Evaluation of a Novel Thiol-Norbornene-Functionalized Gelatin Hydrogel for Bioprinting of Mesenchymal Stem Cells.

Introduction: Three-dimensional bioprinting can be considered as an advancement of the classical tissue engineering concept. For bioprinting, cells have to be dispersed in hydrogels. Recently, a novel semi-synthetic thiolene hydrogel system based on norbornene-functionalized gelatin (GelNB) and thiolated gelatin (GelS) was described that resulted in the photoclick hydrogel GelNB/GelS. In this study, we evaluated the printability and biocompatibility of this hydrogel system towards adipose-tissue-derived mesenchymal stem cells (ASCs). Methods: GelNB/GelS was synthesized with three different crosslinking densities (low, medium and high), resulting in different mechanical properties with moduli of elasticity between 206 Pa and 1383 Pa. These hydrogels were tested for their biocompatibility towards ASCs in terms of their viability, proliferation and differentiation. The extrusion-based bioprinting of ASCs in GelNB/GelS-high was performed to manufacture three-dimensional cubic constructs. Results: All three hydrogels supported the viability, proliferation and chondrogenic differentiation of ASCs to a similar extent. The adipogenic differentiation of ASCs was better supported by the softer hydrogel (GelNB/GelS-low), whereas the osteogenic differentiation was more pronounced in the harder hydrogel (GelNB/GelS-high), indicating that the differentiation fate of ASCs can be influenced via the adaption of the mechanical properties of the GelNB/GelS system. After the ex vivo chondrogenic differentiation and subcutaneous implantation of the bioprinted construct into immunocompromised mice, the production of negatively charged sulfated proteoglycans could be observed with only minimal inflammatory signs in the implanted material. Conclusions: Our results indicate that the GelNB/GelS hydrogels are very well suited for the bioprinting of ASCs and may represent attractive hydrogels for subsequent in vivo tissue engineering applications.

In vivo evaluation of bioprinted prevascularized bone tissue.

Bioprinting can be considered as a progression of the classical tissue engineering approach, in which cells are randomly seeded into scaffolds. Bioprinting offers the advantage that cells can be placed with high spatial fidelity within three-dimensional tissue constructs. A decisive factor to be addressed for bioprinting approaches of artificial tissues is that almost all tissues of the human body depend on a functioning vascular system for the supply of oxygen and nutrients. In this study, we have generated cuboid prevascularized bone tissue constructs by bioprinting human adipose-derived mesenchymal stem cells (ASCs) and human umbilical vein endothelial cells (HUVECs) by extrusion-based bioprinting and drop-on-demand (DoD) bioprinting, respectively. The computer-generated print design could be verified in vitro after printing. After subcutaneous implantation of bioprinted constructs in immunodeficient mice, blood vessel formation with human microvessels of different calibers could be detected arising from bioprinted HUVECs and stabilization of human blood vessels by mouse pericytes was observed. In addition, bioprinted ASCs were able to synthesize a calcified bone matrix as an indicator of ectopic bone formation. These results indicate that the combined bioprinting of ASCs and HUVECs represents a promising strategy to produce prevascularized artificial bone tissue for prospective applications in the treatment of critical-sized bone defects.

In vivo evaluation of an electrospun gelatin nonwoven mat for regeneration of epithelial tissues.

