Abstract: Adult adipose-derived stem cells (ASCs) are considered to be an alternative cell source for cell-based cartilage repair because of their multiple differentiation potentials. This article addresses the chondrogenic differentiation of ASCs seeded into poly-lactide-co-glycolide (PLGA) scaffolds after implantation in a subcutaneous pocket of nude mice. Human ASCs were seeded into PLGA (polylactic acid:polyglycolic acid = 90:10) scaffolds and cultured in transforming growth factor beta 1 (TGF-beta1)-containing medium for 3 weeks in vitro. Then specimens were implanted into a subcutaneous pocket of severe combined immunodeficiency mice and harvested after 8 weeks. Chondrospecific messenger RNA (mRNA) expression was analyzed using reverse transcriptase polymerase chain reaction. Corresponding extracellular matrix (ECM) synthesis was demonstrated using immunohistochemical staining. Chondrospecific marker molecules such as collagen type II and type X, cartilage oligomeric matrix protein, and aggrecan subsequently increased during the 3 weeks period in vitro. After a further 8 weeks, in vivo samples pretreated with TGF-beta1 continued expressing collagen type II and aggrecan mRNA, and collagen type II was found within the ECM using immunohistochemistry. Chondrospecific mRNA was not detected in control samples. ASC-seeded PLGA scaffolds express a stable chondrogenic phenotype in a heterotopic model of cartilage transplantation and represent a suitable tool for tissue engineering of cartilage.

Abstract: INTRODUCTION: Although autologous chondrocyte implantation (ACI) has become well established for the treatment of full-thickness cartilage defects of the knee joint, nevertheless clinical results of retropatellar lesions are still inferior compared to those of defects located on femoral condyles. We report the clinical results obtained in 70 patients treated with ACI for full-thickness defects of the patella, with special reference to defect location and size, age, body mass index and sports activity. METHODS: At a follow-up of 38.4 months (range 14-64, follow-up rate 83.3%), patients' subjective functional knee scores (IKDC, Lysholm) were analysed, as were the results of objective examination (according to ICRS). RESULTS: Mean patient age at the time of surgery was 34.3 years (+/-10.1). The mean Lysholm score at the time of follow-up was 73.0 (+/-22.4) and the subjective IKDC score was 61.6 (+/-21.5); normal and nearly normal clinical results according to the objective criteria of the International Cartilage Research Society (ICRS) were achieved in 67.1% of the patients, while abnormal results were achieved in 20.0% of the patients and severely abnormal results, in 12.9%. While different surgical techniques did not seem to have any significant influence on the treatment results, both defect size and defect location within the patella were found to be significantly associated with clinical outcome. The corollaries to this are that larger cartilage lesions of the patella are associated with an inferior outcome (p = 0.007) and that cartilage defects located on the lateral patellar facet are correlated with a better clinical outcome than those located on the medial facet or those involving both facets (p = 0.017). CONCLUSION: This study demonstrates that within a group of patients treated with ACI for retropatellar cartilage lesion there are significant differences in clinical outcome, which are important and should be taken into account of when a decision has to be made on whether or not ACI is indicated.

Abstract: Survival of ex vivo constructed tissues after transplantation is limited by insufficient oxygen and nutrient supply. Therefore, strategies aiming at improvement of neovascularization of engineered tissues are a key issue in tissue engineering applications. This in vitro study aimed at exploring the usability of osteogenically differentiated human mesenchymal stem cells (MSCs) as carriers of the angiogenic growth factor genes vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) for therapeutic angiogenesis in bone tissue engineering. The ex vivo adenoviral vector mediated transduction into osteogenically differentiated MSCs revealed a highly efficient and long lasting expression of the transgenes. Biological activity of VEGF and Ang-1 secreted from transduced cells was confirmed by analyzing the sprouting, proliferation and apoptosis of human umbilical vein endothelial cells (HUVECs) in response to conditioned medium obtained from transduced cells. The transduced osteogenically differentiated MSCs described in this report may be suitable for inducing neovascularization in bone tissue engineering applications.

Abstract: The activity of a tartrat-resistant acid phosphatase 5B (TRACP 5b), a marker of osteoclast function, was quantified in osteoarthritic bone specimens from patients treated with Alendronate. Prior to total hip replacement, 12 patients were randomized in a bisphosphonate and a control group. The bisphosphonate group received daily oral Alendronate for 50 days before operation. After operation, the femoral heads were harvested. Samples of the anterior femoral head (A1) and the intertrochanteric area (A2) were taken, analyzed with an immunoassay and stained for TRACP 5-positive-cells. The immunoassay revealed that TRACP-5b activity of the bisphosphonate group was significantly increased in A1 compared to A2, but not of the control group. Bisphosphonate treatment decreased enzyme activity compared to the controls: 0.41 U/mg vs. 0.31 U/mg in A1 and 0.26 U/mg vs. 0.18 U/mg in A2 (p<0.05). The histological examination shows significantly less TRACP-positive cells in bisphosphonate-treated bone sections, confirming the results. Our data suggest that bisphosphonates reduce TRACP 5b activity in the intertrochanteric area rather than in the anterior femoral head. Consequently, they are more effective in areas of well-supplied bone than in osteoarthritic bone tissue.

