Osteoporosis is accompanied by reduced CD274 expression in human bone marrow-derived mesenchymal stem cells.

Underlying pathomechanisms of osteoporosis are still not fully elucidated. Cell-based therapy approaches pose new possibilities to treat osteoporosis and its complications. The aim of this study was to quantify differences in human bone marrow-derived mesenchymal stem cells (hBMSCs) between healthy donors and those suffering from clinically manifest osteoporosis. Cell samples of seven donors for each group were selected retrospectively from the hBMSC cell bank of the Trauma Department of Hannover Medical School. Cells were evaluated for their adipogenic, osteogenic and chondrogenic differentiation potential, for their proliferation potential and expression of surface antigens. Furthermore, a RT2 Osteoporosis Profiler PCR array, as well as quantitative real-time PCR were carried out to evaluate changes in gene expression. Cultivated hBMSCs from osteoporotic donors showed significantly lower cell surface expression of CD274 (4.98 % ± 2.38 %) than those from the control group (26.03 % ± 13.39 %; p = 0.007), as assessed by flow cytometry. In osteoporotic patients, genes involved in inhibition of the anabolic WNT signalling pathway and those associated with stimulation of bone resorption were significantly upregulated. Apart from these changes, no significant differences were found for the other cell surface antigens, adipogenic, osteogenic and chondrogenic differentiation ability as well as proliferation potential. These findings supported the theory of an influence of CD274 on the regulation of bone metabolism. CD274 might be a promising target for further investigations of the pathogenesis of osteoporosis and of cell-based therapies involving MSCs.

In vivo heterotopic culturing of prefabricated tendon grafts with mechanical stimulation in a sheep model.

BACKGROUND: The goal of this study is to investigate the biomechanical and histological properties of in vivo heterotopically prefabricated cruciate ligament replacement grafts with and without mechanical stimulation. The clinical goal is to heterotopically prefabricate a bone-tendon-bone graft for anterior cruciate ligament reconstruction, which allows rapid ingrowth and early full weight bearing. METHODS: In a sheep model, eight quadriceps tendon grafts were harvested and introduced into culture chambers at their proximal and distal ends. In group S, four tendon-chamber constructs were mechanically stimulated by direct attachment to the quadriceps tendon and patella. In group NS, the same constructs were cultured without proximal attachment. All sheep were sacrificed six weeks postoperatively and the constructs were examined biomechanically and histologically. The healthy contralateral ACL and quadriceps tendon were used as controls. RESULTS: Macroscopically, no obvious ossification could be observed at the ends of the tendon-chamber constructs six weeks postoperatively. Histologically, the tendon tissue from the mechanically stimulated constructs revealed higher counts of cells and capillaries. However, there was less regular cell distribution and collagen fiber orientation compared to the control group. In addition, osteoblasts and osteogenesis were observed in the prefabricated constructs both with and without mechanical stimulation. Biomechanically, there were no significant differences in stiffness, elongation and ultimate failure load between the groups. CONCLUSION: In vivo heterotopic culture of prefabricated tendon grafts may have the potential to stimulate osteoblasts and induce osteogenesis. Future studies with longer follow-up and modifications of the surgical technique and culture conditions are desirable.

[Complex joint reconstruction and joint transplantation with the FLOCSAT concept-planning and surgical implementation]. / Komplexe Gelenkrekonstruktion und Gelenktransplantation mit dem FLOCSAT-Konzept ­ Planung und chirurgische Umsetzung.

Cartilage defects in adult patients are so far incurable. Fresh osteochondral allograft (OCA) transplantation is based on the insertion of mature, living, mechanically sound hyaline cartilage into an osteochondral defect where it undergoes osseointegration. Intact hyaline cartilage of OCA does not cause immune reactions in the recipient. Many reports show that small OCA have good osseointegration and show good long-term results. These observations have been incorporated into the development of the fresh large (> 10 cm2) osteochondral shell allograft transplantation (FLOCSAT) concept, which is based on the following principles: 1) the thickness of the osseous layer should be kept as thin as possible (target < 6-8 mm) so that the transplant remains stable and fixable. This results in reduced segments of vascularization, simplified ossification and reduced immunogenic bone volume. 2) The bone surface is processed and enlarged (oscillating saw: pie crust technique, drill holes) and areas of sclerosis are simultaneously broken off. 3) Cell reduction and washing out of the bony layer with a pulsatile jet lavage. 4) Prevention of impaction and dessication: cartilage with its living chondrocytes are very sensitive to mechanical contusion and dessication. When introducing the transplant, the tissue must therefore be continually moistened and the pressure acting on the cartilage must be controlled. 5) Stable fixation: extensive uniplanar osteochondral transplants cannot be inserted by the press-fit method; therefore, fixation is carried out with small implants. In this publication we demonstrate how severe and complex posttraumatic or degenerative delayed problems can be solved using FLOCSAT.