One major objective in epithelial tissue engineering is to identify a suitable biomaterial that supports epithelial tissue formation. Therefore, the purpose of this study is to elucidate a novel electrospun gelatin nonwoven mat (NWM) for epithelial tissue engineering purposes in vivo. This NWM was seeded with either human gingival keratinocytes (GK, in coculture with gingival fibroblast) or human skin epithelial keratinocytes (EK, in coculture with skin dermal fibroblasts). These constructs were ex vivo cultured for 4 days before subcutaneous implantation into athymic nude mice. After 7 days, the constructs were explanted and investigated by immunohistology. Our results show that GK form a stratified epithelium on the surface of the NWM, mostly independent of a fibroblastic counterpart. Like native mucosa, the regenerated epithelium showed expression of epidermal growth factor receptor, cytokeratin-14 and -1, and involucrin. Only the expression of the basement membrane constituent laminin 5 was more pronounced in cocultures. Comparing GK and skin EK, we found that skin EK form a less developed epithelial tissue. Furthermore, the NWM allows not only for epithelial tissue formation by GK, but also for infiltration of human fibroblasts and mouse immune cells, thus representing a biomaterial with potential regenerative capacity for oral mucosa tissue engineering applications. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1605-1614, 2019.

Gelatin nonwovens-based epithelial morphogenesis involves a signaling axis comprising EGF-receptor, MAP kinases ERK 1/2, and ß1 integrin.

In biomaterials research, biomechanics which support tissue regeneration steadily gains of importance. Hence, we have previously shown that gelatin-based electrospun nonwoven mats (NWMs) with a distinct modulus of elasticity (3.2 kPa) promotes epithelial morphogenesis. Since molecular mechanisms of this morphogenesis are still unknown, the present study aims at identifying molecules, involved herein. Epithelia established on the NMWs showed persistence of the activated state of the epidermal growth factor receptor (EGF-R), phosphorylated at the src-specific tyrosine 845 (EGF-RT845 ) throughout the observation period of 10 days. To elucidate whether the observed morphogenesis mechanistically involves EGF-R signaling, we inhibited EGF-R, by employing the EGF-RT845 specific inhibitor Gefitinib (IRESSA®). Gefitinib administration yielded a reduced expression of the ß1 integrin subunit, a well-known cell-matrix interaction receptor, concomitant with downregulation of p42/44 ERK1/2 MAP-kinase activity. To elucidate whether the observed downregulation of ß1 is EGF-RT845 -dependent or emerging from ERK1/2 signaling, we exposed epithelia, grown on the NWMs, with the ERK1/2-directed inhibitor U0126. In the absence of Gefitinib, inhibition of p42/44 MAP-kinase activity resulted in decreased ß1 integrin protein levels, thus indicating that ß1 expression is dependent on ERK1/2 and not EGF-RT845 . Our results showed the first time that an EGF-R-ß1 integrin-signaling axis, including ERK1/2, promotes NWM-elasticity-based epithelial morphogenesis. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 663-677, 2019.

Nanofibered Gelatin-Based Nonwoven Elasticity Promotes Epithelial Histogenesis.

Regarding tissue regeneration, mechanics of biomaterials gains progressive importance. Therefore, this study reports on in situ crosslinked electrospun gelatin nonwoven mats (NWMs) whose distinct modulus of elasticity (ME) promotes epithelial tissue formation in a graded manner. NWMs, comprising fiber diameters in various distributions, yield an ME of about 2.1, 3.2, and 10.9 kPa. A two-step approach of preclinical in vitro validation identifies the elasticity of 3.2 kPa as superior to the other, regarding the histogenetic epithelial outcome. Hence, this 3.2 kPa candidate NWM is colonized with oral mucosal epithelial keratinocytes in the absence or presence of mesenchymal fibroblasts and/or endothelial cells. Evaluation of epithelial histogenesis at days 1 to 10 occurs by colorimetric and fluorescence-based immunohistochemistry (IHCH) of specific biomarkers. These include cytokeratins (CK) 14, CK1, and involucrin that indicate different stages of epithelial differentiation, as well as the basement membrane constituent collagen type IV and Ki-67 as a proliferation marker. Intriguingly, histogenesis and IHCH reveal the best resemblance of the native epithelium by the NWM alone, irrespective of other cell counterparts. These findings prove the gelatin NWM a convenient cell matrix, and evidence that NWM mechanics is important to promote epithelial histogenesis in view of prospective clinical applications.

