Lehrstuhl für Orthopädie
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Background
The key for successful delivery in minimally-invasive hip replacement lies in the exact knowledge about the surgical anatomy. The minimally-invasive direct anterior approach to the hip joint makes it necessary to clearly identify the tensor fasciae latae muscle in order to enter the Hueter interval without damaging the lateral femoral cutaneous nerve. However, due to the inherently restricted overview in minimally-invasive surgery, this can be difficult even for experienced surgeons.
Methods and Surgical Technique
In this technical note, we demonstrate for the first time how to use the tensor fasciae latae perforator as anatomical landmark to reliably identify the tensor fasciae latae muscle in orthopaedic surgery. Such perforators are used for flaps in plastic surgery as they are constant and can be found at the lateral third of the tensor fasciae latae muscle in a direct line from the anterior superior iliac spine.
Conclusion
As demonstrated in this article, a simple knowledge transfer between surgical disciplines can minimize the complication rate associated with minimally-invasive hip replacement.
Primary osteoporosis is an age-related disease characterized by an imbalance in bone homeostasis. While the resorptive aspect of the disease has been studied intensely, less is known about the anabolic part of the syndrome or presumptive deficiencies in bone regeneration. Multipotent mesenchymal stem cells (MSC) are the primary source of osteogenic regeneration. In the present study we aimed to unravel whether MSC biology is directly involved in the pathophysiology of the disease and therefore performed microarray analyses of hMSC of elderly patients (79-94 years old) suffering from osteoporosis (hMSC-OP). In comparison to age-matched controls we detected profound changes in the transcriptome in hMSC-OP, e.g. enhanced mRNA expression of known osteoporosis-associated genes (LRP5, RUNX2, COL1A1) and of genes involved in osteoclastogenesis (CSF1, PTH1R), but most notably of genes coding for inhibitors of WNT and BMP signaling, such as Sclerostin and MAB21L2. These candidate genes indicate intrinsic deficiencies in self-renewal and differentiation potential in osteoporotic stem cells. We also compared both hMSC-OP and non-osteoporotic hMSC-old of elderly donors to hMSC of similar to 30 years younger donors and found that the transcriptional changes acquired between the sixth and the ninth decade of life differed widely between osteoporotic and non-osteoporotic stem cells. In addition, we compared the osteoporotic transcriptome to long term-cultivated, senescent hMSC and detected some signs for pre-senescence in hMSC-OP. Our results suggest that in primary osteoporosis the transcriptomes of hMSC populations show distinct signatures and little overlap with non-osteoporotic aging, although we detected some hints for senescence-associated changes. While there are remarkable inter-individual variations as expected for polygenetic diseases, we could identify many susceptibility genes for osteoporosis known from genetic studies. We also found new candidates, e.g. MAB21L2, a novel repressor of BMP-induced transcription. Such transcriptional changes may reflect epigenetic changes, which are part of a specific osteoporosis-associated aging process.
Regulating and reverting the adipo-osteogenic lineage decision of trabecular human bone marrow stromal cells (hBMSCs) represents a promising approach for osteoporosis therapy and prevention. Fibroblast growth factor 1 (FGF1) and its subfamily member FGF2 were scored as lead candidates to exercise control over lineage switching processes (conversion) in favor of osteogenesis previously. However, their impact on differentiation events is controversially discussed in literature. Hence, the present study aimed to investigate the effects of these FGFs on the adipogenic and osteogenic differentiation and conversion of primary hBMSCs. Moreover, involved downstream signaling mechanisms should be elucidated and, finally, the results should be evaluated with regard to the possible therapeutic approach.
This study clearly revealed that culture in the presence of FGF1 strongly prevented the adipogenic differentiation of hBMSCs as well as the adipogenic conversion of pre-differentiated osteoblastic cells. Lipid droplet formation was completely inhibited by a concentration of 25 ng/µL. Meanwhile, the expression of genetic markers for adipogenic initiation, peroxisome proliferator-activated receptor gamma 2 (PPARg2) and CCAAT/enhancer binding protein alpha (C/EBPa), as well as subsequent adipocyte maturation, fatty acid binding protein 4 (FABP4) and lipoprotein lipase (LPL), were significantly downregulated. Yet, the genetic markers of osteogenic commitment and differentiation were not upregulated during adipogenic differentiation and conversion under FGF supplementation, not supporting an event of osteogenic lineage switching.
Moreover, when examining the effects on the osteogenic differentiation of hBMSCs and the osteogenic conversion of pre-differentiated adipocytic cells, culture in the presence of FGF1 markedly decreased extracellular matrix (ECM) mineralization. Additionally, the gene expression of the osteogenic marker alkaline phosphatase (ALP) was significantly reduced and ALP enzyme activity was decreased. Furthermore, genetic markers of osteogenic commitment, like the master regulator runt-related transcription factor 2 (RUNX2) and bone morphogenetic protein 4 (BMP4), as well as markers of osteogenic differentiation and ECM formation, like collagen 1 A1 (COL1A1) and integrin-binding sialoprotein (IBSP), were downregulated. In contrast, genes known to inhibit ECM mineralization, like ANKH inorganic pyrophosphate transport regulator (ANKH) and osteopontin (OPN), were upregulated. ANKH inhibition revealed that its transcriptional elevation was not crucial for the reduced matrix mineralization, perhaps due to decreased expression of ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) that likely annulled ANKH upregulation. Like FGF1, also the culture in the presence of FGF2 displayed a marked anti-adipogenic and anti-osteogenic effect.
The FGF receptor 1 (FGFR1) was found to be crucial for mediating the described FGF effects in adipogenic and osteogenic differentiation and conversion. Yet, adipogenic conversion displayed a lower involvement of the FGFR1. For adipogenic differentiation and osteogenic differentiation/conversion, downstream signal transduction involved the extracellular signal-regulated kinases 1 and 2 (ERK1/2) and the mitogen-activated protein kinase (MAPK)/ERK kinases 1 and 2 (MEK1/2), probably via the phosphorylation of FGFR docking protein FGFR substrate 2a (FRS2a) and its effector Ras/MAPK. The c-Jun N-terminal kinase (JNK), p38-MAPK, and protein kinase C (PKC) were not crucial for the signal transduction, yet were in part responsible for the rate of adipogenic and/or osteogenic differentiation itself, in line with current literature.
Taken together, to the best of our knowledge, our study was the first to describe the strong impact of FGF1 and FGF2 on both the adipogenic and osteogenic differentiation and conversion processes of primary hBMSCs in parallel. It clearly revealed that although both FGFs were not able to promote the differentiation and lineage switching towards the osteogenic fate, they strongly prevented adipogenic differentiation and lineage switching, which seem to be elevated during osteoporosis. Our findings indicate that FGF1 and FGF2 entrapped hBMSCs in a pre-committed state. In conclusion, these agents could be applied to potently prevent unwanted adipogenesis in vitro. Moreover, our results might aid in unraveling a pharmacological control point to eliminate the increased adipogenic differentiation and conversion as potential cause of adipose tissue accumulation and decreased osteoblastogenesis in bone marrow during aging and especially in osteoporosis.
1,25-dihydroxyvitamin D3 (1,25D3) was reported to induce premature organismal aging in fibroblast growth factor-23 (Fgf23) and klotho deficient mice, which is of main interest as 1,25D3 supplementation of its precursor cholecalciferol is used in basic osteoporosis treatment. We wanted to know if 1,25D3 is able to modulate aging processes on a cellular level in human mesenchymal stem cells (hMSC). Effects of 100 nM 1,25D3 on hMSC were analyzed by cell proliferation and apoptosis assay, beta-galactosidase staining, VDR and surface marker immunocytochemistry, RT-PCR of 1,25D3-responsive, quiescence-and replicative senescence-associated genes. 1,25D3 treatment significantly inhibited hMSC proliferation and apoptosis after 72 h and delayed the development of replicative senescence in long-term cultures according to beta-galactosidase staining and P16 expression. Cell morphology changed from a fibroblast like appearance to broad and rounded shapes. Long term treatment did not induce lineage commitment in terms of osteogenic pathways but maintained their clonogenic capacity, their surface marker characteristics (expression of CD73, CD90, CD105) and their multipotency to develop towards the chondrogenic, adipogenic and osteogenic pathways. In conclusion, 1,25D3 delays replicative senescence in primary hMSC while the pro-aging effects seen in mouse models might mainly be due to elevated systemic phosphate levels, which propagate organismal aging.