Abstract: Proximal humerus fractures represent an increasing challenge for the health system due to epidemiological changes. As estimated by a Finnish study group the number of fractures may triple by the year 2030. The majority of patients with these fractures are older than 60 years and in this population most of the proximal humerus fractures have been related to osteoporosis. Nondisplaced fractures and fractures with minimal displacement and adequate stability are usually successfully treated non-operatively. The main challenge in the operative treatment of displaced and unstable proximal humerus fractures is to achieve effective stabilization of an adequately reduced fracture to maximize the functional patient outcome. Especially in osteoporotic bone and comminuted fractures operative stabilization is challenging and the management of displaced and unstable fractures remains controversial. The most important factor for favourable results in the treatment of complex three-part or four-part humerus fractures is anatomic reduction. Minimal exposure, high primary stability, and load transfer through the implant are important for avoiding complications such as secondary dislocation, osteonecrosis, and stiffness. Recently invented implants with angular stability provide better biomechanical properties and enhanced anchorage especially in the osteoporotic bone. These implants therefore have a potential for achieving better results in the treatment of these complex injuries.

Abstract: INTRODUCTION: Mesenchymal stromal cells (MSC) represent an attractive cell population for tissue engineering purposes. As MSC are described as immunoprivileged, non-autologous applications seem possible. A basic requirement is the survival of MSC after transplantation in the host. The purpose of the current paper was to evaluate the survival of undifferentiated and osteogenically induced human MSC from different origins after transplantation in immunocompetent mice. METHODS: Human MSC were isolated from bone marrow (BMSC) and adipose tissue (ASC). After cultivation on mineralized collagen, MSC were transplanted subcutaneously into immunocompetent mice (n=12). Undifferentiated MSC (group A) were compared with osteogenic-induced MSC (group B). Human-specific in situ hybridization and anti-vimentin staining was used to follow MSC after transplantation. Quantitative evaluation of lymphocytes and macrophages was performed as a measure of immunologic rejection. Unloaded scaffolds served as controls (group C). Specimens were harvested at 4 and 8 weeks. RESULTS: Undifferentiated BMSC and ASC were detected in the majority of cases after xenogenic transplantation (group A, a total of 22 out of 24 cases), while osteogenic-induced MSC (group B) could be detected in only three of 24 cases. Quantification of lymphocytes and macrophages revealed significantly higher cell numbers in group B compared with group A (P<0.05). DISCUSSION: Our results suggest that undifferentiated MSC are candidates for non-autologous cell transplantation, while osteogenic-induced MSC seem to be eliminated by the host's immune system. This observation seems independent of the origin of MSC and applies to BMSC and ASC.

Abstract: PURPOSE: The combination of ipsilateral femoral neck and shaft fractures remains a treatment challenge in orthopedic surgery because both fracture types constitute separate entities and require specific treatment concepts. MATERIAL AND METHODS: In a case control study, incidence, treatment strategies, and outcomes of this injury were analyzed. All patients with femoral fractures treated between 1 January 2001 and 31 July 2007 at a level I trauma center were included in the study. RESULTS: Twenty-one out of 1,935 patients (1.1%) sustained 22 combined fractures of the femoral neck and shaft. Also considering the combination of femoral shaft fractures with fractures of the acetabulum and the distal femur (knee), the proportion of chain injuries of the femur was 3.1%. The rate of multiply injured patients in the group of patients with ipsilateral femoral neck and shaft fractures was 64%. The majority of the patients could be treated with a single implant for both fracture components. The leading fracture component was the femoral neck fracture in eight cases. All fractures consolidated after 4.7 months on average; one pseudarthrosis of the femoral neck was observed. All fractures were discovered in the course of primary diagnostic measures; in 73% of the patients, a computed tomography (CT) body scan was done. Fifty-nine percent of the patients with ipsilateral femoral neck and shaft fractures received primary definitive operative care. Complications included two torsional failures that needed correction and one case of postoperative infection that was easily treated. CONCLUSION: Treatment of ipsilateral femoral neck and shaft fractures is still demanding, but diagnosis has improved with regular use of CT body scans in the management of multiply injured patients. Furthermore, possibilities for operative treatment have been advanced by the introduction of the long proximal femoral nail and the antegrade femoral nail, two implants supporting stabilization of these fracture entities.