Calmodulin Regulated Spectrin Associated Protein 1 mRNA is Directly Regulated by miR-126 in Primary Human Osteoblasts.

Vascularization is essential for bone development, fracture healing, and bone tissue engineering. We have previously described that coculture of primary human osteoblasts (hOBs) and human umbilical vein endothelial cells (HUVECs) improves differentiation of both cell types. Investigating the role of microRNAs (miRNAs) in this system, we found that miR-126 is highly upregulated in hOBs following coculturing with HUVECs. In this study we performed miR-126 gain-of-function and loss-of-function experiments in hOBs followed by microarray analysis in order to identify targets of miR-126. The transcript cluster IDs were sieved by applying cut-off criteria and by selecting transcripts which were upregulated following miR-126 downregulation and vice versa. The calmodulin regulated spectrin associated protein 1 (CAMSAP1) mRNA was confirmed to be differentially regulated by miR-126. Using the luciferase reporter assay it was demonstrated that CAMSAP1 is directly targeted by miR-126. In this study, we show that miR-126 and CAMSAP1 directly interact in hOBs. This finding has potential implications for tissue engineering applications. J. Cell. Biochem. 118: 1756-1763, 2017. © 2016 Wiley Periodicals, Inc.

Co-culture of adipose-derived stem cells and endothelial cells in fibrin induces angiogenesis and vasculogenesis in a chorioallantoic membrane model.

Neovascularization of adipose tissue equivalents is a crucial step in successful adipose tissue engineering, since insufficient vascularization results in graft resorption in an in vivo situation. A possible cellular approach to overcome this limitation is the co-implantation of adipose-derived stem cells (ASCs) with endothelial cells to stimulate the formation of a vascular network. We investigated the potential of ASCs derived from human abdominal fat tissue co-cultured with endothelial progenitor cells (EPCs) from human peripheral blood to stimulate neovascularization of fibrin constructs on the chorioallantoic membrane (CAM) of fertilized chicken eggs, in direct comparison to human umbilical vein endothelial cells (HUVECs). After 9 days of incubation, cell-fibrin constructs were explanted and histologically evaluated with respect to ingrowth of avian blood vessels into the construct and formation of human blood vessels by co-implanted endothelial cells. When administered on the CAM, ASCs successfully guided host vasculature into the construct (angiogenesis) and guided formation of capillary-like structures by co-implanted human endothelial cells (vasculogenesis), with HUVECs being superior to EPCs, leading to a perfused avian and human capillary network within the fibrin construct. However, the results also showed that perfused human blood vessels were only observed near the CAM compared to unperfused capillary-like structures near the top of the construct, indicating that perfusion of the cell-fibrin construct takes longer than 9 days. In conclusion, as blood vessel formation is an essential step during adipogenic differentiation, the data support our hypothesis that cellular communication between transplanted ASCs and endothelial cells is beneficial for vasculogenesis. Copyright © 2013 John Wiley & Sons, Ltd.

Growth differentiation factor 11 supports migration and sprouting of endothelial progenitor cells.