Resection of musculoskeletal sarcoma can result in large bone defects where regeneration is needed in a quantity far beyond the normal potential of self-healing. In many cases, these defects exhibit a limited intrinsic regenerative potential due to an adjuvant therapeutic regimen, seroma, or infection. Therefore, reconstruction of these defects is still one of the most demanding procedures in orthopaedic surgery. The constraints of common treatment strategies have triggered a need for new therapeutic concepts to design and engineer unparalleled structural and functioning bone grafts. To satisfy the need for long-term repair and good clinical outcome, a paradigm shift is needed from methods to replace tissues with inert medical devices to more biological approaches that focus on the repair and reconstruction of tissue structure and function. It is within this context that the field of bone tissue engineering can offer solutions to be implemented into surgical therapy concepts after resection of bone and soft tissue sarcoma. In this paper we will discuss the implementation of tissue engineering concepts into the clinical field of orthopaedic oncology.
Knowledge of local bone quality is essential for surgeons to determine operation techniques. A device for intraoperative measurement of local bone quality has been developed by the AO-Research Foundation (DensiProbe®). We used this device to experimentally measure peak breakaway torque of trabecular bone in the proximal femur and correlated this with local bone mineral density (BMD) and failure load. Bone mineral density of 160 cadaver femurs was measured by ex situ dual-energy X-ray absorptiometry. The failure load of all femurs was analyzed by side-impact analysis. Femur fractures were fixed and mechanical peak torque was measured with the DensiProbe® device. Correlation was calculated whereas correlation coefficient and significance was calculated by Fisher’s Z-transformation. Moreover, linear regression analysis was carried out. The unpaired Student’s t-test was used to assess the significance of differences. The Ward triangle region had the lowest BMD with 0.511 g/cm2 (±0.17 g/cm2), followed by the upper neck region with 0.546 g/cm2 (±0.16 g/cm2), trochanteric region with 0.685 g/cm2 (±0.19 g/cm2) and the femoral neck with 0.813 g/cm2 (±0.2 g/cm2). Peak torque of DensiProbe® in the femoral head was 3.48 Nm (±2.34 Nm). Load to failure was 4050.2 N (±1586.7 N). The highest correlation of peak torque measured by Densi Probe® and load to failure was found in the femoral neck (r=0.64, P<0.001). The overall correlation of mechanical peak torque with T-score was r=0.60 (P<0.001). A correlation was found between mechanical peak torque, load to failure of bone and BMD in vitro. Trabecular strength of bone and bone mineral density are different aspects of bone strength, but a correlation was found between them. Mechanical peak torque as measured may contribute additional information about bone strength, especially in the perioperative testing.
Background: Revision in failed shoulder arthroplasty often requires removal of the humeral component with a significant risk of fracture and bone loss. Newer modular systems allow conversion from anatomic to reverse shoulder arthroplasty with retention of a well-fixed humeral stem. We report on a prospectively evaluated series of conversions from hemiarthroplasty to reverse shoulder arthroplasty.
Methods: In 14 cases of failed hemiarthroplasty due to rotator cuff deficiency and painful pseudoparalysis (in 13 women), revision to reverse shoulder arthroplasty was performed between October 2006 and 2010, with retention of the humeral component using modular systems. Mean age at the time of operation was 70 (56-80) years. Pre- and postoperative evaluation followed a standardized protocol including Constant score, range of motion, and radiographic analysis. Mean follow-up time was 2.5 (2-5.5) years.
Results: Mean Constant score improved from 9 (2-16) to 41 (17-74) points. Mean lengthening of the arm was 2.6 (0.9-4.7) cm without any neurological complications. One patient required revision due to infection. Interpretation Modular systems allow retainment of a well-fixed humeral stem with good outcome. There is a risk of excessive humeral lengthening.
Background: We present a descriptive and retrospective analysis of revision total hip arthroplasties (THA) using the MRP-TITAN stem (Peter Brehm, Weisendorf, GER) with distal diaphyseal fixation and metaphyseal defect augmentation. Our hypothesis was that the metaphyseal defect augmentation (Impaction Bone Grafting) improves the stem survival.
Methods: We retrospectively analyzed the aggregated and anonymized data of 243 femoral stem revisions. 68 patients with 70 implants (28.8%) received an allograft augmentation for metaphyseal defects; 165 patients with 173 implants (71.2%) did not, and served as controls. The mean follow-up was 4.4 +/- 1.8 years (range, 2.1-9.6 years). There were no significant differences (p > 0.05) between the study and control group regarding age, body mass index (BMI), femoral defects (types I-III as described by Paprosky), and preoperative Harris Hip Score (HHS). Postoperative clinical function was evaluated using the HHS. Postoperative radiologic examination evaluated implant stability, axial implant migration, signs of implant loosening, periprosthetic radiolucencies, as well as bone regeneration and resorption.
Results: There were comparable rates of intraoperative and postoperative complications in the study and control groups (p > 0.05). Clinical function, expressed as the increase in the postoperative HHS over the preoperative score, showed significantly greater improvement in the group with Impaction Bone Grafting (35.6 +/- 14.3 vs. 30.8 +/- 15.8; p <= 0.05). The study group showed better outcome especially for larger defects (types II C and III as described by Paprosky) and stem diameters >= 17 mm. The two groups did not show significant differences in the rate of aseptic loosening (1.4% vs. 2.9%) and the rate of revisions (8.6% vs. 11%). The Kaplan-Meier survival for the MRP-TITAN stem in both groups together was 93.8% after 8.8 years. [Study group 95.7% after 8.54 years; control group 93.1% after 8.7 years]. Radiologic evaluation showed no significant change in axial implant migration (4.3% vs. 9.3%; p = 0.19) but a significant reduction in proximal stress shielding (5.7% vs. 17.9%; p < 0.05) in the study group. Periprosthetic radiolucencies were detected in 5.7% of the study group and in 9.8% of the control group (p = 0.30). Radiolucencies in the proximal zones 1 and 7 according to Gruen occurred significantly more often in the control group without allograft augmentation (p = 0.05).
Conclusion: We present the largest analysis of the impaction grafting technique in combination with cementless distal diaphyseal stem fixation published so far. Our data provides initial evidence of improved bone regeneration after graft augmentation of metaphyseal bone defects. The data suggests that proximal metaphyseal graft augmentation is beneficial for large metaphyseal bone defects (Paprosky types IIC and III) and stem diameters of 17 mm and above. Due to the limitations of a retrospective and descriptive study the level of evidence remains low and prospective trials should be conducted.
Background: To describe changes in health-related quality of life (HRQoL) of postmenopausal women with osteoporosis treated with teriparatide for up to 18 months and followed-up for a further 18 months, and to assess the influence of recent prior and incident fractures.
Methods: The European Forsteo Observational Study (EFOS) is an observational, prospective, multinational study measuring HRQoL using the EQ-5D. The primary objective was to assess changes in HRQoL during 36 months in the whole study population. A secondary post-hoc analysis examined fracture impact on HRQoL in four subgroups classified based on recent prior fracture 12 months before baseline and incident clinical fractures during the study. Changes from baseline were analysed using a repeated measures model.
Results: Of the 1581 patients, 48.4% had a recent prior fracture and 15.6% of these patients had an incident fracture during follow-up. 10.9% of the 816 patients with no recent prior fracture had an incident fracture. Baseline mean EQ-VAS scores were similar across the subgroups. In the total study cohort (n = 1581), HRQoL (EQ-VAS and EQ-5D index scores) improved significantly from baseline to 18 months and this improvement was maintained over the 18-month post-teriparatide period. Improvements were seen across all five EQ-5D domains during teriparatide treatment that were maintained after teriparatide was discontinued. Subjects with incident clinical fractures had significantly less improvement in EQ-VAS than those without incident fractures. Recent prior fracture did not influence the change in EQ-VAS during treatment.