Abstract: BACKGROUND: The matrix component in autologous chondrocyte implantation plays an important role. In this study the influence of an additional fibrin component in cartilage constructs based on polyglycolide polymers (PGA) was investigated. METHODS: Human chondrocytes of femoral heads were isolated and cultured using a serum-free technique. The cells were seeded on PGA-91 scaffolds with and without an additional fibrin component; the constructs were cultured for 2 weeks in vitro. Besides cell viability, DNA content, pH, aggrecan production, mRNA expression of aggrecan, and collagen types I and II were determined by real-time PCR. Furthermore, cartilage grafts were histologically analyzed. RESULTS: All constructs contained viable, metabolically active cells in the investigated time period. There was no cell proliferation within the graft, and the DNA content was decreased over time. The pH level constantly remained within a physiologic range. The Alcian blue staining of the constructs showed the homogeneous cell distribution and a cell-associated proteoglycan production. Aggrecan concentration in the supernatants of fibrin-containing constructs was significantly lower compared to fibrin-free grafts (-24%), a result that correlated with diminished aggrecan mRNA expression (-80%). mRNA expression of collagen type II increased in the fibrin-free constructs over time and was 57% higher than in the fibrin-containing grafts. The immunohistochemical detection of collagen type II was possible in all constructs. CONCLUSION: Cartilage constructs based on carbohydrate matrices are suitable for matrix-associated chondrocyte implantation. The results of this study suggest a partially inhibitory effect of an additional fibrin component in PGA constructs for chondrogenic differentiation.

Abstract: BACKGROUND: The possible functional role of basic fibroblast growth factor (bFGF) in regulating the mitotic and metabolic activity of primary human articular chondrocytes was investigated. METHODS: [EF1]Chondrocytes were enzymatically isolated from femoral head cartilage, and were cultured in vitro in monolayer. bFGF-dependent cell proliferation, production of collagen type II and aggrecan were monitored 10 days after isolation. Furthermore, effect of bFGF on cell cycle, cell morphology, and mRNA expression of integrins and chondrogenic markers determined by real time PCR were analyzed. RESULTS: bFGF concentrations in supernatants of primary human articular chondrocytes peaked immediately after isolation and then declined. In a dose-dependent manner, bFGF enhanced cell amplification and viability. BFGF induced a decrease in the apoptotic cell population, while the number of proliferating cells remained unchanged. Supplementation of cell culture with bFGF reduced collagen type II mRNA by 49%, but increased expression of the integrin alpha(2) by 70%. bFGF did not significantly regulate the integrins alpha(1), alpha(5), alpha(10), alpha(v) and type I collagen. bFGF reduced the amount of collagen type II by 53%, which was correlated with diminished mRNA production. Monolayer cultured chondrocytes secreted significant amounts of aggrecan that decreased over time. Secretion of this cartilage-specific marker was further reduced by the addition of bFGF. DISCUSSION: These findings highlight the potential role of bFGF as an endogenous chondrocyte mediator that can enhance cell amplification and regulate cell differentiation.

Abstract: BACKGROUND: Human mesenchymal stromal cells (MSC) and PBMC play significant roles in repair processes following inflammation. Mechanisms of recruitment are still under investigation. METHODS AND RESULTS: MIP-1alpha induced the chemotactic migration of MSC but not of PBMC. Correlating with this, 7.7% of MSC expressed the chemokine receptor CCR-1, as shown by FACS analysis. In contrast, PBMC did not express CCR-1 or CCR-2 but did express CXCR-4 (81.9%) and CCR-7 (42.2%). Setum induced the chemotaxis of both cell types, and zymosan activation increased the migration of PBMC but not of MSC. Corresponding with this, C5a induced the migration of PBMC but not of MSC. Dose-dependent and -specific adhesion to fibronectin, fibrinogen, collagen type I and collagen type II could be demonstrated for MSC; in contrast, PBMC did not adhere to any of the investigated proteins. Real-time PCR of receptor expression revealed a 12.2-fold higher expression of alphav in MSC compared with PBMC. Incubation of MSC with tumor necrosis factor-alpha (TNFalpha) induced NFkappaB activation and increased the chemotactic response to serum and adhesion to fibronedtin. DISCUSSION: Chemotaxis and adhesion are crucial and differing cell fundtons of MSC and PBMC.

Abstract: OBJECTIVES: This article addresses the interaction of transforming growth factor beta1 (TGF-beta1) and bone morphogenic protein 2 (BMP-2) during osteo-chondrogenic differentiation of adipose-derived adult stem cells (ASC). TGF-beta1 was expected to modulate the BMP-2-induced effects through transcriptional regulation of Dlx-5, Msx-2 and Runx-2. MATERIALS AND METHODS: Encapsulated ASC were cultured for 14 days in medium containing TGF-beta1 and/or BMP-2. mRNA expression of the extracellular matrix molecules col2a1, cartilage oligomeric matrix protein, col10a1, alkaline phosphatase (AP) and transcription factors Msx-2, Dlx-5 and Runx-2 was analysed. Release of glycosaminoglycans, collagen types II and X into the extracellular matrix was demonstrated. RESULTS: BMP-2 and TGF-beta1 induced a chondrogenic phenotype in ASC. Combined growth factor treatment had a synergistic effect on col10a1 and an additive effect on col2a1 mRNA expression. Synthesis of glycosaminoglycans was enhanced by combined growth factor treatment. Addition of TGF-beta1 inhibited BMP-2 induced AP expression and activity and both proteins promoted chondrogenic maturation. CONCLUSIONS: Prevention of BMP-2-induced osteogenic transdifferentiation by TGF-beta1 seemed not to be mediated by transcriptional regulation of Dlx-5. Due to these findings, simultaneous stimulation of ASC with BMP-2 and TGF-beta1 seemed to be beneficial for complete differentiation of ASC into chondrocytes.

