Immune Cell Induced Migration of Osteoprogenitor Cells Is Mediated by TGF-ß Dependent Upregulation of NOX4 and Activation of Focal Adhesion Kinase.

The cytokines secreted by immune cells have a large impact on the tissue, surrounding a fracture, e.g., by attraction of osteoprogenitor cells. However, the underlying mechanisms are not yet fully understood. Thus, this study aims at investigating molecular mechanisms of the immune cell-mediated migration of immature primary human osteoblasts (phOBs), with transforming growth factor beta (TGF-ß), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4) and focal adhesion kinase (FAK) as possible regulators. Monocyte- and macrophage (THP-1 cells ± phorbol 12-myristate 13-acetate (PMA) treatment)-conditioned media, other than the granulocyte-conditioned medium (HL-60 cells + dimethyl sulfoxide (DMSO) treatment), induce migration of phOBs. Monocyte- and macrophage (THP-1 cells)-conditioned media activate Smad3-dependent TGF-ß signaling in the phOBs. Stimulation with TGF-ß promotes migration of phOBs. Furthermore, TGF-ß treatment strongly induces NOX4 expression on both mRNA and protein levels. The associated reactive oxygen species (ROS) accumulation results in phosphorylation (Y397) of FAK. Blocking TGF-ß signaling, NOX4 activity and FAK signaling effectively inhibits the migration of phOBs towards TGF-ß. In summary, our data suggest that monocytic- and macrophage-like cells induce migration of phOBs in a TGF-ß-dependent manner, with TGF-ß-dependent induction of NOX4, associated production of ROS and resulting activation of FAK as key mediators.

Computer-assisted navigation is beneficial both in primary and revision surgery with modular rotating-hinge knee arthroplasty.

PURPOSE: The objective of the present study was to explore the effect of navigation on the reconstruction of the mechanical leg axis, implant positioning and the restoration of the joint line in hinged knee arthroplasty in vivo. We present the first 1- to 3-year clinical and radiological results following computer-navigated implantation of the EnduRo modular rotating-hinge knee arthroplasty system (Aesculap AG, Tuttlingen, Germany) as a primary or revision implant. METHODS: Thirty-one patients were analysed retrospectively. Indication was revision surgery in 18 patients and complex primary surgery in 13. The clinical and radiological results of 31 patients with a minimum follow-up of 12 months (mean 22.2 ± 6.2 months) were recorded. Age at follow-up was 55.2 ± 9.9 years. RESULTS: The absolute varus-valgus deviation from the neutral mechanical leg axis was determined at 5.1° ± 5.1° preoperatively and 2.1° ± 1.4° postoperatively. No intraoperative complications or problems with the navigation system were observed. At latest follow-up, no component loosening was detected. Based on the Knee Society Score, a knee score of 64.9 ± 17.7 points and a function score of 67.2 ± 27.3 points were achieved. CONCLUSIONS: Encouraging short-term clinical and radiological results with the computer-navigated implantation of the modular rotating-hinge EnduRo knee arthroplasty system were found in both primary and revision surgery. The navigation facilitated the reconstruction of the leg axis, implant positioning and the restoration of the joint line. LEVEL OF EVIDENCE: IV.

Long-term results using the straight tapered femoral cementless hip stem in total hip arthroplasty: a minimum of twenty-year follow-up.

We report the first long-term results of a prospective cohort study after total hip arthroplasty using the cementless Bicontact hip stem. Between 1987 and 1990, 250 total hip arthroplasties in 236 patients were performed using the cementless Bicontact hip stem. The average follow-up was 22.8 years (20.4-24.8) and average age at index surgery was 58.1 years. Eighty-one patients died and 9 were lost to follow-up. We noted 11 stem revisions revealing an overall Kaplan Meier survival rate of 95.0% (CI 95%: 91.1-97.2%). The average Harris Hip Score revealed 81 points (range 24-93). The Bicontact hip stem demonstrated high survival rates despite high ages and osteopenic changes, which are equivalent to other long-term reports of cementless stem fixation.

Animal serum-free expansion and differentiation of human mesenchymal stromal cells.