Conclusions: EFOS is the first longitudinal study in women with severe postmenopausal osteoporosis in the real world setting to show a substantial improvement in HRQoL during teriparatide treatment that was sustained during subsequent treatment with other medications. The increase in HRQoL was lower in the subgroups with incident fracture but was not influenced by recent prior fracture. The results should be interpreted in the context of the design of an observational study.
Fracture healing is impaired in aged and osteoporotic individuals. Because adequate mechanical stimuli are able to increase bone formation, one therapeutical approach to treat poorly healing fractures could be the application of whole-body vibration, including low-magnitude high-frequency vibration (LMHFV). We investigated the effects of LMHFV on fracture healing in aged osteoporotic mice. Female C57BL/6NCrl mice (n=96) were either ovariectomised (OVX) or sham operated (non-OVX) at age 41 weeks. When aged to 49 weeks, all mice received a femur osteotomy that was stabilised using an external fixator. The mice received whole-body vibrations (20 minutes/day) with 0.3 G: peak-to-peak acceleration and a frequency of 45 Hz. After 10 and 21 days, the osteotomised femurs and intact bones (contra-lateral femurs, lumbar spine) were evaluated using bending-testing, micro-computed tomography (μCT), histology and gene expression analyses. LMHFV disturbed fracture healing in aged non-OVX mice, with significantly reduced flexural rigidity (-81%) and bone formation (-80%) in the callus. Gene expression analyses demonstrated increased oestrogen receptor β (ERβ, encoded by Esr2) and Sost expression in the callus of the vibrated animals, but decreased β-catenin, suggesting that ERβ might mediate these negative effects through inhibition of osteoanabolic Wnt/β-catenin signalling. In contrast, in OVX mice, LMHFV significantly improved callus properties, with increased flexural rigidity (+1398%) and bone formation (+637%), which could be abolished by subcutaneous oestrogen application (0.025 mg oestrogen administered in a 90-day-release pellet). On a molecular level, we found an upregulation of ERα in the callus of the vibrated OVX mice, whereas ERβ was unaffected, indicating that ERα might mediate the osteoanabolic response. Our results indicate a major role for oestrogen in the mechanostimulation of fracture healing and imply that LMHFV might only be safe and effective in confined target populations.
Juvenile Dermatomyositis (JDM) is a rare autoimmune disease in children and adolescents. In these patients calcinosis might be the most characteristic symptom. However there are only few reported cases of intramuscular calcinosis in Dermatomyositis. We report a case of calcinosis universalis (CU) of the elbow in JDM successfully treated with broaching. The patient, a 24-year-old woman, suffered from a long history of JDM. On examination she presented with a fistula lateral to the olecranon and pain of the right elbow joint. Plain X-rays displayed a diffuse pattern of multiple periarticular, subcutaneous, and intramuscular calcifications. The patient underwent surgery for histological and microbiological sampling as well as broaching. Intraoperatively sinus formation and subfascial hard calcium deposition were found. Due to the risk of collateral tissue damage, incomplete broaching was performed. A local infection with Staphylococcus was diagnosed and treated with antibiotics. On six-week and 30-month follow-up the patient was free of pain and had very good function. Calcifications on standard radiographs had almost resolved entirely. This case report gives a summary on calcinosis in Dermatomyositis and adds a new case of recalcitrant CU to the literature. Broaching surgery proved to be a reliable treatment option in symptomatic calcinosis.
There is a variation of the total number of distinct bones in the human in the literature. This difference is mainly caused by the variable existence of sesamoid bones. Sesamoid bones at the first MTP are seen regularly. In contrast additional sesamoid bones at the divond to fifth MTP are rare. We report a case of additional sesamoid bones at every metatarsophalangeal joint (MTP) of both feet.
A 22-year-old female Caucasian presented with weight-dependent pain of the divond MTP of the left foot. In the radiographs of both feet additional sesamoid bones at every MTP could be seen. This case reports a very rare variation in human anatomy. A similar case has not been displayed to the academic society and therefore should be acknowledged.
Introduction
The bursa subacromialis (BS) provides the gliding mechanism of the shoulder and regenerates itself after surgical removal. Therefore, we explored the presence of mesenchymal stem cells (MSCs) within the human adult BS tissue and characterized the BS cells compared to MSCs from bone marrow (BMSCs) on a molecular level.
Methods
BS cells were isolated by collagenase digest from BS tissues derived from patients with degenerative rotator cuff tears, and BMSCs were recovered by adherent culture from bone-marrow of patients with osteoarthritis of the hip. BS cells and BMSCs were compared upon their potential to proliferate and differentiate along chondrogenic, osteogenic and adipogenic lineages under specific culture conditions. Expression profiles of markers associated with mesenchymal phenotypes were comparatively evaluated by flow cytometry, immunohistochemistry, and whole genome array analyses.
Results
BS cells and BMSCs appeared mainly fibroblastic and revealed almost similar surface antigen expression profiles, which was \(CD44^+, CD73^+, CD90^+, CD105^+, CD106^+\),\(STRO-1^+, CD14^−, CD31^−, CD34^− ,
CD45^−, CD144^−\). Array analyses revealed 1969 genes upregulated and 1184 genes downregulated in BS cells vs. BMSCs, indicating a high level of transcriptome similarity. After 3 weeks of differentiation culture, BS cells and BMSCs showed a similar strong chondrogenic, adipogenic and osteogenic potential, as shown by histological, immunohistochemical and RT-PCR analyses in contrast to the respective negative controls.
Conclusions
Our in vitro characterizations show that BS cells fulfill all characteristics of mesenchymal stem cells, and therefore merit further attention for the development of improved therapies for various shoulder pathologies.
This predefined analysis of the European Forsteo Observational Study (EFOS) aimed to describe clinical fracture incidence, back pain, and health-related quality of life (HRQoL) during 18 months of teriparatide treatment and 18 months post-teriparatide in the subgroup of 589 postmenopausal women with osteoporosis aged ≥75 years. Data on clinical fractures, back pain (visual analogue scale, VAS), and HRQoL (EQ-5D) were collected over 36 months. Fracture data were summarized in 6-month intervals and analyzed using logistic regression with repeated measures. A repeated-measures model analyzed changes from baseline in back pain VAS and EQ-VAS. During the 36-month observation period, 87 (14.8 %) women aged ≥75 years sustained a total of 111 new fractures: 37 (33.3 %) vertebral fractures and 74 (66.7 %) nonvertebral fractures. Adjusted odds of fracture was decreased by 80 % in the 30 to <36–month interval compared with the first 6-month interval (P < 0.009). Although the older subgroup had higher back pain scores and poorer HRQoL at baseline than the younger subgroup, both age groups showed significant reductions in back pain and improvements in HRQoL postbaseline. In conclusion, women aged ≥75 years with severe postmenopausal osteoporosis treated with teriparatide in normal clinical practice showed a reduced clinical fracture incidence by 30 months compared with baseline. An improvement in HRQoL and, possibly, an early and significant reduction in back pain were also observed, which lasted for at least 18 months after teriparatide discontinuation when patients were taking other osteoporosis medication. The results should be interpreted in the context of an uncontrolled observational study.
In situ guided tissue regeneration, also addressed as in situ tissue engineering or endogenous regeneration, has a great potential for population-wide “minimal invasive” applications. During the last two decades, tissue engineering has been developed with remarkable in vitro and preclinical success but still the number of applications in clinical routine is extremely small. Moreover, the vision of population-wide applications of ex vivo tissue engineered constructs based on cells, growth and differentiation factors and scaffolds, must probably be deemed unrealistic for economic and regulation-related issues. Hence, the progress made in this respect will be mostly applicable to a fraction of post-traumatic or post-surgery situations such as big tissue defects due to tumor manifestation. Minimally invasive procedures would probably qualify for a broader application and ideally would only require off the shelf standardized products without cells. Such products should mimic the microenvironment of regenerating tissues and make use of the endogenous tissue regeneration capacities. Functionally, the chemotaxis of regenerative cells, their amplification as a transient amplifying pool and their concerted differentiation and remodeling should be addressed. This is especially important because the main target populations for such applications are the elderly and diseased. The quality of regenerative cells is impaired in such organisms and high levels of inhibitors also interfere with regeneration and healing. In metabolic bone diseases like osteoporosis, it is already known that antagonists for inhibitors such as activin and sclerostin enhance bone formation. Implementing such strategies into applications for in situ guided tissue regeneration should greatly enhance the efficacy of tailored procedures in the future.