Abstract: BACKGROUND: Asthma is characterized by a T(H)2 immune response. CD4(+)CD25(hi) regulatory T cells (Tregs) have been proposed to prevent allergic diseases through suppression of T(H)2 responses. OBJECTIVE: We sought to investigate the role of CD4(+)CD25(hi) T cells in children with asthma. METHODS: CD4(+)CD25(hi) Tregs and forkhead/winged-helix transcription factor FOXP3 mRNA levels were quantified in peripheral blood and bronchoalveolar lavage fluid (BALF) of 18 children with asthma, 10 children with chronic cough, and 13 control subjects without lung diseases. CD4(+)CD25(hi) T cells were isolated from peripheral blood and BALF of asthmatic patients and control subjects, and their capacity to suppress proliferation and cytokine/chemokine production of autologous responder T cells was analyzed. RESULTS: CD4(+)CD25(hi) T cells were decreased in BALF of asthmatic children compared with values in children with cough or control subjects. In children with asthma, inhaled corticosteroid treatment was associated with increased percentages of CD4(+)CD25(hi) T cells in peripheral blood and BALF. Isolated BALF and peripheral blood CD4(+)CD25(hi) T cells from nonasthmatic subjects suppressed proliferation and cytokine/chemokine production by CD4(+)CD25(-) responder T cells. BALF CD4(+)CD25(hi) T cells from asthmatic subjects failed to suppress proliferation and production of T(H)2-associated cytokines and chemokines by CD4(+)CD25(-) responder T cells, which was restored after use of inhaled corticosteroids. CONCLUSION: These findings provide the first evidence that pulmonary CD4(+)CD25(hi) Tregs are impaired in pediatric asthma. CLINICAL IMPLICATIONS: Pulmonary Tregs might represent a therapeutic target in pediatric asthma.

Abstract: Mesenchymal stem cells (MSCs) can be isolated from various tissues and represent an attractive cell population for tissue-engineering purposes. MSCs from bone marrow (bone marrow stromal cells [BMSCs]) are negative for immunologically relevant surface markers and inhibit proliferation of allogenic T cells in vitro. Therefore, BMSCs are said to be available for allogenic cell therapy. Although the immunological characteristics of BMSCs have been the subject of various investigations, those of stem cells isolated from adipose tissue (ASCs) have not been adequately described. In addition, the influence of osteogenic differentiation in vitro on the immunological characteristics of BMSCs and ASCs is the subject of this article. Before and after osteogenic induction, the influence of BMSCs and ASCs on the proliferative behavior of resting and activated allogenic peripheral blood mononuclear cells (PBMCs) was studied as a measure of the immune response (mixed lymphocyte culture). At the same points, the expression of immunologically relevant surface markers (e.g., major histocompatibility complex (MHC)-I, MHC-II, CD40, CD40L) was measured, and correlations between the different sets of results were sought. The pattern of surface antigen expression of BMSCs is the same as that of ASCs. Analogous to BMSCs, undifferentiated cells isolated from adipose tissue lack expression of MHC-II; this is not lost in the course of the osteogenic differentiation process. In co-culture with allogenic PBMCs, both cell types fail to lead to any significant stimulation, and they both retain these characteristics during the differentiation process. BMSCs and ASCs suppress proliferation on activated PBMCs before and after osteogenic differentiation. Our results confirm that MSCs are immune modulating cells. These properties are retained even after osteogenic induction in vitro and seem to be similar in BMSCs and ASCs. Our results suggest that allogenic transplantation of BMSCs and ASCs would be possible, for example, in the context of tissue engineering.

