Jochen Eulert

Chondrogenic differentiation of mesenchymal progenitor cells encapsulated in ultrahigh-viscosity alginate.

One major problem of current cartilage repair techniques is that three‐dimensional encapsulated mesenchymal progenitor cells frequently differentiate into hypertrophic cells that express type X collagen and osteogenic marker genes. Studies on wild‐type cells of murine mesenchymal C3H10T1/2 progenitor cells as well as on cells transfected with cDNA encoding for bone morphogenetic protein (BMP)‐2 or ‐4 in alginate revealed that the formation of markers for osteogenesis and chondrogenic hypertrophy apparently depended on the BMP‐transfection. Cells were encapsulated in ultrahigh‐viscosity, clinical grade alginate and differentiation was studied over a period of 17 days. Consistent with results published previously (Biomaterials, 2002;23:2003) staining with haematoxylin–eosin or Alcian blue, immunohistochemical analysis, and quantitative RT‐PCR confirmed the expression of chondrogenic markers (chondroitin‐4‐ and ‐6‐sulfate as well as type II collagen). Production of chondrogenic markers was particularly high in BMP‐4 transfected cells. Hypertrophic chondrogenesis did not occur in BMP‐4 transfected cells, as revealed by measurement of type X collagen, but could be demonstrated for wild‐type cells and to some extent for BMP‐2 transfected cells. The osteogenic markers, type I collagen, alkaline phosphatase, and Cbfal were upregulated in all cell lines even though the levels and the time of upregulation differed significantly. In any case, the markers were less and only very shortly expressed in BMP‐4 transfected cells as revealed quantitatively by real time RT‐PCR. Thus, the in vitro results suggested that BMP‐4 is a very promising candidate for suppressing chondrogenic hypertrophy, while simultaneously enhancing the production of chondrogenic components.

Hypertrophy is induced during the in vitro chondrogenic differentiation of human mesenchymal stem cells by bone morphogenetic protein-2 and bone morphogenetic protein-4 gene transfer

IntroductionThe present study compares bone morphogenetic protein (BMP)-4 and BMP-2 gene transfer as agents of chondrogenesis and hypertrophy in human primary mesenchymal stem cells (MSCs) maintained as pellet cultures.MethodsAdenoviral vectors carrying cDNA encoding human BMP-4 (Ad.BMP-4) were constructed by cre-lox combination and compared to previously generated adenoviral vectors for BMP-2 (Ad.BMP-2), green fluorescent protein (Ad.GFP), or firefly luciferase (Ad.Luc). Cultures of human bone-marrow derived MSCs were infected with 5 × 102 viral particles/cell of Ad.BMP-2, or Ad.BMP-4, seeded into aggregates and cultured for three weeks in a defined, serum-free medium. Untransduced cells or cultures transduced with marker genes served as controls. Expression of BMP-2 and BMP-4 was determined by ELISA, and aggregates were analyzed histologically, immunohistochemically, biochemically and by RT-PCR for chondrogenesis and hypertrophy.ResultsLevels of BMP-2 and BMP-4 in the media were initially 30 to 60 ng/mL and declined thereafter. BMP-4 and BMP-2 genes were equipotent inducers of chondrogenesis in primary MSCs as judged by lacuna formation, strong staining for proteoglycans and collagen type II, increased levels of GAG synthesis, and expression of mRNAs associated with the chondrocyte phenotype. However, BMP-4 modified aggregates showed a lower tendency to progress towards hypertrophy, as judged by expression of alkaline phosphatase, annexin 5, immunohistochemical staining for type X collagen protein, and lacunar size.ConclusionsBMP-2 and BMP-4 were equally effective in provoking chondrogenesis by primary human MSCs in pellet culture. However, chondrogenesis triggered by BMP-2 and BMP-4 gene transfer showed considerable evidence of hypertrophic differentiation, with, the cells resembling growth plate chondrocytes both morphologically and functionally. This suggests caution when using these candidate genes in cartilage repair.