The skeleton is a preferred homing site for breast cancer metastasis. To date, treatment options for patients with bone metastases are mostly palliative and the disease is still incurable. Indeed, key mechanisms involved in breast cancer osteotropism are still only partially understood due to the lack of suitable animal models to mimic metastasis of human tumor cells to a human bone microenvironment. In the presented study, we investigate the use of a human tissue-engineered bone construct to develop a humanized xenograft model of breast cancer-induced bone metastasis in a murine host. Primary human osteoblastic cell-seeded melt electrospun scaffolds in combination with recombinant human bone morphogenetic protein 7 were implanted subcutaneously in non-obese diabetic/severe combined immunodeficient mice. The tissue-engineered constructs led to the formation of a morphologically intact 'organ' bone incorporating a high amount of mineralized tissue, live osteocytes and bone marrow spaces. The newly formed bone was largely humanized, as indicated by the incorporation of human bone cells and human-derived matrix proteins. After intracardiac injection, the dissemination of luciferase-expressing human breast cancer cell lines to the humanized bone ossicles was detected by bioluminescent imaging. Histological analysis revealed the presence of metastases with clear osteolysis in the newly formed bone. Thus, human tissue-engineered bone constructs can be applied efficiently as a target tissue for human breast cancer cells injected into the blood circulation and replicate the osteolytic phenotype associated with breast cancer-induced bone lesions. In conclusion, we have developed an appropriate model for investigation of species-specific mechanisms of human breast cancer-related bone metastasis in vivo.
[Purpose] A wide variety of accelerometer tools are used to estimate human movement, but there are no adequate data relating to gait symmetry parameters in the context of knee osteoarthritis. This study's purpose was to evaluate a 3D-kinematic system using body-mounted sensors (gyroscopes and accelerometers) on the trunk and limbs. This is the first study to use spectral analysis for data post processing. [Subjects] Twelve patients with unilateral knee osteoarthritis (OA) (10 male) and seven age-matched controls (6 male) were studied. [Methods] Measurements with 3-D accelerometers and gyroscopes were compared to video analysis with marker positions tracked by a six-camera optoelectronic system (VICON 460, Oxford Metrics). Data were recorded using the 3D-kinematic system. [Results] The results of both gait analysis systems were significantly correlated. Five parameters were significantly different between the knee OA and control groups. To overcome time spent in expensive post-processing routines, spectral analysis was performed for fast differentiation between normal gait and pathological gait signals using the 3D-kinematic system. [Conclusions] The 3D-kinematic system is objective, inexpensive, accurate and portable, and allows long-term recordings in clinical, sport as well as ergonomic or functional capacity evaluation (FCE) settings. For fast post-processing, spectral analysis of the recorded data is recommended.
The canonical Wnt/beta-catenin pathway plays a key role in the regulation of bone remodeling in mice and humans. Two transmembrane proteins that are involved in decreasing the activity of this pathway by binding to extracellular antagonists, such as Dickkopf 1 (Dkk1), are the low-density lipoprotein receptor related protein 5 (Lrp5) and Kremen 2 (Krm2). Lrp 5 deficiency (Lrp5(-/-)) as well as osteoblast-specific overexpression of Krm2 in mice (Col1a1-Krm2) result in severe osteoporosis occurring at young age. In this study, we analyzed the influence of Lrp5 deficiency and osteoblast-specific overexpression of Krm2 on fracture healing in mice using flexible and semi-rigid fracture fixation. We demonstrated that fracture healing was highly impaired in both mouse genotypes, but that impairment was more severe in Col1a1-Krm2 than in Lrp5(-/-) mice and particularly evident in mice in which the more flexible fixation was used. Bone formation was more reduced in Col1a1-Krm2 than in Lrp5(-/-) mice, whereas osteoclast number was similarly increased in both genotypes in comparison with wild-type mice. Using microarray analysis we identified reduced expression of genes mainly involved in osteogenesis that seemed to be responsible for the observed stronger impairment of healing in Col1a1-Krm2 mice. In line with these findings, we detected decreased expression of sphingomyelin phosphodiesterase 3 (Smpd3) and less active beta-catenin in the calli of Col1a1-Krm2 mice. Since Krm2 seems to play a significant role in regulating bone formation during fracture healing, antagonizing KRM2 might be a therapeutic option to improve fracture healing under compromised conditions, such as osteoporosis.
Background and purpose - Due to the relative lack of reports on the medium- to long-term clinical and radiographic results of modular femoral cementless revision, we conducted this study to evaluate the medium- to long-term results of uncemented femoral stem revisions using the modular MRP-TITAN stem with distal diaphyseal fixation in a consecutive patient series.
Patients and methods - We retrospectively analyzed 163 femoral stem revisions performed between 1993 and 2001 with a mean follow-up of 10 (5-16) years. Clinical assessment included the Harris hip score (HHS) with reference to comorbidities and femoral defect sizes classified by Charnley and Paprosky. Intraoperative and postoperative complications were analyzed and the failure rate of the MRP stem for any reason was examined.
Results - Mean HHS improved up to the last follow-up (37 (SD 24) vs. 79 (SD 19); p < 0.001). 99 cases (61%) had extensive bone defects (Paprosky IIB-III). Radiographic evaluation showed stable stem anchorage in 151 cases (93%) at the last follow-up. 10 implants (6%) failed for various reasons. Neither a breakage of a stem nor loosening of the morse taper junction was recorded. Kaplan-Meier survival analysis revealed a 10-year survival probability of 97% (95% CI: 95-100).
Interpretation - This is one of the largest medium- to longterm analyses of cementless modular revision stems with distal diaphyseal anchorage. The modular MRP-TITAN was reliable, with a Kaplan-Meier survival probability of 97% at 10 years.
Background: Anti-resorptive bisphosphonates (BP) are used for the treatment of osteoporosis and bone metastases. Clinical studies indicated a benefit in survival and tumor relapse in subpopulations of breast cancer patients receiving zoledronic acid, thus stimulating the debate about its anti-tumor activity. Amino-bisphosphonates in nM concentrations inhibit farnesyl pyrophosphate synthase leading to accumulation of isopentenyl pyrophosphate (IPP) and the ATP/ pyrophosphate adduct ApppI, which induces apoptosis in osteoclasts. For anti-tumor effects μM concentrations are needed and a sensitizer for bisphosphonate effects would be beneficial in clinical anti-tumor applications. We hypothesized that enhancing intracellular pyrophosphate accumulation via inhibition of probenecid-sensitive channels and transporters would sensitize tumor cells for bisphosphonates anti-tumor efficacy.
Methods: MDA-MB-231, T47D and MCF-7 breast cancer cells were treated with BP (zoledronic acid, risedronate, ibandronate, alendronate) and the pyrophosphate channel inhibitors probenecid and novobiocin. We determined cell viability and caspase 3/7 activity (apoptosis), accumulation of IPP and ApppI, expression of ANKH, PANX1, ABCC1, SLC22A11, and the zoledronic acid target gene and tumor-suppressor KLF2.
Results: Treatment of MDA-MB-231 with BP induced caspase 3/7 activity, with zoledronic acid being the most effective. In MCF-7 and T47D either BP markedly suppressed cell viability with only minor effects on apoptosis. Co-treatment with probenecid enhanced BP effects on cell viability, IPP/ApppI accumulation as measurable in MCF-7 and T47D cells, caspase 3/7 activity and target gene expression. Novobiocin co-treatment of MDA-MB-231 yielded identical results on viability and apoptosis compared to probenecid, rendering SLC22A family members as candidate modulators of BP effects, whereas no such evidence was found for ANKH, ABCC1 and PANX1.