BACKGROUND AIMS: Mesenchymal stromal cells (MSC) are attracting increasing interest for possible application in cell therapies. Fetal calf serum (FCS) is widely utilized for cell culture, but its use in the context of clinical applications is associated with too many risks. Therefore we tested FCS-free media for the expansion and differentiation of MSC in compliance with the European good manufacturing practice (GMP) regulations for medicinal products. METHODS: MSC expansion medium was modified by replacing FCS with human plasma and platelet extract. Cells were characterized according to the defined minimal criteria for multipotent MSC. For chondrogenic differentiation, serum-free micromass cultures were employed. For adipogenic and osteogenic differentiation, the FCS was replaced by human plasma. After 28 days of incubation in differentiation media, cells were analyzed by cytochemical and immunohistochemical staining. Furthermore, mRNA expression of chondrogenic, adipogenic and osteogenic markers was investigated by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). RESULTS: Expansion and differentiation of MSC under FCS-free conditions yielded cells with chondrogenic, adipogenic and osteogenic phenotypes and a characteristic gene expression. Chondrocytes in micromass pellets revealed an accumulation of proteoglycans and type II collagen as well as a significantly increased mRNA expression of chondrogenic marker genes. The adipocytes displayed Oil red O staining and expressed peroxisome proliferator-activated receptor gamma(2) (ppARgamma2) and lipoprotein lipase (LPL) mRNA. The osteoblasts were positive for von Kossa staining and expressed mRNA of osteogenic marker genes. The results did not indicate any spontaneous differentiation. CONCLUSIONS: Human plasma is a suitable FCS replacement for the expansion and differentiation of MSC, providing a feasible alternative for tissue engineering with GMP-compatible protocols.

Isolation of functionally distinct mesenchymal stem cell subsets using antibodies against CD56, CD271, and mesenchymal stem cell antigen-1.

BACKGROUND: Conventionally, mesenchymal stem cells are functionally isolated from primary tissue based on their capacity to adhere to a plastic surface. This isolation procedure is hampered by the unpredictable influence of co-cultured hematopoietic and/or other unrelated cells and/or by the elimination of a late adhering mesenchymal stem cells subset during removal of undesired cells. To circumvent these limitations, several antibodies have been developed to facilitate the prospective isolation of mesenchymal stem cells. Recently, we described a panel of monoclonal antibodies with superior selectivity for mesenchymal stem cells, including the monoclonal antibodies W8B2 against human mesenchymal stem cell antigen-1 (MSCA-1) and 39D5 against a CD56 epitope, which is not expressed on natural killer cells. DESIGN AND METHODS: Bone marrow derived mesenchymal stem cells from healthy donors were analyzed and isolated by flow cytometry using a large panel of antibodies against surface antigens including CD271, MSCA-1, and CD56. The growth of mesenchymal stem cells was monitored by colony formation unit fibroblast (CFU-F) assays. The differentiation of mesenchymal stem cells into defined lineages was induced by culture in appropriate media and verified by immunostaining. RESULTS: Multicolor cell sorting and CFU-F assays showed that mesenchymal stem cells were approximately 90-fold enriched in the MSCA-1(+)CD56(-) fraction and approximately 180-fold in the MSCA-1(+)CD56(+) fraction. Phenotype analysis revealed that the expression of CD10, CD26, CD106, and CD146 was restricted to the MSCA-1(+)CD56(-) mesenchymal stem cells subset and CD166 to MSCA-1(+)CD56(+/-) mesenchymal stem cells. Further differentiation of these subsets showed that chondrocytes and pancreatic-like islets were predominantly derived from MSCA-1(+)CD56(+/-) cells whereas adipocytes emerged exclusively from MSCA-1(+)CD56(-) cells. The culture of single sorted MSCA-1(+)CD56(+) cells resulted in the appearance of phenotypically heterogeneous clones with distinct proliferation and differentiation capacities. CONCLUSIONS: Novel mesenchymal stem cells subsets with distinct phenotypic and functional properties were identified. Our data suggest that the MSCA-1(+)CD56(+) subset is an attractive starting population for autologous chondrocyte transplantation.