Cytokine response of human macrophage-like cells after contact with polyethylene and pure titanium particles

The aim of this study was to establish a human macrophage cell culture system to examine the effect of polyethylene (PE) and titanium particles on cytokine release by macrophage-like cells (MLC) and to quantify this response with respect to the nature and concentration of particles. Human monocytic leukemia cells were differentiated under standard conditions with vitamin D3 and granulocyte macrophage-colony-stimulating factor. Cells were characterized by fluorescence-activated cell-sorter Scan of CD 14 expression analysis as well as a phagocytosis test exploiting fluorescence-labeled particles of bacteria] walls. To achieve a relevant contact between the floating PE particles (approximately 1 microm in size) and MLC, a rotation device was used (15 rotations/min) during incubation. The same was done with the titanium particles. Cell culture supernatants were then analyzed for interleukin (IL)-1beta, IL-8, and tumor necrosis factor (TNF)-alpha using the enzyme-linked immunosorbent assay technique in the absence or presence of particles. Rotation of incubated MLC alone did not influence the secretion of TNF-alpha, but it enhanced secretion of IL-1beta and IL-8 about 30-fold compared to background levels. Both PE and titanium particles significantly enhanced MLC cytokine release, the amount of which depended on the concentration of particles. Using 40 X 10(8) PE particles (0.7 x 10(8) titanium particles) and 10(6) MLC, the maximal release of IL-1beta was about 20-fold (7-fold titanium particles) higher than that of the rotating control sample. The stimulation of IL-8 release was 4-fold (3-fold titanium particles) and of TNF-alpha. 300-fold (170-fold titanium particles) compared to controls. MLC were viable (>90% cell survival) at concentrations less than 108 x 10(8) polyethylene particles per 10(6) MLC and 16 x 10(8) titanium particles per 10(6) MLC. Rotation per se as well as exposure to increasing concentrations of PE and titanium particles stimulates cytokine release (TNF-alpha, IL-1beta, IL-8) by macrophages in vitro. This in vitro model resembles the in vivo situation near arthroplasties, where implant particles make contact with inflammatory cells, such as macrophages. Cytokine release by macrophages may impair osteoblast function as well as stimulate bone resorption by osteoclasts and macrophages, thereby causing aseptic loosening of arthroplasties. Our in vitro model provides a reproducible human cell system that might shed light on the pathogenesis of particle disease and might serve as a reproducible in vitro test system for the biocompatibility of foreign materials.

Pulse treatment with zoledronic acid causes sustained commitment of bone marrow derived mesenchymal stem cells for osteogenic differentiation

The aminobisphosphonate zoledronic acid (ZA) is a bone seeking specific inhibitor of protein farnesylation and geranylgeranylation, which causes inhibition of osteoclast function and apoptosis. It is widely used as an osteoclast targeted antiresorptive treatment of metastatic bone disease, Pagets disease and osteoporosis. Mesenchymal stem cells (MSC) and osteoblast precursors can also be targets of bisphosphonates, but the clinical relevance of these effects is under debate. We show here that ZA in vitro causes inhibition of proliferation and induction of apoptosis in hMSC, when applied in concentrations of 20 and 50 microM for more than 24 h which can be rescued by treatment with 10 microM geranylgeranyl pyrophosphate (GGPP). However, pulse stimulation for 3 and 6 h with these concentrations and subsequent culture for up to 2 weeks under osteogenic conditions exerts sustained regulation of osteogenic marker genes in hMSC. The effect on gene regulation translates into marked enhancement of mineralization, as shown by alizarin red and alkaline phosphatase staining after 4 weeks of osteogenic culture. ZA, when applied as a pulse stimulus, might therefore also stimulate osteogenic differentiation in vivo, since muM plasma concentrations can be achieved by intravenous application of 5 mg in patients. These data set the stage for the future dissection of the effects of ZA and other aminobisphosphonates on cells beyond osteoclasts, with respect to cell differentiation in benign metabolic and to antitumor efficacy in metastatic bone diseases, as well as adverse events due to putative substance accumulation in bone during long-term treatment.

The need for a dual rating system in total knee arthroplasty.