Conclusions: In summary, we demonstrate effects of various bisphosphonates on caspase 3/7 activity, cell viability and expression of tumor suppressor genes in breast cancer cells. Blocking probenecid- and novobiocin-sensitive channels and transporters enhances BP anti-tumor effects and renders SLC22A family members good candidates as BP modulators. Further studies will have to unravel if treatment with such BP-sensitizers translates into preclinical and clinical efficacy.
CCN family member 1 (CCN1), also known as cysteine-rich angiogenic inducer 61 (CYR61), belongs to the extracellular matrix-associated CCN protein family. The diverse functions of these proteins include regulation of cell migration, adhesion, proliferation, differentiation and survival/apoptosis, induction of angiogenesis and cellular senescence. Their functions are partly overlapping, largely non-redundant, cell-type specific, and depend on the local microenvironment. To elucidate the role of CCN1 in the crosstalk between stromal cells and myeloma cells, we performed co-culture experiments with primary mesenchymal stem cells (MSC) and the interleukin-6 (IL-6)-dependent myeloma cell line INA-6. Here we show that INA-6 cells display increased transcription and induction of splicing of intron-retaining CCN1 pre-mRNA when cultured in contact with MSC. Protein analyses confirmed that INA-6 cells co-cultured with MSC show increased levels of CCN1 protein consistent with the existence of a pre-mature stop codon in intron 1 that abolishes translation of unspliced mRNA. Addition of recombinant CCN1-Fc protein to INA-6 cells was also found to induce splicing of CCN1 pre-mRNA in a concentration-dependent manner. Only full length CCN1-Fc was able to induce mRNA splicing of all introns, whereas truncated recombinant isoforms lacking domain 4 failed to induce intron splicing. Blocking RGD-dependent integrins on INA-6 cells resulted in an inhibition of these splicing events. These findings expand knowledge on splicing of the proangiogenic, matricellular factor CCN1 in the tumor microenvironment. We propose that contact with MSC-derived CCN1 leads to splicing and enhanced transcription of CCN1 which further contributes to the translation of angiogenic factor CCN1 in myeloma cells, supporting tumor viability and myeloma bone disease.
Premature implant loosening following total knee arthroplasty (TKA) can have several causes. In this article we report on a rare case of a 74 year old male patient suffering tibial component loosening 14 month after primary TKA. The patient did neither have any malignancies nor joint arthroplasty before. Upon clinical examination the range of motion in the diseased knee was painfully restricted to 80° of knee flexion, with the patient increasingly suffering sleeping and resting pain, and also at weight bearing. In standard radiographs, loosening of the TKA due to a large osteolysis at the tibial component was evident. Local computed tomography (CT) of the right knee revealed loosening of the tibial component due to a presumably malign bone tumor. For determination of the final diagnosis a representative biopsy of the tumor was taken by open surgery prior to the tumor resection. Histopathologic evaluation of the biopsy revealed a periprosthetic myxoid chondrosarcoma of the proximal tibia. Pre-operative staging examination included CT scans of lung and abdomen, as well as a bone scintigraphy which revealed no signs of tumor metastasis in the body. Surgical management comprised wide tumor resection and implantation of a hinged tumor knee arthroplasty with replacements of the distal femur and proximal tibia, as well as a patella tendon replacement using a synthetic ligament. Revision surgery was necessary twice due to impaired wound healing and critical soft tissue coverage, and treatment included a gastrocnemius muscle flap with skin mesh graft covering. Unfortunately long-term follow-up examinations could not be obtained, as the patient deceased due to an alveolitis during rehabilitation. In summary, the specifics of this rare case of aseptic TKA loosening, and the unusual circumstances of chondrosarcoma diagnosis and treatment are informative for those providing surgical treatment of similar cases.
WISP3 is a member of the CCN family which comprises six members found in the 1990’s: Cysteine-rich,angiogenic inducer 61 (CYR61, CCN1), Connective tissue growth factor (CTGF, CCN2), Nephroblastoma overexpressed (NOV, CNN3) and the Wnt1 inducible signalling pathway protein 1-3 (WISP1-3, CCN4-6).They are involved in the adhesion, migration, mitogenesis, chemotaxis, proliferation, cell survival, angiogenesis, tumorigenesis, and wound healing by the interaction with different integrins and heparan sulfate proteoglycans. Until now the only member correlated to the musculoskeletal autosomal disease Progressive Pseudorheumatoid Dysplasia (PPD) is WISP3. PPD is characterised by normal embryonic development followed by cartilage degradation over time starting around the age of three to eight years. Animal studies in mice exhibited no differences between knock out or overexpression compared to wild type litter mates, thus were not able to reproduce the symptoms observed in PPD patients. Studies in vitro and in vivo revealed a role for WISP3 in antagonising BMP, IGF and Wnt signalling pathways. Since most of the knowledge of WISP3 was gained in epithelial cells, cancer cells or chondrocyte cell lines, we investigated the roll of WISP3 in primary human mesenchymal stem cells (hMSCs) as well as primary chondrocytes.
WISP3 knock down was efficiently established with three short hairpin RNAs in both cell types, displaying a change of morphology followed by a reduction in cell number. Simultaneous treatment with recombinant WISP3 was not enough to rescue the observed phenotype nor increase the endogenous expression of WISP3. We concluded that WISP3 acts as an essential survival factor, where the loss resulted in the passing of cell cycle control points followed by apoptosis. Nevertheless, Annexin V-Cy3 staining and detection of active caspases by Western blot and immunofluorescence staining detected no clear evidence for apoptosis. Furthermore, the gene expression of the death receptors TRAILR1 and TRAILR2,important for the extrinsic activation of apoptosis, remained unchanged during WISP3 mRNA reduction. Autophagy as cause of cell death was also excluded, given that the autophagy marker LC3 A/B demonstrated to be uncleaved in WISP3-deficient hMSCs. To reveal correlated signalling pathways to WISP3 a whole genome expression analyses of WISP3-deficient hMSCs compared to a control (scramble) was performed. Microarray analyses exhibited differentially regulated genes involved in cell cycle control, adhesion, cytoskeleton and cell death. Cell death observed by WISP3 knock down in hMSCs and chondrocytes might be explained by the induction of necroptosis through the BMP/TAK1/RIPK1 signalling axis. Loss of WISP3 allows BMP to bind its receptor activating the Smad 2/3/4 complex which in turn can activate TAK1 as previously demonstrated in epithelial cells. TAK1 is able to block
caspase-dependent apoptosis thereby triggering the assembly of the necrosome resulting in cell death by necroptosis.
Together with its role in cell cycle control and extracellular matrix adhesion, as demonstrated in human mammary epithelial cells, the data supports the role of WISP3 as tumor suppressor and survival factor in cells of the musculoskeletal system as well as epithelial cells.
Introduction
To stimulate healing of large bone defects research has concentrated on the application of mesenchymal stem cells (MSCs).
Methods
In the present study, we induced the overexpression of the growth factors bone morphogenetic protein 2 (BMP-2) and/or Indian hedgehog (IHH) in human MSCs by adenoviral transduction to increase their osteogenic potential. GFP and nontransduced MSCs served as controls. The influence of the respective genetic modification on cell metabolic activity, proliferation, alkaline phosphatase (ALP) activity, mineralization in cell culture, and osteogenic marker gene expression was investigated.
Results
Transduction had no negative influence on cell metabolic activity or proliferation. ALP activity showed a typical rise-and-fall pattern with a maximal activity at day 14 and 21 after osteogenic induction. Enzyme activity was significantly higher in groups cultured with osteogenic media. The overexpression of BMP-2 and especially IHH + BMP-2 resulted in a significantly higher mineralization after 28 days. This was in line with obtained quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analyses, which showed a significant increase in osteopontin and osteocalcin expression for osteogenically induced BMP-2 and IHH + BMP-2 transduced cells when compared with the other groups. Moreover, an increase in runx2 expression was observed in all osteogenic groups toward day 21. It was again more pronounced for BMP-2 and IHH + BMP-2 transduced cells cultured in osteogenic media.