BACKGROUND: Neovascularization plays an important role in tissue engineering applications. In animal models, it was demonstrated that implantation of endothelial progenitor cells (EPCs) from cord blood led to the formation of a complex functional neovasculature, whereas EPCs isolated from peripheral blood (pbEPCs) showed a limited vasculogenic potential, which may be attributed to age-related dysfunction. Growth differentiation factor 11 (GDF11) was recently identified as a rejuvenation factor, which was able to reverse age-related dysfunction of stem cells. Therefore, we hypothesized that GDF11 may improve the vasculogenesis-related phenotype of pbEPCs. MATERIALS AND METHODS: pbEPCs were isolated from adult peripheral blood. Transforming growth factor (TGF)-ß type-I receptor expression was analyzed by immunostaining. pbEPCs were treated with recombinant GDF11 for various time periods. Thereafter, phosphorylation of Smad2/Smad3, adhesion, proliferation, cell survival, migration, and in vitro sprout formation was investigated. RESULTS: pbEPCs express the TGF-ß type-I receptors ALK4 and ALK5, but not ALK7. Treatment of pbEPCs with recombinant GDF11 resulted in activation of the Smad2/Smad3 pathway and in increased migration, which was inhibited by the TGF-ß1 superfamily type-I activin receptor-like kinase inhibitor SB431542, demonstrating that the TGF-ß receptor-Smad2/Smad3 pathway is involved in GDF11 induced migration. Moreover, in vitro sprout formation was increased as well by GDF11 treatment. However, other parameters such as adherence, proliferation, and apoptosis were not affected by GDF11. CONCLUSIONS: This study provides evidence that GDF11 improves vasculogenesis-related growth parameters in pbEPCs and may represent a therapeutic option to ameliorate the angiogenic and vasculogenic properties of pbEPCs.

Comparison between endothelial progenitor cells and human umbilical vein endothelial cells on neovascularization in an adipogenesis mouse model.

Volume stability and growth of tissue engineered adipose tissue equivalents using adipose-derived stem cells (ASCs) rely strongly on angiogenesis and neovascularization to support the maintenance of cells. An attractive cellular approach is based on coimplantation of endothelial cells to create a vascular network. Endothelial progenitor cells (EPCs) are a promising cell population, since they can be easily isolated from autologous human peripheral blood and thus represent a clinically feasible option. We have previously shown in in vitro and semi-in vivo studies that ASCs exert beneficial effects on EPCs in terms of enhanced tube formation and formation of blood vessels, respectively. In this study, we investigated the in vivo effects of coimplantation on endothelial cell-mediated neovascularization and ASC-mediated adipose tissue formation. For this purpose, human ASCs and human EPCs (or HUVECs as direct comparison to EPCs) were suspended alone or in coculture in fibrin and subcutaneously injected into the back of athymic nude mice and explanted after 1, 3 or 6months. Our results show that monocultures of EPCs or HUVECs were not able to perform vasculogenesis and constructs exhibited complete resorption already after 1month. However, a remarkable difference between EPCs and HUVECs was detected when coimplanted with ASCs. While coimplanted HUVECs gave rise to a stable neovasculature which was characterized by perfusion with erythrocytes, coimplanted EPCs showed no ability to form vascular structures. In the case of HUVEC-derived neovasculature, coimplanted ASCs displayed perivascular properties by stabilizing these neovessels. However, formation of human adipose tissue was independent of coimplanted endothelial cells. Our results indicate that HUVECs are superior to EPCs in terms of promoting in vivo neovascularization and recruiting perivascular cells for vessel stabilization when coimplanted with ASCs.

miR-126 regulates platelet-derived growth factor receptor-α expression and migration of primary human osteoblasts.

Adequate vascularization is an essential requirement for bone development, fracture healing and bone tissue engineering. We have previously described the coculture of primary human osteoblasts (hOBs) and human endothelial cells (HUVECs), designed to investigate the interactions between these cells. In this system, we showed that cocultivation of these two cell types leads to a downregulation of platelet-derived growth factor receptor-α (PDGFR-α) in hOBs, which was a consequence of reduced mRNA stability. In the current study we investigated the possible involvement of microRNAs in this process. Firstly, we performed a microarray analysis of osteoblastic miRNAs following cocultivation with HUVECs, revealing an upregulation of miR-126. This result was confirmed by RT-qPCR, and we observed that the increase is dependent on direct cell-to-cell contacts. Gain-of-function and loss-of-function experiments showed that miR-126 is a negative regulator of PDGFR-α mRNA. Additionally, migration of hOBs was inhibited by miR-126 overexpression and stimulated by miR-126 inhibition. Addition of PDGFR-α blocking antibody to hOB culture also inhibited hOB migration. There was no effect of miR-126 modulation on osteoblast proliferation, apoptosis rate or differentiation. In conclusion, we report that the miR-126/PDGFR-α system regulates the migratory behavior of human osteoblasts, without exerting effects on cell survival and differentiation.