The sequential course of the knee score and functional score of the Knee Society rating system of 276 press fit condylar modular unconstrained total knee arthroplasties performed for osteoarthritis between June 1988 and December 1992 was documented prospectively. The knee score increased significantly and stayed on a constant level from 2 years on, whereas the function score reached a maximum at 2 years and declined subsequently. Multiple regression analysis was performed testing the statistical significance and correlation of preoperative predictors with criteria at followup to determine their influence on outcome. Preoperative predictors were knee score and function score, body mass index, age, gender, patient category, and implant factors. Criteria studied were pain, knee score, and function score at 2 years followup. The function score is influenced significantly by the walking distance, age, body mass index, and patient category correlating moderately. The knee score is not affected by any of these factors. Pain was found to correlate low with the walking distance. Rating systems are influenced by numerous factors linked to the patients general health and condition. Their impact on the overall result can be controlled by separate rating of the knee score and function score as the dual Knee Society rating system does. Scoring systems adding up knee and functional rating to an overall result should not be used. There is a need for additional improvement of total knee arthroplasty rating such as patient based evaluation and establishing reliability and validity.

Randomized trial comparing early postoperative irradiation vs. the use of nonsteroidal antiinflammatory drugs for prevention of heterotopic ossification following prosthetic total hip replacement.

PURPOSE A randomized trial was undertaken to assess the comparative efficacy of early postoperative irradiation with either 5 or 7 Gy vs. the use of nonsteroidal antiinflammatory drug (NSAID) for prevention of heterotopic ossification (HO) following prosthetic total hip replacement (THP). METHODS AND MATERIALS Between 1993 and 1994, 301 patients were randomized to receive postoperative irradiation (5 or 7 Gy) or NSAID. One hundred and thirteen patients were treated with NSAID (indomethacin 2 x 50 mg/day for 1 week), 93 patients were irradiated with a single 7 Gy fraction, 95 patients with a single 5 Gy fraction. The treatment volume included the soft tissues between the periacetabular region of pelvis and the intertrochanteric portion of the femur. X-rays of treated hips were obtained immediately and 6 months after surgery. Heterotopic ossification was scored according to the Brooker Grading system. One hundred patients receiving no prophylactic therapy after total hip arthroplasty between 1988 and 1992, were analyzed and defined as historical control group. RESULTS Incidence of heterotopic ossification was 16.0% in NSAID-group (Brooker Score I: 8.0%; II: 6.2%; III: 1.8%; IV: 0%), 30.1% in 5 Gy group (Brooker Score I: 24.7%; II: 4.3%; III: 1.1%; IV: 0%), and 11.1% in 7 Gy group (Brooker Score I: 11.6%; II: 0%; III: 0%; IV: 0%). Regarding overall heterotopic ossification there was a significant difference between the NSAID group and the 5 Gy group (p < .015), respectively, between the 7 Gy group and the 5 Gy group (p < .0001). No significant difference was noted in the influence of overall HO between the NSAID and the 7 Gy group (p > 0.3). Analyzing the clinically significant HO (Brooker Score III and IV) patients irradiated with 7 Gy developed less HO than those treated with NSAID (p = 0.003). Incidence of HO was greater in the untreated historical control group (Brooker Score I: 26%; II: 15%; III: 19%; IV: 5%) than in all three prophylacticly treated groups. CONCLUSION Prophylactic irradiation of the operative site after hip replacement with single a 7 Gy fraction is the most effective postoperative treatment schedule in prevention of clinically significant heterotopic ossification. This therapy modality is more effective than irradiation with a single 5 Gy fraction or use of NSAID.

PREOPERATIVE IRRADIATION VERSUS THE USE OF NONSTEROIDAL ANTI-INFLAMMATORY DRUGS FOR PREVENTION OF HETEROTOPIC OSSIFICATION FOLLOWING TOTAL HIP REPLACEMENT: THE RESULTS OF A RANDOMIZED TRIAL