Conclusions
In summary, viral transduction did not negatively influence cell metabolic activity and proliferation. The overexpression of BMP-2 in combination with or without IHH resulted in an increased deposition of mineralized extracellular matrix, and expression of osteogenic marker genes. Viral transduction therefore represents a promising means to increase the osteogenic potential of MSCs and the combination of different transgenes may result in synergistic effects.
Background: Desmoplastic fibroma (DF) is an extremely rare locally aggressive bone tumor with an incidence of 0.11% of all primary bone tumors. The typical clinical presentation is pain and swelling above the affected area. The most common sites of involvement are the mandible and the metaphysis of long bones. Histologically and biologically, desmoplastic fibroma mimics extra-abdominal desmoid tumor of soft tissue.
Case Presentation and Literature Review: A case of a 27-year old man with DF in the ilium, including the clinical, radiological and histological findings over a 4-year period is presented here. CT scans performed in 3-year intervals prior to surgical intervention were compared with respect to tumor extension and cortical breakthrough. The patient was treated with curettage and grafting based on anatomical considerations. Follow-up CT scans over 18-months are also documented here. Additionally, a review and analysis of 271 cases including the presented case with particular emphasis on imaging patterns in MRI and CT as well as treatment modalities and outcomes are presented.
Conclusion: In patients with desmoplastic fibroma, CT is the preferred imaging technique for both the diagnosis of intraosseus tumor extension and assessment of cortical involvement, whereas MRI is favored for the assessment of extraosseus tumor growth and preoperative planning. While tumor resection remains the preferred treatment for DF, curettage and grafting prove to be an acceptable alternative treatment modality with close follow-up when resection is not
possible. Curettage and grafting have been shown to provide good clinical results and are associated with long recurrence free intervals.
Background
Chronic osteomyelitis due to direct bone trauma or vascular insufficiency is a frequent problem in orthopaedic surgery. In contrast, acute haematogenous osteomyelitis represents a rare entity that almost exclusively affects prepubescent children or immunodeficient adults.
Case Presentation
In this article, we report the case of acute pneumococcal osteomyelitis of the humerus in an immunocompetent and otherwise healthy 44-year-old male patient presenting with minor inflammation signs and misleading clinical features.
Conclusions
The diagnosis had to be confirmed by open biopsy which allowed the initiation of a targeted therapy. A case of pneumococcal osteomyelitis of a long bone, lacking predisposing factors or trauma, is unique in adults and has not been reported previously.
Introduction: To date, no single most-appropriate factor or delivery method has been identified for the purpose of mesenchymal stem cell (MSC)-based treatment of cartilage injury. Therefore, in this study we tested whether gene delivery of the growth factor Indian hedgehog (IHH) was able to induce chondrogenesis in human primary MSCs, and whether it was possible by such an approach to modulate the appearance of chondrogenic hypertrophy in pellet cultures in vitro. Methods: First-generation adenoviral vectors encoding the cDNA of the human IHH gene were created by cre-lox recombination and used alone or in combination with adenoviral vectors, bone morphogenetic protein-2 (Ad.BMP- 2), or transforming growth factor beta-1 (Ad.TGF-b1) to transduce human bone-marrow derived MSCs at 5 × 102 infectious particles/cell. Thereafter, 3 × 105 cells were seeded into aggregates and cultured for 3 weeks in serumfree medium, with untransduced or marker gene transduced cultures as controls. Transgene expressions were determined by ELISA, and aggregates were analysed histologically, immunohistochemically, biochemically and by RT-PCR for chondrogenesis and hypertrophy. Results: IHH, TGF-b1 and BMP-2 genes were equipotent inducers of chondrogenesis in primary MSCs, as evidenced by strong staining for proteoglycans, collagen type II, increased levels of glycosaminoglycan synthesis, and expression of mRNAs associated with chondrogenesis. IHH-modified aggregates, alone or in combination, also showed a tendency to progress towards hypertrophy, as judged by the expression of alkaline phosphatase and stainings for collagen type X and Annexin 5. Conclusion: As this study provides evidence for chondrogenic induction of MSC aggregates in vitro via IHH gene delivery, this technology may be efficiently employed for generating cartilaginous repair tissues in vivo.
Reconstruction of the extensor mechanism is essential for good extremity function after endoprosthetic knee replacement following tumor resection. Only a few biological methods have been able to reliably restore a functional extensor mechanism, but they are often associated with significant complication rates. Reattachment of the patellar tendon to the prosthesis using an alloplastic patellar ligament (Trevira cord) can be an appropriate alternative. In vivo and in vitro studies have already shown that complete fibrous ingrowth in polyethylene chords can be seen after a period of six months. However, until now, no biomechanical study has shown the efficacy of an alloplastic cord and its fixation device in providing sufficient stability and endurance in daily life-activity until newly formed scar tissue can take over this function. In a special test bench developed for this study, different loading regimes were applied to simulate loads during everyday life. Failure loads and failuremodes were evaluated. The properties of the cord were compared before and after physiological conditioning. It was shown that rubbing was the mode of failure under dynamic loading. Tensile forces up to 2558N did not result in material failure. Thus, using an artificial cord together with this fixation device, temporary sufficient stable fixation can be expected.
Human adult cartilage is an aneural and avascular type of connective tissue, which consequently reflects reduced growth and repair rates. The main cell type of cartilage are chondrocytes, previously derived from human mesenchymal stem cells (hMSCs). They are responsible for the production and maintainance of the cartilaginous extracellular matrix (ECM), which consists mainly of collagen and proteoglycans. Signal transmission to or from chondrocytes, generally occurs via interaction with signalling factors connected to the cartilaginous ECM. In this context, proteins of the CCN family were identified as important matricellular and multifunctional regulators with high significance during skeletal development and fracture repair. In this thesis, main focus lies on WISP1/CCN4, which is known as a general survival factor in a variety of cell types and seems to be crucial during lineage progression of hMSCs into chondrocytes. We intend to counter the lack of knowledge about the general importance of WISP1-signalling within the musculoskeletal system and especially regarding cell death and survival by a variety of molecular and cell biology methods. First, we established a successful down-regulation of endogenous WISP1 transcripts within different cell types of the human musculoskeletal system through gene-silencing. Interestingly, WISP1 seems to be crucial to the survival of all examined cell lines and primary hMSCs, since a loss of WISP1 resulted in cell death. Bioinformatical analyses of subsequent performed microarrays (WISP1 down-regulated vs. control samples) confirmed this observation in primary hMSCs and the chondrocyte cell line Tc28a2. Distinct clusters of regulated genes, closely related to apoptosis induction, could be identified. In this context, TRAIL induced apoptosis as well as p53 mediated cell death seem to play a crucial role during the absence of WISP1 in hMSCs. By contrast, microarray analysis of WISP1 down-regulated chondrocytes indicated rather apoptosis induction via MAPK-signalling. Despite apoptosis relevant gene regulations, microarray analyses also identified clusters of differentially expressed genes of other important cellular activities, e.g. a huge cluster of interferon-inducible genes in hMSCs or gene regulations affecting cartilage homeostasis in chondrocytes. Results of this thesis emphasize the importance of regulatory mechanisms that influence cell survival of primary hMSCs and chondrocytes in the enforced absence of WISP1. Moreover, findings intensified the assumed importance for WISP1-signalling in cartilage homeostasis. Thus, this thesis generated an essential fundament for further examinations to investigate the role of WISP1-signalling in cartilage homeostasis and cell death.