[Implantation of a Modular Distal Femoral Replacement with Compressive Osseointegration as a Salvage Procedure in a Complex Femoral Posttraumatic Setting]. / Implantation einer modularen Knietotalendoprothese mit distalem Femurersatz mit kompressiver Osteointegration als Rückzugsoption bei komplexer femoraler posttraumatischer Ausgangssituation.

BACKGROUND: Large bone defects and losses play a crucial role in both tumour surgery and in complex primary and revision total knee replacement. The established options of cemented or uncemented long intramedullary stems are limited by large bone defects and are at risk from relatively high exposure to aseptic loosening. There is no general valid agreement on implant fixation of the distal femur. A further option is the cementless fixation method with compressive osseointegration, based on the so-called Wolff law of bone remodelling. This method was developed in order to reduce the high loosening rates in revision arthroplasty due to intense stress shielding and is intended to be applied in patients with huge bone losses. The so-called Compress® system (or CPS) allows such a distal femur reconstruction. It has mainly been applied and evaluated in tumor endoprosthetics. There are currently few data on the application of this system in complex distal femoral posttraumatic deformity or revision arthroplasty. PATIENTS: A case report of two male patients aged 59/56 years with a 1-year follow-up is presented. Both patients had a complex post-traumatic femoral deformity with bone loss, prior surgery and an ipsilateral hip replacement. Implantation was performed of a modular total knee replacement, consisting of a cemented modular tibia base plate and distal femoral replacement with cementless implant fixation by compressive osseointegration. Both patients were clinically and radiologically evaluated prospectively. RESULTS: Good clinical and radiological results were demonstrated in both patients after distal femoral replacement by compressive osseointegration. There was no need for further or revision surgery. Both patients were rapidly able to resume their jobs. The survival rates for CPS were comparable to published values with conventional procedures. There are yet no long-term results or extensive data for revision arthroplasty or posttraumatic cases. CONCLUSIONS: Besides distal femoral replacement with compressive osseointegration in oncological arthroplasty, the indication of complex distal femoral settings with large bone defects can be evaluated for daily clinical routine. Especially if there is ipsilateral total hip replacement, this option might be used to avoid interprosthetic stress risers.

Bone Marrow-Harvesting Technique Influences Functional Heterogeneity of Mesenchymal Stem/Stromal Cells and Cartilage Regeneration.

BACKGROUND: Connective tissue progenitors (CTPs) from native bone marrow (BM) or their culture-expanded progeny, often referred to as mesenchymal stem/stromal cells, represents a promising strategy for treatment of cartilage injuries. But the cartilage regeneration capacity of these cells remains unpredictable because of cell heterogeneity. HYPOTHESIS: The harvest technique of BM may highly influence stem cell heterogeneity and, thus, cartilage formation because these cells have distinct spatial localization within BM from the same bone. STUDY DESIGN: Controlled laboratory study. METHODS: CTPs obtained from the femur of patients undergoing total hip replacement by 2 harvest techniques-BM aspiration and BM collection-after bone rasping were immunophenotyped by flow cytometry and evaluated for chondrogenic ability. The spatial localization of different CTP subsets in BM was verified by immunohistochemistry. RESULTS: Cells from the BM after rasping were significantly more chondrogenic than the donor-matched aspirate, whereas no notable difference in their osteogenic or adipogenic potential was observed. The authors then assessed whether distinct immunophenotypically defined CTP subsets were responsible for the different chondrogenic capacity. Cells directly isolated from BM after rasping contained a higher percentage (mean, 7.2-fold) of CD45-CD271+CD56+ CTPs as compared with BM aspirates. The presence of this subset in the harvested BM strongly correlated with chondrogenic ability, showing that CD271+CD56+ cells are enriched in chondroprogenitors. Furthermore, evaluation of these CTP subsets in BM revealed that CD271+CD56+ cells were localized in the bone-lining regions whereas CD271+CD56- cells were found in the perivascular regions. Since the iliac crest remains a frequent site of BM harvest for musculoskeletal regeneration, the authors also compared the spatial distribution of these subsets in trabeculae of femoral head and iliac crest and found CD271+CD56+ bone-lining cells in both tissues. CONCLUSION: Chondrogenically distinct CTP subsets have distinct spatial localization in BM; hence, the harvest technique of BM determines the efficiency of cartilage formation. CLINICAL RELEVANCE: The harvest technique of BM may be of major importance in determining the clinical success of BM mesenchymal stem/stromal cells in cartilage repair.