[Partial and complete joint transplantation with fresh osteochondral allografts-the FLOCSAT concept]. / Partielle und komplette Gelenktransplantation mit frischen osteochondralen Allografts ­ das FLOCSAT-Konzept.

Cartilage defects in adult patients do not heal well. Fresh osteochondral allograft (OCA) transplantation is based on mature, living, mechanically sound hyaline cartilage attached to a bone interface, which is brought into an osteochondral defect, where it becomes osseointegrated. According to current knowledge, intact hyaline cartilage tissue is immune privileged and does not, in contrast to bone, meniscus or ligaments, cause an immune reaction. The technique has the unique advantage of transplanting viable, mature and mechanically stable hyaline cartilage into the affected area. An OCA is the only biological surgical technique for chondral and osteochondral lesions after failed cell-based techniques or autologous osteochondral transplantation.Fresh osteochondral allografts with mainly small cylindrical transplants show survival of 20 years and more. Based on this experience the FLOCSAT (fresh large [> 10 cm2] osteochondral shell allograft transplantation) concept with the thinnest possible (< 6-8 mm) bone thickness has been developed. Cells survive in special media at 4 °C for 4 weeks or more and are transplanted with a minimum of 70% living cells in a live/dead assay and cell density > 200 cells/mm2.FLOCSATs can replace parts or entire joints as uniplanar or multiplanar(n-planar)-FLOCSAT, as unipolar, bipolar or tripolar(n-polar)-FLOCSAT, and in combination with meniscus and/or ligaments (nMnL)-FLOCSAT (n number of structures).The FLOCSAT concept was applied successfully to knee, ankle and elbow joints. All transplants showed sound osseointegration. Cartilage failure was the reason for unsuccessful outcome. Challenges remain regarding graft availability, precise size matching, complex logistics, demanding surgical technique in complex geometries, and open questions in immunology and chimerism.

Two-stage late reconstruction with a fresh large osteochondral shell allograft transplantation (FLOCSAT) for a large ostechondral defect in a non-union after a lateral tibia plateau fracture 2-year follow up.

This is the description of a 58-year-old female patient presenting 8 months after a horse riding accident with significant pain and inability to walk independently. Imaging revealed a large osseous defect of the lateral tibia plateau which was not united posteriorly. The patient refused knee replacement and we developed a patient specific two-step procedure for her. Step 1: Filling of the defect with a large cortico-cancellous autograft from the posterior iliac crest; step 2: Transplantation of a fresh large osteochondral shell allograft (FLOCSAT). The postoperative protocol included continuous passive motion (CPM), partial weight bearing for three months, and physiotherapy. Based on the concept of immuno-privileged cartilage tissue, the patient did not get any immuno-suppressive therapy. Pain-, activity of daily living, Lysholm and Tegner scores were evaluated before defect filling surgery with autograft, before allograft transplantation, and at 12 and 24 months after allograft transplantation. There were no complications. Radiographic analyses with plain films and CT scans revealed solid osseous integration within 3 month. The patient regained excellent functionality in both, activities of daily living and sports (back to horse riding, trampolin jumping). Knee arthroscopy after 1year showed excellent condition of the lateral meniscus and the cartilage of the lateral tibia plateau. Chimerism/DNA analysis of a cartilage biopsy showed, that at 1year 32% of the donor cells have been already replaced by the patient's own cells. To our knowledge, this is the first case of a patient who sustained such a large defect during a tibia plateau fracture, and got successfully treated with a fresh large osteochondral shell allograft transplantation in a two-step procedure.

Nlrp1 inflammasome is downregulated in trauma patients.

