involving proteoglycan receptors, TLR4 and NF-κB, and shed light on the mechanism by which VLP stimulate immunity in the absence of adjuvantsin vivo. LC may resist activation in normal epithelium abundant in TGF-β, but not in situations in which TGF-β concentrations are reduced. [ABSTRACT FROM AUTHOR]