Navigated TKA After Osteotomy Versus Primary Navigated TKA: A Matched-Pair Analysis.

This article presents clinical and radiological outcome analysis of navigated total knee arthroplasty (TKA) following osteotomy compared with primary navigated TKA implantation. The study group (29 legs) received navigated TKA (Columbus with deep-dish, cruciate-retaining inlay, Aesculap AG, Tuttlingen, Germany) following distal femoral (6 legs) or high tibial (23 legs) osteotomy, and the control group (29 legs) received a primary navigated TKA. All patients were examined clinically and radiologically in a retrospective matched-pair analysis. Both groups showed comparable clinical scores (Oxford Knee Score, Tegner and Lysholm scores, and Knee Society Score). Radiological evaluations offered no relevant differences. The study group showed a significant mediolateral ligamentous instability (3 legs ≤5°, 1 leg 6°-9°, 25 legs ≥10° mediolateral deviation) compared with the control group (14 legs ≤5°, 9 legs 6°-9°, 6 legs >10°; P<.001). Significantly higher mediolateral ligamentous instability was seen in otherwise comparable clinical and radiological results in patients with navigated TKA implantation following osteotomy, compared with primary TKA. [Orthopedics; 2016. 39(3):S77-S82.].

Crucial Role of Vitamin D in the Musculoskeletal System.

Vitamin D is well known to exert multiple functions in bone biology, autoimmune diseases, cell growth, inflammation or neuromuscular and other immune functions. It is a fat-soluble vitamin present in many foods. It can be endogenously produced by ultraviolet rays from sunlight when the skin is exposed to initiate vitamin D synthesis. However, since vitamin D is biologically inert when obtained from sun exposure or diet, it must first be activated in human beings before functioning. The kidney and the liver play here a crucial role by hydroxylation of vitamin D to 25-hydroxyvitamin D in the liver and to 1,25-dihydroxyvitamin D in the kidney. In the past decades, it has been proven that vitamin D deficiency is involved in many diseases. Due to vitamin D's central role in the musculoskeletal system and consequently the strong negative impact on bone health in cases of vitamin D deficiency, our aim was to underline its importance in bone physiology by summarizing recent findings on the correlation of vitamin D status and rickets, osteomalacia, osteopenia, primary and secondary osteoporosis as well as sarcopenia and musculoskeletal pain. While these diseases all positively correlate with a vitamin D deficiency, there is a great controversy regarding the appropriate vitamin D supplementation as both positive and negative effects on bone mineral density, musculoskeletal pain and incidence of falls are reported.

Phenotypic and functional heterogeneity of human bone marrow- and amnion-derived MSC subsets.

Bone marrow-derived mesenchymal stromal/stem cells (MSCs) are nonhematopoietic cells that are able to differentiate into osteoblasts, adipocytes, and chondrocytes. In addition, they are known to participate in niche formation for hematopoietic stem cells and to display immunomodulatory properties. Conventionally, these cells are functionally isolated from tissue based on their capacity to adhere to the surface of culture flasks. This isolation procedure is hampered by the unpredictable influence of secreted molecules, the interactions between cocultured hematopoietic and other unrelated cells, and by the arbitrarily selected removal time of nonadherent cells before the expansion of MSCs. Finally, functionally isolated cells do not provide biological information about the starting population. To circumvent these limitations, several strategies have been developed to facilitate the prospective isolation of MSCs based on the selective expression, or absence, of surface markers. In this report, we summarize the most frequently used markers and introduce new targets for antibody-based isolation procedures of primary bone marrow- and amnion-derived MSCs.

TGF-beta enhances the integrin alpha2beta1-mediated attachment of mesenchymal stem cells to type I collagen.