Human adipose-derived stem cells enhance the angiogenic potential of endothelial progenitor cells, but not of human umbilical vein endothelial cells.

One of the current challenges in the field of adipose tissue engineering is to promote sufficient vascularization to prevent cell death and to support adipose tissue formation. Thus, a novel strategy to enhance neovascularization of tissue-engineered adipose tissue might be the coimplantation of adipose-derived stem cells (ASCs) with endothelial progenitor cells (EPCs). However, no knowledge is given about the cellular interaction in vitro of human ASCs derived from subcutaneous fat tissue and EPCs derived from human peripheral blood. In this study, the first aim was to characterize ASCs and EPCs. Secondly, the two-dimensional Matrigel assay and the three-dimensional spheroid sprouting assay were applied for analyzing the ASC-EPC interaction in regard to formation of capillary-like structures by EPCs by ASC-conditioned medium (CM) or coculture of both cell types and compared to cocultures of ASCs and human umbilical vein endothelial cells (HUVECs). ASC-CM had no influence on the formation of capillary-like structures by EPCs. However, coculture with ASCs significantly enhanced the formation of capillary-like structures by EPCs; an effect that was not observed in cocultures of ASCs with HUVECs. Importantly, this increase in capillary-like structure formation by EPCs due to cell-cell contact was associated with significantly increased vascular endothelial growth factor (VEGF) secretion and VEGF-A mRNA expression, while inhibition of VEGF receptor tyrosine kinases completely abolished this effect. In conclusion, these data suggest that cellular communication occurs between ASCs and EPCs triggered by cell-cell contact or at least close proximity, which is partially mediated by secreted VEGF leading to the enhancement of angiogenic properties in EPCs, but not in HUVECs.

Human endothelial progenitor cells induce extracellular signal-regulated kinase-dependent differentiation of mesenchymal stem cells into smooth muscle cells upon cocultivation.

Neovascularization represents an important issue in tissue-engineering applications, since survival of implanted cells strongly relies on sufficient oxygen and nutrient supply. We have recently observed that human bone marrow-derived mesenchymal stem cells (MSCs) support neovessel formation originating from coimplanted endothelial cells (ECs) in vivo, suggesting that MSCs may function as perivascular cells by investing and stabilizing nascent EC-derived neovessels. In this study, we investigated EC-induced mural cell differentiation of MSCs in vitro. For this purpose, endothelial progenitor cells (EPCs) from two different origins, namely adult peripheral blood (pbEPCs) and neonatal cord blood (cbEPCs), or human umbilical vein endothelial cells (HUVECs), were cocultured with human MSCs to analyze the effect on MSC differentiation toward a smooth muscle cell (SMC)/pericyte phenotype. EPCs as well as HUVECs increased alpha-smooth muscle actin expression in MSCs upon cocultivation in a time-dependent manner. This effect was strongly dependent on direct cell-to-cell contact and extracellular signal-regulated kinase (ERK) signaling, but was not mediated by heterotypic gap junction communication. Beyond enhanced SMC marker gene expression in MSCs, EPCs also enhanced the functional characteristics of cocultured MSCs by increasing their ability to attach to EC tubes in vitro. In conclusion, our study has shown that EPCs from adult peripheral blood as well as from cord blood commit cocultivated MSCs toward an SMC/pericyte phenotype in a cell-contact- and ERK-dependent manner.

Bi-directional exchange of membrane components occurs during co-culture of mesenchymal stem cells and nucleus pulposus cells.

