Alexander T. Mehlhorn

COMPARISON OF MESENCHYMAL STEM CELLS FROM BONE MARROW AND ADIPOSE TISSUE FOR BONE REGENERATION IN A CRITICAL SIZE DEFECT OF THE SHEEP TIBIA AND THE INFLUENCE OF PLATELET-RICH PLASMA

Aim of the present study was to compare the osteogenic potential of bone marrow derived mesenchymal stem cells (BMSC) and adipose-tissue derived stem cells (ASC) and to evaluate the influence of platelet-rich plasma (PRP) on the osteogenic capacity of ASC in a large animal model. Ovine BMSC (BMSC-group) and ASC (ASC-group) were seeded on mineralized collagen sponges and implanted into a critical size defect of the sheep tibia (n=5 each). In an additional group, platelet-rich plasma (PRP) was used in combination with ASC (PRP-group). Unloaded mineralized collagen (EMPTY-group) served as control (n=5 each). Radiographic evaluation was performed every 2 weeks, after 26 weeks histological analysis was performed. Radiographic evaluation revealed a significantly higher amount of newly formed bone in the BMSC-group compared to the ASC-group at week 10 and compared to EMPTY-group from week 12 (all p<0.05). A superiority on radiographic level concerning bone formation of the PRP-group versus the empty control group was found (p<0.05), but not for the ASC-group. Histological analysis confirmed radiographic evaluation finding analogous significances. In conclusion, ASC seem to be inferior to BMSC in terms of their osteogenic potential but that can partially be compensated by the addition of PRP.

Survival of human mesenchymal stromal cells from bone marrow and adipose tissue after xenogenic transplantation in immunocompetent mice

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 hosts immune system. This observation seems independent of the origin of MSC and applies to BMSC and ASC.

BM cells giving rise to MSC in culture have a heterogeneous CD34 and CD45 phenotype

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.

Chondrogenesis of Adipose-Derived Adult Stem Cells in a Poly-Lactide-Co-Glycolide Scaffold

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.

Development and Characterization of a Spheroidal Coculture Model of Endothelial Cells and Fibroblasts for Improving Angiogenesis in Tissue Engineering

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.

bFGF influences human articular chondrocyte differentiation

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.

Mesenchymal Stem Cell-Based HLA-Independent Cell Therapy for Tissue Engineering of Bone and Cartilage

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.

Survival and neurologic outcome after traumatic out-of-hospital cardiopulmonary arrest in a pediatric and adult population: a systematic review

IntroductionThis systematic review is focused on the in-hospital mortality and neurological outcome of survivors after prehospital resuscitation following trauma. Data were analyzed for adults/pediatric patients and for blunt/penetrating trauma.MethodsA systematic review was performed using the data available in Ovid Medline. 476 articles from 1/1964 - 5/2011 were identified by two independent investigators and 47 studies fulfilled the requirements (admission to hospital after prehospital resuscitation following trauma). Neurological outcome was evaluated using the Glasgow outcome scale.Results34 studies/5391 patients with a potentially mixed population (no information was found in most studies if and how many children were included) and 13 paediatric studies/1243 children (age ≤ 18 years) were investigated. The overall mortality was 92.8% (mixed population: 238 survivors, lethality 96.7%; paediatric group: 237 survivors, lethality 86.4% = p < 0.001).Penetrating trauma was found in 19 studies/1891 patients in the mixed population (69 survivors, lethality: 96.4%) and in 3 pediatric studies/91 children (2 survivors lethality 97.8%).44.3% of the survivors in the mixed population and 38.3% in the group of children had a good neurological recovery. A moderate disability could be evaluated in 13.1% in the mixed population and in 12.8% in children. A severe disability was found in 29.5% of the survivors in the mixed patients and in 38.3% in the group of children. A persistent vegetative state was the neurological status in 9.8% in the mixed population and in 10.6% in children.For each year prior to 2010, the estimated log-odds for survival decreased by 0.022 (95%-CI: [0.038;0.006]). When jointly analyzing the studies on adults and children, the proportion of survivors for children is estimated to be 17.8% (95%-CI: [15.1%;20.8%]). The difference of the paediatric compared to the adult proportion is significant (p < 0.001).ConclusionsChildren have a higher chance of survival after resuscitation of an out-of-hospital traumatic cardiac arrest compared to adults but tend to have a poorer neurological outcome at discharge.

In vivo quantification of intraarticular cytokines in knees during natural and surgically induced cartilage repair

BACKGROUND AIMS Cartilage defects are considered to be an initial event in the progress of osteoarthritis. Reliable data about in vivo regulation of cytokines in natural and surgically induced cartilage repair are still missing. METHODS Knee lavage fluids of 47 patients were collected prospectively between August 2006 and September 2007. Five patients without cartilage lesions served as a control group. In 42 patients the cartilage defects were treated by microfracturing (19) or autologous chondrocyte implantation (ACI) (23). Total protein content and concentrations of aggrecan, basic fibroblast growth factor (bFGF), Insulin-like growth factor and interleukin (IL)-1beta were determined. Clinical status was evaluated using the Lysholm score. RESULTS High-level expression in all knees was found for aggrecan, low-level constitutive expression for bFGF and IGF-I, while concentrations of IL-1beta in the control group remained below detection levels. The concentration of IGF-I in the knees with cartilage lesions was significantly higher (P<0.05) than in the control group. bFGF concentrations depended on cartilage lesion size; levels in the knees of patients undergoing ACI (6.1 cm(2)), were significantly higher compared with the control group (P<0.05) and the group of patients undergoing microfracturing (3.4 cm(2), P<0.001). Levels of aggrecan did not change after surgical cartilage repair, whereas concentrations of bFGF, IL-1beta and IGF-I significantly increased (P<0.01). Levels of IL-1beta significantly correlated with systemic C-reactive protein (CRP). The Lysholm score showed a medium significant negative correlation with IGF-I levels. CONCLUSIONS Aggrecan is constitutively expressed in knee joints. bFGF and IGF-I seem to play a pivotal role in natural and surgical cartilage repair. Operative intervention is additionally associated with IL-1beta-related inflammation-like reactions.

Mobile decision support for transplantation patient data

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.