The heterodimeric integrins are important receptors for the attachment of cells to their extracellular matrix. Here, we studied the attachment of human mesenchymal stem cells (MSCs) to type I collagen (col-1), which is part of the extracellular matrix in bone, skin, and connective tissues. Furthermore, we examined how TGF-beta influences the integrin expression and attachment of MSC. Using flow cytometry, immunoblot, and RT-PCR, we report that MSC express several integrin subunits, including the alpha(2)beta(1) integrin (VLA-2, CD49b/CD29). TGF-beta increases the expression of integrin subunits alpha(2), alpha(6), and beta(1) in MSC, thereby enhancing the attachment of MSC to col-1. The TGF-beta-mediated up-regulation of the expression of the integrin subunits alpha(2) and alpha(6) is mainly mediated in MSC by Smad2.

The mesenchymal stem cell antigen MSCA-1 is identical to tissue non-specific alkaline phosphatase.

We have recently identified 2 distinct CD271(bright)MSCA-1(dim)CD56(+) and CD271(bright)MSCA-1(bright)CD56(-) MSC subsets in primary femur-derived bone marrow (BM), which differ in their expression pattern and morphology as well as in their clonogenic and differentiation capacity. Here, we show that MSCA-1 is identical to tissue non-specific alkaline phosphatase (TNAP), an ectoenzyme known to be expressed at high levels in liver, bone, and kidney as well as in embryonic stem (ES) cells. SDS-PAGE of WERI-RB-1 cell lysate and supernatant from phosphatidylinositol-specific phospholipase C (PI-PLC)-treated WERI-RB-1 cells resulted in the appearance of a prominent 68-kDa band. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDITOF MS) sequence analysis revealed TNAP-specific peptides. Screening of the MSCA-1-specific antibody W8B2 on HEK-293 cells transfected with the full-length coding sequence of TNAP showed specific reactivity with transfected but not with parent cell line. In addition, TNAP-specific mRNA expression was selectively detected in the transfectant line. In agreement with these findings, enzymatic activity of TNAP was exclusively detected in sorted MSCA-1(+) BM cells but not in the MSCA-1(-) negative fraction. Surface marker analysis revealed coexpression of the embryonic marker SSEA-3 but not SSEA-4, TRA-1-60, and TRA-1-81. In endometrium, TNAP is expressed at intermediate levels on CD146(+) cells and at high levels in the luminal space of glandular epithelia. Our results demonstrate that TNAP is a selective marker for the prospective isolation of BM-derived MSC and MSC-like cells in endometrium.

Phenotypic characterization of distinct human bone marrow-derived MSC subsets.

Very recently, we identified two distinct mesenchymal stem cell (MSC) subsets in primary bone marrow (BM) that differ in their expression pattern (CD271(bright)MSCA-1(dim)CD56(+) and CD271(bright)MSCA-1(bright)CD56(-)) and morphology as well as in their clonogenic and differentiation capacity. Here we analyzed the cell surface antigen expression in these subsets in more detail and compared the profiles with the expression pattern on cultured MSCs. Most of the tested antigens, including CD13, CD15, CD73, CD140b, CD144, CD146, and CD164, are expressed at similar levels in both primary BM populations. However, a number of markers were differentially expressed. Of these, CD166 (ALCAM), CD200, and CD106 (VCAM-1) showed an almost selective expression on either CD271(bright)MSCA-1(dim)CD56(+) (increased CD166 and CD200 expression) or CD271(bright)MSCA-1(bright)CD56(-) (increased CD106 expression) MSCs, respectively. Additional markers with elevated expression on CD56(+) MSCs include F9-3C2F1, HEK-3D3, HEK5-1B3, and W1C3 antigens, whereas CD10, CD26, CD106, 7C5G1, 9A3G2, 56A1C2, 66E2D11, HEK-3D6, HEK4-1A1, HEK4-2D6, W1D6, W4A5, W7C6, and W8B2 (MSCA-1) antigens showed increased expression in the CD56(-) population. The majority of the analyzed markers found on primary MSCs were also expressed on cultured MSCs. However, in contrast to primary MSCs, HEK7-1C4, W1C3, W1D6, and W4A5 antigens were absent on the cultured counterparts. 7G5G1 and 9A3G2 antigens showed reduced, and HEK-3D6, F9-3C2, and HEK-3D3 showed increased expression on cultured cells. The extended knowledge about the phenotype of the two subsets and the identification of novel MSC markers may result in the isolation of attractive starting populations for applications in regenerative medicine.