Mesenchymal stem cell (MSC)-based therapies have been proposed as novel treatments for intervertebral disc (IVD) degeneration. We have previously demonstrated that when MSCs are co-cultured with nucleus pulposus (NP) cells with direct cell-cell contact, they differentiate along the NP lineage and simultaneously stimulate the degenerate NP cell population to regain a normal (non-degenerate) phenotype, an effect which requires cell-cell communication. However, the mechanisms by which NP cells and MSCs interact in this system are currently unclear. Thus, in this study we investigated a range of potential mechanisms for exchange of cellular components or information that may direct these changes, including cell fusion, gap-junctional communication and exchange of membrane components by direct transfer or via microvesicle formation. Flow cytometry of fluorescently labeled MSCs and NP cells revealed evidence of some cell fusion and formation of gapjunctions, although at the three timepoints studied these phenomena were detectable only in a small proportion of cells. While these mechanisms may play a role in cell-cell communication, the data suggests they are not the predominant mechanism of interaction. However, flow cytometry of fluorescently dual-labeled cells showed that extensive bi-directional transfer of membrane components is operational during direct co-culture of MSCs and NP cells. Furthermore, there was also evidence for secretion and internalization of membrane-bound microvesicles by both cell types. Thus, this study highlights bi-directional intercellular transfer of membrane components as a possible mechanism of cellular communication between MSC and NP cells.

Co-culture induces mesenchymal stem cell differentiation and modulation of the degenerate human nucleus pulposus cell phenotype.

AIMS: While mesenchymal stem cell (MSC)-based therapies for repair of the degenerate intervertebral disc (IVD) have been proposed, the interaction of MSCs with cells of the degenerate IVD has not been fully investigated. Therefore, it is unclear whether implanted MSCs would differentiate into nucleus pulposus (NP) cells and/or stimulate endogenous NP cells. Here, we investigate the differences in interaction between human MSCs and NP cells from both nondegenerate and degenerate discs during in vitro co-culture with direct cell-cell contact. MATERIALS & METHODS: Human bone marrow-derived MSCs (labeled with CFDA) were co-cultured with direct cell-cell contact in monolayer with NP cells obtained from nondegenerate or degenerate human NP tissue from lumbar IVDs at 50:50 ratios for 7 days. Differentiation of MSCs and changes of matrix-associated genes in NP cells were assessed by quantitative real-time PCR. RESULTS: MSCs differentiated to an NP-like phenotype following direct co-culture with both nondegenerate and degenerate NP, as shown by a significant upregulation of SOX9, type VI collagen, aggrecan and versican gene expression together with a simultaneous upregulation of CDMP-1, TGF-ß1, IGF-1 and CTGF. Direct co-culture of normal NP cells with MSCs had no effect on the phenotype of normal NP cells, while co-culture with degenerate NP cells resulted in enhanced matrix gene expression in degenerate NP cells, accompanied by increases in both TGF-ß and CDMP-1 gene expression. CONCLUSION: Importantly for MSC-based therapies for repair of the degenerate IVD, these data suggest that cellular interactions between MSCs and degenerate NP cells may both stimulate MSC differentiation to an NP-like phenotype and also stimulate the endogenous NP cell population to regain a nondegenerate phenotype and consequently enhance matrix synthesis for self-repair.

Matrix metabolism rate differs in functionally distinct tendons.

Tendon matrix integrity is vital to ensure adequate mechanical properties for efficient function. Although historically tendon was considered to be relatively inert, recent studies have shown that tendon matrix turnover is active. During normal physiological activities some tendons are subjected to stress and strains much closer to their failure properties than others. Tendons with low safety margins are those which function as energy stores such as the equine superficial digital flexor tendon (SDFT) and human Achilles tendon (AT). We postulate therefore that energy storing tendons suffer a higher degree of micro-damage and thus have a higher rate of matrix turnover than positional tendons. The hypothesis was tested using tissue from the equine SDFT and common digital extensor tendon (CDET). Matrix turnover was assessed indirectly by a combination of measurements for matrix age, markers of degradation, potential for degradation and protein expression. Results show that despite higher cellularity, the SDFT has lower relative levels of mRNA for collagen types I and III. Non-collagenous proteins, although expressed at different levels per cell, do not appear to differ between tendon types. Relative levels of mRNA for MMP1, MMP13 and both pro-MMP3 and MMP13 protein activity were significantly higher in the CDET. Correspondingly levels of cross-linked carboxyterminal telopeptide of type I collagen (ICTP) were higher in the CDET and tissue fluorescence lower suggesting more rapid turnover of the collagenous component. Reduced or inhibited collagen turnover in the SDFT may account for the high level of degeneration and subsequent injury compared to the CDET.