Mechanical forces are translated into biochemical signals and contribute to cell differentiation and phenotype maintenance. Mesenchymal stem cells and their tissuespecific offspring, as osteoblasts and chondrocytes, cells of cardiovascular tissues and lung cells are sensitive to mechanical loading but molecules and mechanisms involved have to be unraveled. It is well established that cellular mechanotransduction is mediated e.g. by activation of the transcription factor SP1 and by kinase signaling cascades resulting in the activation of the AP1 complex. To investigate cellular mechanisms involved in mechanotransduction and to analyze substances, which modulate cellular mechanosensitivity reporter gene constructs, which can be transfected into cells of interest might be helpful. Suitable small-scale bioreactor systems and mechanosensitive reporter gene constructs are lacking. To analyze the molecular mechanisms of mechanotransduction and its crosstalk with biochemically induced signal transduction, AP1 and SP1 luciferase reporter gene constructs were cloned and transfected into various cell lines and primary cells. A newly developed bioreactor and small-scale 24-well polyurethane dishes were used to apply cyclic stretching to the transfected cells. 1 Hz cyclic stretching for 30 min in this system resulted in a significant stimulation of AP1 and SP1 mediated luciferase activity compared to unstimulated cells. In summary we describe a small-scale cell culture/bioreactor system capable of analyzing subcellular crosstalk mechanisms in mechanotransduction, mechanosensitivity of primary cells and of screening the activity of putative mechanosensitizers as new targets, e.g. for the treatment of bone loss caused by both disuse and signal transduction related alterations of mechanotransduction.
Ruptures of the anterior cruciate ligament (ACL) and defects of the rotator cuff represent the most common ligament and tendon injuries in knee and shoulder. Both injuries represent significant implications for the patients. After an injury, the ACL and the rotator cuff both exhibit poor intrinsic healing capacities. In order to prevent further defects such as arthritis of the knee and fatty infiltration of the rotator cuff, surgical interaction is essential. In both cases, the currently used surgical techniques are far from optimal because even after the therapy many patients report problems ranging from pain and reduced mobility to complete dysfunction of the involved joint and muscles. Tissue engineering may be a possible solution. It is a promising field of regenerative medicine and might be an advantageous alternative for the treatment of musculoskeletal injuries and diseases in the near future. In this thesis, different tissue engineering based approaches were investigated. For the reconstruction of damaged or diseased ligaments and tendons, the use of MSCs and gene therapy with growth factors is especially suitable and possesses a great therapeutic potential. Therefore, the first method studied and tested in this thesis was the development of a biomaterial based construct for the repair of a ruptured ACL. The second approach represents a cell based strategy for the treatment of the fatty infiltration in the rotator cuff. The third approach was a combined cell, biomaterial, and growth factor based strategy for ACL ruptures. Biomaterial based ACL construct The implant is currently tested in a preclinical in vivo study in mini pigs. This proof-of-principle study is performed to validate the functional capability of the collagen fiber based implant under load in vivo and its population with fibroblasts which produce a ligamentogenic matrix. Cell based treatment of the fatty infiltration in the rotator cuff Regarding the treatment of the fatty infiltration of the rotator cuff in a rabbit model, the in vivo results are also promising. The group treated with autologous MSCs (+MSC group) showed a lower fat content than the untreated group (–MSC group) 6 weeks after the treatment. Furthermore, the SSP muscle of the MSC-treated animals revealed macroscopically and microscopically only few differences compared to the healthy control group. The exact underlying mechanisms leading to the positive results of the treatment are not yet fully understood and have therefore to be further investigated in the future. Cell, biomaterial, and growth factor based treatment of ACL ruptures Studies described in current literature show that collagen hydrogel scaffolds are not ideal for a complete ligament or tendon reconstruction, because of their insufficient mechanical stability. Introduced as an alternative and superior therapy, the combined strategy used in this thesis proves that the cultivation of BMP-12, -13, and IGF-1 transduced MSCs and ACL fibroblasts in a collagen hydrogel is successful. The results of the performed in vitro study reveal that the cells exhibit a fibroblastic appearance and produce a ligamentogenic matrix after 3 weeks. Furthermore, the adenoviral transduction of MSCs and ACL fibroblasts showed no negative effects on proliferation or viability of the cells nor was apoptosis caused. Therefore, the application of these cells represents a possible future therapy for a partial ligament and tendon rupture where the mechanical stability of the remaining ligament or tendon is sufficient and the healing can be improved substantially by this therapy. In general, prospective randomized clinical trials still have to prove the postulated positive effect of MSCs for the treatment of various musculoskeletal diseases, but the results obtained here are already very promising. Ideally, the treatment with MSCs is superior compared to the standard surgical procedures. Because of current safety issues the use of genetically modified cells cannot be expected to be applied clinically in the near future. In summary, the different tissue engineering approaches for novel therapies for musculoskeletal injuries and diseases invested in this thesis showed very promising results and will be further developed and tested in preclinical and clinical trials.
Chondrogenic differentiation of human mesenchymal stem cells and articular cartilage reconstruction
(2008)
Articular cartilage defects are still one of the major challenges in orthopedic and trauma surgery. Today, autologous chondrocyte transplantation (ACT), as a cell-based therapy, is an established procedure. However, one major limitation of this technique is the loss of the chondrogenic phenotype during expansion. Human mesenchymal stem cells (hMSCs) have an extensive proliferation potential and the capacity to differentiate into chondrocytes when maintained under specific conditions. They are therefore considered as candidate cells for tissue engineering approaches of functional cartilage tissue substitutes. First in this study, hMSCs were embedded in a collagen type I hydrogel to evaluate the cartilaginous construct in vitro. HMSC collagen hydrogels cultivated in different culture media showed always a marked contraction, most pronounced in chondrogenic differentiation medium supplemented with TGF-ß1. After stimulation with chondrogenic factors (dexamethasone and TGF-ß1) hMSCs were able to undergo chondrogenesis when embedded in the collagen type I hydrogel, as evaluated by the temporal induction of cartilage-specific gene expression. Furthermore, the cells showed a chondrocyte-like appearance and were homogeneously distributed within a proteoglycan- and collagen type II-rich extracellular matrix, except a small area in the center of the constructs. In this study, chondrogenic differentiation could not be realized with every hMSC preparation. With the improvement of the culture conditions, e.g. the use of a different FBS lot in the gel fabrication process, a higher amount of cartilage-specific matrix deposition could be achieved. Nevertheless, the large variations in the differentiation capacity display the high donor-to-donor variability influencing the development of a cartilaginous construct. Taken together, the results demonstrate that the collagen type I hydrogel is a suitable carrier matrix for hMSC-based cartilage regeneration therapies which present a promising future alternative to ACT. Second, to further improve the quality of tissue-engineered cartilaginous constructs, mechanical stimulation in specific bioreactor systems are often employed. In this study, the effects of mechanical loading on hMSC differentiation have been examined. HMSC collagen hydrogels were cultured in a defined chondrogenic differentiation medium without TGF-ß1 and subjected to a combined mechanical stimulation protocol, consisting of perfusion and cyclic uniaxial compression. Bioreactor cultivation neither affected overall cell viability nor the cell number in collagen hydrogels. Compared with non-loaded controls, mechanical loading promoted the gene expression of COMP and biglycan and induced an up-regulation of matrix metalloproteinase 3. These results circumstantiate that hMSCs are sensitive to mechanical forces, but their differentiation to chondrocytes could not be induced. Further studies are needed to identify the specific metabolic pathways which are altered by mechanical stimulation. Third, for the development of new cell-based therapies for articular cartilage repair, a reliable cell monitoring technique is required to track the cells in vivo non-invasively and repeatedly. This study aimed at analyzing systematically the performance and biological impact of a simple and efficient labeling protocol for hMSCs. Very small superparamagnetic iron oxide particles (VSOPs) were used as magnetic resonance (MR) contrast agent. Iron uptake was confirmed histologically with prussian blue staining and quantified by mass spectrometry. Compared with unlabeled cells, VSOP-labeling did neither influence significantly the viability nor the proliferation potential of hMSCs. Furthermore, iron incorporation did not affect the differentiation capacity of hMSCs. The efficiency of the labeling protocol was assessed with high resolution MR imaging at 11.7 Tesla. VSOP-labeled hMSCs were visualized in a collagen type I hydrogel indicated by distinct hypointense spots in the MR images, resulting from an iron specific loss of signal intensity. This was confirmed by prussian blue staining. In summary, this labeling technique has great potential to visualize hMSCs and track their migration after transplantation for articular cartilage repair with MR imaging.