Abstract: BACKGROUND: Mesenchymal stromal cells (MSC) isolated from adult human BM are characterized by their fibroblast-like morphology, adherent growth and capacity to differentiate into adipocytes, osteocytes, chondrocytes, cardiomyocytes and neuroprogenitors. After culturing these cells in vitro, they express the cell-surface molecules CD44, CD90, SH2 and SH3, and are negative for CD34 and the hematopoietic marker CD45. The aim of this study was to characterize the in vivo phenotype of MSC relative to the expression of CD34 and CD45. METHODS: BM mononuclear cells were stained with Ab against both molecules and separated into the CD34(+), CD34(-), CD45(+) CD34(+), CD45(high+) CD34(-), CD45(med,low+) CD34(-) and CD45(-) CD34(-) subpopulations, which were then cultured under the same conditions and analyzed for growth of MSC. RESULTS: A small population of MSC arose from the CD45(+) CD34(+) fraction, although the majority was obtained from the CD45(-) CD34(-) subpopulation. MSC from all fractions could be differentiated into adipocytes and osteocytes. In addition, MSC from the CD34(+) and CD34(-) fractions were shown to differentiate into chondrocytes. After in vitro culture, MSC from both fractions possessed the same phenotype, which was negative for CD34 and CD45. DISCUSSION: MSC from the CD45(+) CD34(+) fraction change their phenotype under in vitro conditions.

Abstract: Cartilage tissue engineering is applied clinically to cover and regenerate articular cartilage defects. Two bioresorbable nonwoven scaffolds, polyglycolic acid (PGA) and poly(lactic-co-glycolic acid) (PLGA) (90/10 copolymer of L-lactide and glycolide), were seeded with human chondrocytes after initial progeny in a monolayer with a serum-free medium. Two subgroups of nontreated and plasma-treated (using low-pressure plasma technique) scaffolds were investigated. The constructs were cultivated after seeding in six-well plates with serum-free medium for 7 days and implanted subcutaneously into nude mice for 6 and 12 weeks. Chondrogenic differentiations were investigated using immunhistology and reverse transcriptase-polymerase chain reaction. Cell adhesion only differed from 50% to 65% without a significant difference between the groups. During further cultivation for 7 days, the aggrecan synthesis of the seeded constructs was always higher in the PGA groups (p < 0.05). The mRNA gene expression for collagen type II was significantly higher in the PGA groups after 6 and 12 weeks (p < 0.05). A decrease in the expression of collagen type I was investigated in all groups. The expression for collagen type X and cartilage oligomeric matrix protein (COMP) increased in all groups over time. After cell proliferation in serum-free medium, the long-term chondrogenic differentiation in PGA scaffolds in vitro is cartilage specific and may be utilized in cartilage tissue engineering applications.

Abstract: Retropatellar cartilage defects treated with autologous chondrocyte implantation (ACI) are still associated with inferior clinical outcome compared to defects being located on the femoral condyles. This is partly because of the biomechanical characteristics of the patellofemoral section of the joint, in which, in contrast to the medial or lateral compartments of the knee joint, prejudicial shearing forces are dominant. The patellar ridge has a particularly important role in the reduction of these shearing forces. The double eye technique was developed as a modification of ACI with preserving the important patellar ridge for the treatment of retropatellar cartilage defects extending beyond the patellar ridge and involving the medial and lateral retropatellar facets. This technique provides for a separate reconstruction of the medial and the lateral facets by means of ACI, but the ridge region is preserved to maintain the original thickness of cartilage at this point. The present paper describes the "double eye" technique as a modification of autologous chondrocyte transplantation (ACI) for treatment of cartilage defects of the patella, that involve both lateral and medial facets, and gives first clinical results of 11 patients. The average follow-up was 41.6 (+/-15.0) months, and the average age at diagnosis was 40.4 (+/-10.1) years. The Lysholm score, the subjective IKDC score, and the ICRS score were the instruments used to measure the outcome. This paper focuses on the introduction of the double eye technique with preservation of the patella ridge in the treatment of retropatellar cartilage lesion. Nevertheless, first clinical results of 11 patients are given, with an average Lysholm score of 75 (+/-14) points and an average subjective IKDC score of 60 (+/-14). Objective evaluation according to the criteria of the IKDC score showed very good or good treatment results in 9 of the 11 cases, with only 2 poor results. In conclusion, with the double eye modification presented in this paper, the potential for successful results in the treatment of combined cartilage defects of the medial and lateral facets of the patella is high; it takes into account the specific biomechanical properties of the patella ridge. The procedure needs further evaluation in clinical studies involving larger numbers of patients so that the indications can be determined more precisely.

Abstract: Survival of ex vivo constructed tissues after transplantation is limited by insufficient oxygen and nutrient supply. Therefore, strategies aiming at the improvement of neovascularization of engineered tissues are a key issue. A method to enhance graft vascularization is to establish a primitive vascular plexus within the graft before transplantation by the use of cellular-based concepts. To explore the utility of endothelial progenitor cells (EPCs) for the ex vivo vascularization of tissue engineered grafts, we analyzed the in vitro angiogenic properties of this cell type in two different angiogenesis models: the 3-dimensional spheroid sprouting assay and the 2-dimensional matrigel assay. In both assays, EPCs were able to form tubelike structures, resembling early capillaries. This process was significantly enhanced by the addition of angiogenic growth factors. Direct comparison between EPCs and mature endothelial cells, represented by human umbilical vein endothelial cells (HUVECs), revealed that both cell types displayed an almost identical angiogenic potential. Other functional in vitro parameters such as angiogenic growth factor induced cell proliferation and cell survival were investigated as well, revealing a significantly decreased level of apoptosis of EPCs in relation to HUVECs under serum-deprived conditions. The observed survival advantage of EPCs along with the observation that EPCs perform very well in the above mentioned in vitro angiogenesis assays, make them an ideal autologous cell source for vascularization of ex vivo generated tissues. The attractiveness of this cell type for tissue engineering applications is strengthened further by the fact that these cells can be easily isolated from the peripheral blood of patients, thereby eliminating donor site morbidity.