Cell phenotypic variation in normal and damaged tendons.

Injuries to tendons are common in both human athletes as well as in animals, such as the horse, which are used for competitive purposes. Furthermore, such injuries are also increasing in prevalence in the ageing, sedentary population. Tendon diseases often respond poorly to treatment and require lengthy periods of rehabilitation. The tendon has a unique extracellular matrix, which has developed to withstand the mechanical demands of such tensile-load bearing structures. Following injury, any repair process is inadequate and results in tissue that is distinct from original tendon tissue. There is growing evidence for the key role of the tendon cell (tenocyte) in both the normal physiological homeostasis and regulation of the tendon matrix and the pathological derangements that occur in disease. In particular, the tenocyte is considered to have a major role in effecting the subclinical matrix degeneration that is thought to occur prior to clinical disease, as well as in the severe degradative events that occur in the tendon at the onset of clinical disease. Furthermore, the tenocyte is likely to have a central role in the production of the biologically inadequate fibrocartilaginous repair tissue that develops subsequent to tendinopathy. Understanding the biology of the tenocyte is central to the development of appropriate interventions and drug therapies that will either prevent the onset of disease, or lead to more rapid and appropriate repair of injured tendon. Central to this is a full understanding of the proteolytic response in the tendon in disease by such enzymes as metalloproteinases, as well as the control of the inappropriate fibrocartilaginous differentiation. Finally, it is important that we understand the role of both intrinsic and extrinsic cellular elements in the repair process in the tendon subsequent to injury.

Towards in situ tissue repair: human mesenchymal stem cells express chemokine receptors CXCR1, CXCR2 and CCR2, and migrate upon stimulation with CXCL8 but not CCL2.

The recruitment of bone marrow CD34- mesenchymal stem- and progenitor cells (MSC) and their subsequent differentiation into distinct tissues is the precondition for in situ tissue engineering. The objective of this study was to determine the entire chemokine receptor expression profile of human MSC and to investigate their chemotactic response to the selected chemokines CCL2, CXCL8 and CXCL12. Human MSC were isolated from iliac crest bone marrow aspirates and showed a homogeneous population presenting a typical MSC-related cell surface antigen profile (CD14-, CD34-, CD44+, CD45-, CD166+, SH-2+). The expression profile of all 18 chemokine receptors was determined by real-time PCR and immunohistochemistry. Both methods consistently demonstrated that MSC express CC, CXC, C and CX(3)C receptors. Gene expression and immunohistochemical analysis documented that MSC express chemokine receptors CCR2, CCR8, CXCR1, CXCR2 and CXCR3. A dose-dependent chemotactic activity of CXCR4 and CXCR1/CXCR2 ligands CXCL12 and CXCL8 (interleukin-8) was demonstrated using a 96-well chemotaxis assay. In contrast, the CCR2 ligand CCL2 (monocyte chemoattractant protein-1, MCP-1) did not recruited human MSC. In conclusion, we report that the chemokine receptor expression profile of human MSC is much broader than known before. Furthermore, for the first time, we demonstrate that human MSC migrate upon stimulation with CXCL8 but not CCL2. In combination with already known data on MSC recruitment and differentiation these are promising results towards in situ regenerative medicine approaches based on guiding of MSC to sites of degenerated tissues.