CYR61 and WISP3 belong to the family of CCN-proteins. These proteins are characterised by 10% cysteine residues whose positions are strictly conserved. The proteins are extracellular signalling molecules that can be associated with the extracellular matrix. CCN-proteins function in a cell- and tissue specific overlapping yet distinct manner. CCN-proteins are expressed and function in several cells and tissues of the musculoskeletal system. In this study the impact of the angiogenic inducer cysteine-rich protein 61 (CYR61/CCN1) on endothelial progenitor cells (EPCs) and mesenchymal stem cells (MSCs) as well as the wnt1 inducible signalling pathway protein 3 (WISP3/CCN6) on MSCs were elucidated. EPCs are promising cells to induce neovascularisation in ischemic regions as tissue engineered constructs. A major drawback is the small amount of cells that can be obtained from patients; therefore a stimulating factor to induce in vitro propagation of EPCs is urgently needed. In this study, mononuclear cells obtained from peripheral blood were treated with 0.5 µg/ml CYR61, resulting in an up to 7-fold increased cell number within one week compared to untreated control cells. To characterise if EPCs treated with CYR61 display altered or maintained EPC phenotype, the expression of the established markers CD34, CD133 and KDR as well as the uptake of acLDL and concurrent staining for ulex lectin was analysed. Both CYR61 treated and untreated control cells displayed EPCs characteristics, indicating that CYR61 treatment induces EPC number without altering their phenotype. Further studies revealed that the stimulating effect of CYR61 on EPCs is due to enhanced adhesion, rather than improved proliferation. Usage of mutated CYR61-proteins showed that the adhesive effect is mediated, at least partly, by the integrin α6β1, while the integrin αυβ3 has no influence. Endogenous expression of CYR61 was not detectable in EPCs, which indicated that control cells are not influenced by endogenous secretion of CYR61 and also could explain the dose-dependent effect of CYR61 that is measured at a low concentration of 0.05 µg/ml. MSCs were treated with 0.5 µg/ml CYR61, a combination of growth factors including VEGF, both together and compared to untreated control cells. Matrigel angiogenesis assay revealed an induction of angiogenesis, detected by induced sprouting of the cells, after CYR61 treatment of the MSC. Induced sprouting and vessel like structure formation after CYR61 treatment was similar to the results obtained after treatment with growth factors including the established angiogenesis inducer VEGF. This result clearly demonstrates the angiogenic potential of CYR61 on MSCs. Further studies revealed a migrative and proliferative effect of CYR61 on MSCs. Both properties are crucial for the induction of angiogenesis thus further strengthening the view of CYR61 as an angiogenic inducer. MSCs and EPCs are promising cells for tissue engineering applications in bone remodelling and reconstruction. MSCs due to their potential to differentiate into other lineages; EPCs induce neovascularisation within the construct. Both cell types respond to CYR61 treatment. Furthermore EPCs home to sides were CYR61 expression is detectable and both are induced by similar stimulators. Therefore CYR61 is a promising factor for tissue engineered bone reconstruction applications. WISP3 is expressed in cartilage in vivo and in chondrocytes in vitro. Loss of function mutations in the WISP3 gene are associated to the inherited human disease progressive pseudorheumatoid dysplasia (PPD), that is characterised by cartilage loss and bone and joint destruction. Since MSCs also express the protein, the aim of this study was to elucidate if recombinant protein targets MSCs. A migratory effect of WISP3 treatment on MSCs and osteogenic differentiated MSCs has been proven in this study. To elucidate if global gene expression patterns are influenced by WISP3, cells were treated with 0.5 µg/ml WISP3 and compared to untreated control MSCs. Gene expression study by using affymetrix technology revealed an induction of interferon inducible genes including CXCL chemokines and members of the TNFSF family. Reevaluation by RT-PCR on identical RNA and an additional time series confirmed the results. Although no established cartilage associated genes were detected as regulated genes within this 24h treatment, anti-angiogenic and immunosuppressive genes indicate a protective role of WISP3 for the cartilage, which is sensitive to inflammatory processes. Both CCN-proteins CYR61 and WISP3 are valuable for the musculoskeletal system. This and previous studies revealed the role of CYR61 for osteogenesis and angiogenesis of tissue engineered applications. WISP3 is responsible for development, protection and maintenance of cartilage. Therefore further studies with the proteins in the musculoskeletal system are of high relevance.
The goal of the project was to establish knock down of mRNA in human mesenchymal stem cells. Since these cells are difficult to transfect, a viral approach is needed to achieve sufficient expression of e. g. shRNA in a high percentage of cells to allow for an efficient silencing of corresponding mRNAs. For this purpose for every gene product of interest, a number of shRNA clones have to be tested to detect an individual shRNA with sufficient efficacy. Lentiviral systems for shRNA approaches have recently become available. The principal advantage of the lentiviral system is that it allows gene silencing in nondividing cells and therefore expands the usefulness of the RNAi-based gene silencing system. Lentivirus-delivered shRNAs are capable of specific, highly stable and functional silencing of gene expression in a variety of cell types. Since the viral transfection of MSCs is a time consuming process that involves transfection of 293 FT cells plus transduction of target cells, for this thesis the following approach was chosen: genes of interest were checked for expression in 293FT cells by RT-PCR. These gene products can be silenced in 293FT cells simply by transfection of shRNA clones and efficacy was subsequently tested by RT-PCR. Beyond this thesis then the project can proceed with effective clones to transduce primary MSCs with individual shRNA clones identified as effective silencing tool in this thesis.
With ageing, the loss of bone mass correlates with the expansion of adipose tissue in human bone marrow thus facilitating bone-related diseases like osteopenia and osteoporosis. The molecular mechanisms underlying these events are still largely unknown. Reduced osteogenesis and concurrently enhanced adipogenesis might not only occur due to the impairment of conventional osteogenic differentiation originating from mesenchymal stem cells (MSCs). Additionally, transdifferentiation of (pre-)osteoblasts into adipocytes could contribute to the fatty conversion. Therefore, the aim of the present study was to prove the existence of transdifferentiation between the adipogenic and osteogenic lineage and to elucidate molecular mechanisms underlying this phenomenon. At first, a cell culture system of primary human MSCs was established that allowed for differentiation into the adipogenic and osteogenic lineage and proved that the MSC-derived adipocytes and pre-osteoblasts were capable of transdifferentiation (reprogramming) from one into the other lineage. Thereby, lineage-specific markers were completely reversed after reprogramming of pre-osteoblasts into adipocytes. The osteogenic transdifferentiation of adipocytes was slightly less efficient since osteogenic markers were present but the adipogenic ones partly persisted. Hence, plasticity also reached into the differentiation pathways of both lineages and the better performance of adipogenic reprogramming further supported the assumption of its occurrence in vivo. The subsequent examination of gene expression changes by microarray analyses that compared transdifferentiated cells with conventionally differentiated ones revealed high numbers of reproducibly regulated genes shortly after initiation of adipogenic and osteogenic reprogramming. Thereof, many genes were correlated with metabolism, transcription, and signal transduction as FGF, IGF, and Wnt signalling, but only few of the established adipogenesis- and none of the osteogenesis-associated marker genes were detected within 24 h after initiation of transdifferentiation. To find possible key control factors of transdifferentiation amongst the huge amount of regulated genes, a novel bioinformatic scoring scheme was developed that ranked genes due to their potential relevance for reprogramming. Besides the reproducibility and level of their regulation, also the possible reciprocity between the adipogenic and osteogenic transdifferentiation pathway was taken into account. Fibroblast growth factor 1 (FGF1) that ranked as one of the leading candidates to govern reprogramming was proven to inhibit adipogenic differentiation as well as adipogenic transdifferentiation in our cell culture system. Further examination of the FGF signalling pathway and other highly ranked genes could help to better understand the age-related fatty degeneration at the molecular level and therefore provide target molecules for therapeutic modulation of the plasticity of both lineages in order to inhibit adipogenic degeneration and to enhance osteogenesis.