Abstract: Patients suffering form epilepsy have an increased risk for fractures. Beside fractures caused by fall or accident muscles forces alone generated during tonic-clonic seizure can result in severe musculoskeletal injury. Contractions of strong paraspinal muscles can lead to compression fracture of the mid-thoracic spine. We report a patient who had suffered from a tonic-clonic seizure during early morning hours. After a cracking sound the patient woke up in a state of post-ictal disorientation, loss of urine and tongue bite. He was admitted to our facilities with the suspected vertebral fracture albeit he just reported of mild lower back pain. Native X-rays and computer-tomography scans showed instable burst fractures of L2 and L4. The fractures were stabilised with a dorsally instrumented internal fixator from L1 to L5 followed by hemi-laminectomy and ventral spondylodesis. Muscle force alone can result in severe skeletal trauma including vertebral fractures.This example emphasizes the importance of critical examination of patients after grand mal seizures. Seizures-induced injuries can appear clinically asymptomatic and can easily be overseen due to absence of trauma and post-ictal impairment of consciousness.

Abstract: Interaction between chondrocytes and extracellular matrix is considered a key factor in the generation of grafts for matrix-associated chondrocyte transplantation. Therefore, our objective was to study the influence of differentiation status on cellular attachment. Adhesion of chondrocytes to collagen type II increased after removal from native cartilage up to the third day in monolayer in a dose-dependent manner. Following dedifferentiation after the second passage, adhesion to collagen types I (-84%) and II (-46%) decreased, whereas adhesion to fibrinogen (+59%) and fibronectin (+43%) increased. A cartilage construct was developed based on a clinically established collagen type I scaffold. In this matrix, more than 80% of the cells could be immobilized by mechanisms of adhesion, filtration, and cell entrapment. Confocal laser microscopy revealed focal adhesion sites as points of cell-matrix interaction, as well as collagen type II expression in the cartilage graft after two weeks of in vitro cultivation. Basic fibroblast growth factor (bFGF) treated chondrocytes showed increased adhesion to collagen types I and II, fibronectin, and fibrinogen. Attachment to these investigated proteins significantly enhanced cell proliferation. Matrix design in cartilage engineering must meet the biological demands of amplified cells, because adhesion of chondrocytes depends on their differentiation status and is regulated by bFGF.

Abstract: Mesenchymal stem cells (MSC) can be obtained from human bone marrow aspirates and, thanks to their differentiation potential and excellent in vitro culture properties, represent an attractive cell line for the regeneration of mesenchymal tissue. Both in vitro and in vivo, they can differentiate into cartilage, bone, tendons and fat cells, and-in contrast to embryonic stem cells-they are not under ethical scrutiny. Cultured on three-dimensional scaffolds according to the tissue engineering concept, they have already been successfully employed for reconstruction of mesenchymal tissues in numerous studies involving both small and large animal models. Recently, immunological properties of MSC have been investigated by several groups. On the basis of the available literature, MSC have to be referred to as immune privileged, and they seem to be available for HLA-independent cell transplantation. While clinical MSC transplantation has also been successfully performed in pilot studies in humans, numerous points still remain to be clarified, underscoring the need for further intensive research before large-scale clinical application can be contemplated. Only then can it be shown whether the associated high expectations are justified.

Abstract: Adipose-derived adult stem cells (ADASCs) or bone marrow-derived mesenchymal stem cells (BMSCs) are considered as alternative cell sources for cell-based cartilage repair due to their ability to produce cartilage-specific matrix. This article addresses the differential expression pattern of extracellular matrix (ECM) molecules in BMSCs or ADASCs following chondrogenic differentiation. Human BMSCs or ADASCs were encapsulated in alginate and cultured in TGF-beta1-containing medium for 2 or 3 weeks. Chondrospecific mRNA expression was analyzed and alternative splicing of alpha(1)-procollagen type II mRNA was monitored via reverse transcriptase-polymerase chain reaction (RT-PCR). Corresponding ECM synthesis was demonstrated using immunohistochemistry. After chondroinduction, expression of collagen type II, type X, COMP and aggrecan mRNA was 3-15-fold higher than in ADASCs. The type IIA splicing form of alpha(1)-procollagen type II was expressed in both populations, and the type IIB splicing form was exclusively detected in BMSCs. In response to TGF-beta, collagen type II and X were secreted more strongly by BMSCs than by ADASCs. BMSCs express a more mature phenotype than ADASCs after chondroinduction. TGF-beta1 induces alternative splicing of the alpha(1)-procollagen type II transcript in BMSCs, but not in ADASCs. These findings may direct the development of a cell-specific culture environment either to prevent hypertrophy in BMSCs or to promote chondrogenic maturation in ADASCs.