UNLABELLED: After a major trauma, IL-1ß-producing capacity of monocytes is reduced. Generation of IL-1ß is important for appropriate immune response after trauma and requires not only synthesis and transcription of inflammasome components but also their activation. Altered IL-1ß-processing due to deregulated NLRP inflammasomes assembly is associated with several inflammatory diseases. However, the precise role of NLRP1 inflammasome in monocytes after trauma is unknown. Here, we investigated if NLRP1 inflammasome components are responsible for depressed monocyte function after trauma. We found in ex vivo in vitro assays that LPS-stimulation of CD14(+)-isolated monocytes from healthy volunteers (HV) results in remarkably higher capacity of the IL-1ß-release compared to trauma patients (TP). During the 10-day time course, this monocyte depression was highest immediately after admission. Inflammasome activation correlating with this inflammatory response was demonstrated by enhanced protein production of cleaved IL-1ß and caspase-1. Furthermore, we found that the gene expression of IL-1ß, caspase-1, and ASC was comparable in TP and HV after LPS-stimulation during the 10-day course, while NLRP1 was markedly reduced in TP. We demonstrated that transfected monocytes from TP, which expressed the lacking components, were recovered in their LPS-induced IL-1ß-release and that lacking of NLRP1 is responsible for the suppressed monocyte activity after trauma. The restoration of NLRP1 inflammasome suggests new mechanistic target for the recovery of dysbalanced immune reaction after trauma. KEY MESSAGE: Suppression in monocyte function occurs early after a major trauma or surgery. Reduced gene expression abrogates NLRP1 inflammasome assembly after trauma. Limited availability of inflammasome components may cause reduced host defense. Restoring NLRP1 in immune-suppressed monocytes recovers NLPR1 activity after trauma. Recovered inflammasome activity may improve the immune response to PAMPs/DAMPs.

Effects of trauma-hemorrhage and IL-6 deficiency on splenic immune function in a murine trauma model.

Splenic immune function is known to be depressed following hemorrhage. The present study investigates the effects of femoral shaft fracture, isolated or in combination with hemorrhage, on early stage cytokine production capacity of splenocytes and observes the role of IL-6 under these conditions. Male IL-6 knockout (IL-6(-/-)) and wild-type mice (WT) were randomly divided into three groups: sham (S), isolated femoral fracture (Fx), and femoral fracture + volume controlled hemorrhage (TH-Fx) (n = 6 per group). Animals were sacrificed four hours after induction of hemorrhage and fracture. Cytokine release (TNF-α, IL-6, and IL-10) of isolated and LPS-stimulated splenocytes was determined by cytometric bead array. Femoral fracture with or without hemorrhage caused a suppression of in vitro cytokine production capacity of splenocytes at an early posttraumatic stage in WT and IL-6(-/-). In the absence of IL-6, the profile of splenic cytokine secretion is significantly altered, identifying this cytokine as a potential therapeutic target to modulate the posttraumatic immune response.

The protein tyrosine phosphatase Rptpzeta is expressed in differentiated osteoblasts and affects bone formation in mice.

Tyrosine phosphorylation of intracellular substrates is one mechanism to regulate cellular proliferation and differentiation. Protein tyrosine phosphatases (PTPs) act by dephosphorylation of substrates and thereby counteract the activity of tyrosine kinases. Few PTPs have been suggested to play a role in bone remodeling, one of them being Rptpzeta, since it has been shown to be suppressed by pleiotrophin, a heparin-binding molecule affecting bone formation, when over-expressed in transgenic mice. In a genome-wide expression analysis approach we found that Ptprz1, the gene encoding Rptpzeta, is strongly induced upon terminal differentiation of murine primary calvarial osteoblasts. Using RT-PCR and Western Blotting we further demonstrated that differentiated osteoblasts, in contrast to neuronal cells, specifically express the short transmembrane isoform of Rptpzeta. To uncover a potential role of Rptpzeta in bone remodeling we next analyzed the skeletal phenotype of a Rptpzeta-deficient mouse model using non-decalcified histology and histomorphometry. Compared to wildtype littermates, the Rptpzeta-deficient mice display a decreased trabecular bone volume at the age of 50 weeks, caused by a reduced bone formation rate. Likewise, Rptpzeta-deficient calvarial osteoblasts analyzed ex vivo display decreased expression of osteoblast markers, indicating a cell-autonomous defect. This was confirmed by the finding that Rptpzeta-deficient osteoblasts had a diminished potential to form osteocyte-like cellular extensions on Matrigel-coated surfaces. Taken together, these data provide the first evidence for a physiological role of Rptpzeta in bone remodeling, and thus identify Rptpzeta as the first PTP regulating bone formation in vivo.