PURPOSE Previous studies showed the effectiveness of early preoperative (4 h before operation) irradiation for prevention of heterotopic ossification (HO) after total hip replacement. This procedure can result in logistic problems, if there is a great distance between the department of radiotherapy and the orthopedic clinic. To avoid these organizational problems a prospective study was undertaken to analyze the effectiveness of preoperative irradiation on the day preceding surgery (16-20 h before operation). METHODS AND MATERIALS Between 1995 and 1996, 100 patients were randomized to receive a prophylactic therapy for prevention of heterotopic ossification. Forty-six patients were irradiated with 7 Gy single dose within 16-20 h before operation. Fifty-four patients were treated with nonsteroidal anti-inflammatory drugs (NSAID) (Voltaren resinat 2 x 75 mg/day for 2 weeks). Heterotopic ossification was scored according to the Brooker Grading system. One hundred patients receiving no prophylactic therapy after total hip arthroplasty between 1988 and 1992 were analyzed and defined as the historical control group. RESULTS Incidence of heterotopic ossification was 47.8% in the 7 Gy preoperative group (Brooker Score I: 36.9%; II: 8.7%; III: 2.2%; IV: 0%) and 11.1% in the NSAID group (Brooker Score I: 9.3%; II: 1.8%; III: 0%; IV: 0%). Regarding overall heterotopic ossification there was a significant difference between the NSAID group and the 7 Gy group (p < 0.01). Analyzing the clinically significant heterotopic ossification (Brooker Score III and IV) there was no significant difference between the two treatment arms (p > 0.05). In the untreated historical control group the incidence of heterotopic ossification was 65% (Brooker Score I: 26%; II: 15%; III: 19%; IV: 5%). Referring to overall and to clinically relevant heterotopic ossification the incidence of HO was greater in the control group than in the prophylactically treated groups (p < 0.05). CONCLUSION Irradiation within 16-20 h before operation and use of NSAID (Voltaren resinat) can reduce the incidence of clinically relevant heterotopic ossification after total hip replacement.

Effects of polyethylene and TiAIV wear particles on expression of RANK, RANKL and OPG mRNA

Background Wear debris has been associated with periprosthetic osteolysis and loosening of total joint arthroplasties. RANKL (receptor activator of NF-κB ligand), RANK (receptor activator of NF-κB) and OPG (osteoprotegerin) are three key molecules which regulate differentiation, survival, fusion, and activation of osteoclasts.Material and methods We evaluated the effect of TiAlV and polyethylene particles on expression of RANK, RANKL and OPG mRNA. We used a human monocytic leukemic cell line (THP-1) in this in vitro study. THP-1 monocytes were differentiated into macrophage-like cells and exposed to polyethylene particles and prosthesis-derived TiAlV particles. The supernantant was used for measurement of TNFα protein and total RNA was extracted from the cells. Expression of the genes coding for OPG, RANKL and RANK was analysed at the mRNA level using a semiquantitative RT-PCR method.Results Both polyethylene and TiAlV particles induced a significant release of TNFα after 6 h of exposure and a significant upregulation of RANK mRNA. OPG mRNA expression was transiently upregulated after exposure to polyethylene and TiAlV particles. These effects were dependent on particle dose. RANKL mRNA was not detectable in our THP-1 model. In contrast, LPS exhibited a different pattern of RANK/RANKL/OPG mRNA expression.Interpretation Our findings provide evidence that both polyethylene and TiAlV particles induce upregulation of RANK expression in cells of the monocytic lineage, which may be important for periprosthetic osteoclastogenesis.

Prophylaxis of heterotopic ossification after total hip arthroplasty: a prospective randomized study comparing indomethacin and meloxicam.

: We performed a randomized, prospective study on the prophylaxis of heterotopic ossification (HO) after total hip arthroplasty (THR), comparing indomethacin and the selective COX-2 inhibitor meloxicam. From the day after surgery, 272 patients were treated with 7.5 mg meloxicam, 15 mg meloxicam, or 2 x 50 mg indomethacin a day, for 14 days. After 6 months, radiographs of patients treated with 7.5 mg meloxicam showed that HO had occurred in one third. This treatment was therefore stopped after 26 patients have been assigned to this group. According to the intention-to-treat principle, patients given 15 mg meloxicam developed HO in 25% (20% Brooker grade I, 4% grade II and 1% grade III) and those given indomethacin in 10% (7% Brooker grade I, 1% grade II and 2% grade III), a statistically significant difference.

Heterotopic ossification after total knee arthroplasty : 54/615 cases after 1-6 years'follow-up

We found heterotopic ossifications in 54 (9%) of 615 cases after total knee arthroplasty. The largest ossifications were located in the anterior distal femur. In 12 cases smaller ossifications were found in other knee regions. The development of heterotopic ossification showed a positive correlation with hypertrophic arthrosis and a negative correlation with rheumatoid arthritis. We propose a new 3-grade classification which refers only to the anterior distal femoral region. Grade III heterotopic ossifications occurred in 4 patients (4 knees) who had clinical symptoms; 2 were successfully reoperated with removal of the ossifications. Prophylaxis should be considered in patients with marked hypertrophic arthrosis or marked periosteal damage to the anterior distal femur.