Abstract: This article addresses the stability of chondrogenic phenotype and the transdifferentiation potential of bone marrow-derived mesenchymal stem cells (MSCs) at distinct stages of differentiation. Differentiated MSCs were expected to maintain cartilage-like gene expression pattern in the absence of any chondrogenic growth factor or in the presence of osteogenic signals. MSCs encapsulated in alginate beads were treated with transforming growth factor (TGF)-beta 3 for 3, 6, or 14 days and then cultured in absence of TGF-beta for the remainder of the 2-week culture period. Additionally, cells were cultured in osteogenic medium after TGF-beta-mediated chondroinduction. Gene expression of col2a1, aggrecan, COMP, alkaline phosphatase (AP), and correlating protein synthesis was analyzed. After short-term stimulation with TGF-beta, MSCs maintained a chondrogenic phenotype. Chondrogenic gene expression and protein synthesis directly correlated with the extent of stimulation time and the concentration of TGF-beta. Pretreatment with TGF-beta could prevent AP mRNA expression of encapsulated MSCs. TGF- beta stimulation within the first 3 days of culture seems to be crucial for the expression of a chondrogenic phenotype. Fully differentiated and encapsulated MSCs are not able to transdifferentiate into osteoblasts. These findings give rise to a better understanding of the behavior of cartilage grafts affected by local factors of osteochondral transplantation sites in vivo.

Abstract: Primary hemiarthroplasty of the shoulder is an accepted procedure to treat complex proximal humeral fractures. The goal of this study was to assess the functional outcome in patients treated with hemiarthroplasty using a custom offset shoulder prosthesis, either for an acute four-part fracture of the proximal humerus or following failed primary treatment of a complex humeral fracture. Thirty seven patients were followed up for a mean of 17 months after shoulder replacement (Group A: four-part-fractures; n = 26, Group B: posttraumatic necrosis/non-union after failed primary treatment; n = 11). The Constant-Murley-Score and radiological score according to Neer's classification were used for postoperative functional and radiological assessment. Following hemiarthroplasty, Group A achieved an average Constant Score of 52 and Group B of 46. The pain relief after hemiarthroplasty was about 53% in Group A and only 33% in Group B. The least satisfying partial function was shoulder mobility in both groups. Radiographic evaluation did not correlate with the Constant Score. Patients secondarily treated with arthroplasty seem to have less chance to achieve a satisfying functional outcome compared to those with immediate hemiarthroplasty. These results emphasise the importance of a careful initial decision to select the most appropriate treatment modality in complex fractures of the proximal humerus.

Abstract: Midfoot injuries of children are rare entities and often caused by high energy trauma mechanisms. Foot fractures in children may pose a diagnostic challenge but they usually have a good prognosis. In special cases computed tomography is necessary to find the right diagnosis in addition to plain X-rays. Based on two cases of midfoot injuries, a type II open Lisfranc fracture dislocation and a dislocation of a Chopart's joint, we describe the causes, diagnosis, and possibilities for treatment of juvenile midfoot injuries.

Abstract: BACKGROUND: Tissue engineering using mesenchymal stromal cells (MSC) represents a promising approach for bone regeneration. Nevertheless, the optimal constructs have yet to be determined. It still remains unclear if there is a benefit of in vitro differentiation of MSC prior to transplantation or if undifferentiated MSC hold the optimal potential concerning new tissue formation. METHODS: After isolation and in vitro expansion, MSC were seeded on mineralized collagen sponges and transplanted in a heterotopic SCID mice model (n=12). While group A contained undifferentiated MSC, in group B cells were cultivated for 14 days in vitro under osteogenic conditions prior to implantation. Results were compared with non-loaded scaffolds (group C). Animals were killed for investigation at 4 and at 8 weeks. RESULTS: In situ hybridization demonstrated integration of MSC for up to 8 weeks in groups A and B. Histology revealed significantly more extracellular matrix synthesis in MSC-seeded scaffolds containing calcium phosphate and collagen type I at 4 and 8 weeks after transplantation compared with unloaded controls. At a biochemical level, higher levels of specific alkaline phosphatase expression were detected in MSC-loaded scaffolds (P<0.05). Scaffolds containing undifferentiated and differentiated MSC did not appear to differ in terms of matrix synthesis and protein expression, while the number of avital cells was significant higher in those probes loaded with differentiated MSC (P<0.01). DISCUSSION: The integration of transplanted cells and MSC-associated matrix synthesis encourages the use of MSC-loaded mineralized collagen for tissue engineering of bone. Furthermore, our data suggest that in vitro differentiation of MSC does not have a positive influence in terms of improved matrix synthesis.

Abstract: Soft tissue injuries associated with pelvic fractures are often responsible for compromised haemodynamics. The objective of this study was to clarify what parameters determine patient outcome. In a cohort study, all patients with a pelvic fracture treated between 1991 and 2001 at a Level I trauma center were analysed for associated intrapelvic injuries, classification, severity of trauma, type of intervention and outcome. Of 552 patients with a pelvic fracture who entered the study, 15.5% presented with associated intrapelvic injuries secondary to the fracture (group I). A subgroup of patients with lacerations of branches of the iliac artery was identified as being at high risk for lethal outcome; they represented 4.3% of all patients with pelvic fracture (group II). The overall mortality reached 4.4%; it increased in group I to 15.5%, and in group II to 33.3%. In the subgroup with pelvic arterial haemorrhage (group II), the severity of injury, the proportion of multiple injured patients, the prevalence of unstable fractures and the incidence of sepsis were significantly increased. The only predictive factor for outcome was the amount of blood transfused, suggesting that fast elimination of the bleeding source decides about patient survival.

Abstract: Neovascularization is a critical step in tissue engineering applications since implantation of voluminous grafts without sufficient vascularity results in hypoxic cell death of central tissues. We have developed a three-dimensional spheroidal coculture system consisting of human umbilical vein endothelial cells (HUVECs) and human primary fibroblasts (hFBs) to improve angiogenesis in tissue engineering applications. Morphological analysis of cryosections from HUVEC/hFB cospheroids revealed a characteristic temporal and spatial organization with HUVECs located in the center of the cospheroid and a peripheral localization of fibroblasts. In coculture spheroids, the level of apoptosis of endothelial cells was strongly decreased upon cocultivation with fibroblasts. Collagen-embedded HUVEC spheroids develop numerous lumenized capillary-like sprouts. This was also apparent for HUVEC/hFB cospheroids, albeit to a lesser extent. Quantification of cumulative sprout length revealed an approximately 35% reduction in endothelial cell sprouting upon cocultivation with fibroblasts in cospheroids. The slight reduction in endothelial cell sprouting was not mediated by a paracrine mechanism but is most likely due to the formation of heterogenic cell contacts between HUVECs and hFBs within the cospheroid. The model system introduced in this study is suitable for the development of a preformed lumenized capillary-like network ex vivo and may therefore be useful for improving angiogenesis in in vivo tissue engineering applications.

Abstract: Human epidermal growth factor (hEGF) and basic fibroblast growth factor (bFGF) influence critical characteristics of chondrocytes. The effects on metabolism and differentiation were evaluated following transfection using specific plasmids coding for both cytokines.Chondrocytes were isolated from femoral head cartilage of patients undergoing a hip arthroplasty for femoral neck fracture. Following collagenase-digestion, cells were cultured in monolayers, and cell proliferation, glucosaminoglycan-production and collagen type II expression were monitored 10 days after isolation.Addition of recombinant hEGF and bFGF resulted in a significant increase in cell proliferation and glucosaminoglycan production. Chondrocytes were transfected with vectors coding for either hEGF or bFGF and the production of these proteins was measured in supernatants by ELISA. Expression kinetics showed different patterns: hEGF was detectable 2.5 days following transfection and peaked at day 5.5, whereas bFGF-production reached its maximum 1.5 days after transfection, declining thereafter. Chondrocytes endogenously produced significant amounts of bFGF within 5 days following isolation. Proliferation of hEGF-transfected cells increased up to 81%; bFGF-transfection caused an increase up to 76%. Similarly, glucosaminoglycan-production was enhanced up to 120% by hEGF-transfection and 37% by bFGF transfection, respectively. Collagen type II production decreased following transfection with both plasmids.Temporary in vitro gene transfer of the growth factors hEGF and bFGF provides a method to stimulate chondrocyte proliferation and induces signs of dedifferentiation, which would limit a reasonable clinical application.

Abstract: In high-critical medical fields instant information delivery is essential. Task-flow analyses within the transplantation unit of the Technische Universität München revealed that valuable time could be saved in pre-transplantation management being able to retrieve data of organ receivers ubiquitously. Inspired by this clinical scenario, a mobile application was designed and implemented providing surgeons with decision-relevant information on potential organ receivers. It assists them in considering the prospects of forthcoming organ transplantations and facilitates decision making and documentation with regard to high security demands. The described system services three organ receiver lists and is used by the surgeons in every transplantation procedure. After a 6-month period of clinical usage, the system has been evaluated in terms of handling, clinical benefit and total time savings. Intuitive, ubiquitous access to decision-relevant patient data and authenticated documentation were the major improvements with average total time savings of 50 min in comparison to the old system.

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