Comparison of different strategies for in vivo seeding of prevascularized scaffolds.

Scaffolds seeded with multipotent precursor cells were hypothesized to heal critically sized bone defects. However, the success of this concept was limited by low cell survival after transplantation due to a lack of nutrients and oxygen. In vivo prevascularization of scaffolds before cell seeding may improve cell survival, yet the best seeding technique and time point of cell application remain elusive. Thus, the aim of this study was to compare different strategies. Demineralized bone matrix scaffolds were implanted around the saphenous arteriovenous (AV) bundle in nude mice. In vivo seeding was performed 0, 5, or 21 days after implantation using enhanced green fluorescent protein (eGFP)-expressing mesenchymal stem cells (MSCs). Cells were applied either by injection or the repetitive dripping technique. In vitro seeded and subcutaneously implanted scaffolds served as controls. Fourteen days after cell application, the fluorescence intensity of transplanted cells and the extent of newly formed vessels were quantified. We found that the AV flow through model as well as cell application increased vessel formation. In vitro seeding resulted in significantly higher cell numbers than in vivo seeding. With increasing time of prevascularization, the number of cells declined dramatically. In vivo seeding by cell injection was superior to the repetitive dripping protocol. On subcutaneously implanted scaffolds, significantly, more cells were found than on axially perfused scaffolds. We conclude that in vitro seeding is more efficient compared to the two novel in vivo seeding techniques of prevascularized scaffolds. With increasing time of prevascularization, the seeding efficiency for the in vivo methods further decreases, presumably due to the ingrowth of connective tissue. Even though, the presence of MSCs and the longer period of prevascularization enhances vessel formation, this conceivable advantage is limited supposedly by the inferior seeding efficiency.

Polypropylene meshes coated with a polysaccharide based bioadhesive for intra-abdominal mesh fixation in a rabbit model.

BACKGROUND: In this study, we evaluate a new bioadhesive for intra-abdominal onlay mesh fixation of a polypropylene-polyvinylchloride graft. METHODS: Three pieces of a commercially available polypropylene/polyvinylfluoride mesh, each 3 × 3 cm in size, and three pieces of the same mesh coated with a polysaccharide bioadhesive were fixated to the surface of the anterior abdominal wall of 30 New Zealand white rabbits. The fixation was performed either by using four transabdominal Prolene(®) 4/0 sutures, four spiral tacks (Protack 5 mm Tyco), or cyanoacrylate glue (Glubran(®) GEM, Viareggio, Italy). Each mesh position and the according kind of fixation were randomized before implantation. The animals were sacrificed 12 weeks postoperatively. After determining the extent of intra-abdominal adhesions, the meshes were excised en bloc with the anterior abdominal wall for tensile strength measurements and histological analysis. RESULTS: All meshes coated with the bioadhesive adhered to the intact peritoneum without extra fixation. Irrespective of the fixation technique coated meshes led to more and stronger adhesions. Mesh shrinkage by scarring was increased in coated meshes fixed with glue and low in uncoated meshes fixed with tacks. Testing the tensile strength, coated meshes fixed with transfascial sutures achieved the best results (16.14 ± 6.1 N), whereas coated meshes fixed with glue showed the lowest strength (10.39 ± 4.81 N). The foreign body reaction was considerably more distinctive using coated mesh. The mesh ingrowth was not influenced by this reaction. CONCLUSIONS: All meshes coated with the new bioadhesive were self-adhesive in that way; they stayed in position when attached to the peritoneum. Although this may facilitate intra-operative mesh fixation, the bioadhesive displayed several disadvantages, such as stronger adhesions and an increased shrinkage of the implant. The tensile strength was not influenced by the use of the bioadhesive. At present, we see no major advantage for polysaccharide bioadhesive applied in this study.

Long-term detection of fluorescently labeled human mesenchymal stem cell in vitro and in vivo by semi-automated microscopy.

The use of seeded scaffolds in regenerative medicine is limited by the low survival of transplanted mesenchymal stem cells (MSC). Current approaches aim at improving cell viability but require an adequate long-term detection of the transplanted cells. Unfortunately, commonly performed labeling techniques have not been validated for this purpose, and studies often reveal inconclusive results. Consequently, we intended to identify the most suitable method for long-term detection of human MSC (hMSC) in vitro and in vivo. hMSC were labeled using the vital stainings PKH26 and carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) as well as enhanced green fluorescent protein (eGFP) transduction. Metabolic activity and relative fluorescence intensity (RFI) were quantified in vitro over 21 days at 8 time points using standardized semi-automated microscopy and flow cytometry. In vivo, cell seeded scaffolds were subcutaneously implanted in nude mice, and RFI was analyzed over 42 days at 5 time points. In vitro, PKH26 and CFDA-SE significantly reduced metabolic activity. RFI of both stainings significantly decreased after 1 day and further faded to <1% after 7 days. In contrast, labeling with eGFP showed no metabolic effect on hMSC, and no significant reduction of RFI over the total period of 21 days. In vivo, RFI of eGFP labeled cells reached a plateau phase after 21 days and displayed a 3.8-fold higher RFI compared with PKH26 and CFDA-SE on day 42 evaluated in 280 field of views per scaffold using three scaffolds for each labeling technique and time point. We conclude that PKH26 and CFDA-SE are unsuitable for long-term detection of hMSC. eGFP transduction, in turn, allows long-term detection of hMSC in vitro and in vivo. Our results suggest that eGFP is currently the best option among the fluorescent labeling techniques to follow the fate of transplanted hMSC.

Effects of different media on proliferation and differentiation capacity of canine, equine and porcine adipose derived stem cells.

Adult stem cells are of particular interest for therapeutic use in the field of regenerative medicine. Adipose-derived mesenchymal stem cells (ASCs) are an attractive stem cell source for all fields of regenerative medicine because adipose tissue - and therewith cells - can easily be harvested from each donor. However, common expansion using fetal bovine serum (FBS) can not be used for clinical applications as xenogenic proteins must be avoided. Adipose tissue from equine, canine and porcine donors was digested with collagenase to isolate ASCs. ASCs were either expanded in a cell culture medium supplemented with FBS or in a serum-free medium (UltraCulture; UC) supplemented with a serum substitute (UltroserG). From all three animal species, the adipogenic and osteogenic differentiation potential of ASCs cultured with different media was analyzed in vitro. Cell proliferation analysis showed a population doubling time of 48-68 h for canine cells, 54-65 h for porcine cells and 54-70 h for equine cells, expanded in different media. Except for porcine ASCs, cells cultured in media supplemented with FBS grew faster than cells expanded in UC medium with UltroserG. Yet, all cells maintained their potential to differentiate into adipocytes and osteoblasts. UltraCulture medium containing UltroserG can for all examined species be recommended if FBS needs to be avoided in the expansion of donor-derived (stem) cells.

Characterization of adipose-derived equine and canine mesenchymal stem cells after incubation in agarose-hydrogel.

Adult stem cells are of particular interest for the therapeutic approach in the field of regenerative medicine. Due to their ease of harvest, adipose-derived mesenchymal stem cells (ASCs) are an attractive stem cell source that has become increasingly popular. Critical aspects of applied cell therapies are the circumstances of transport from the laboratory towards the site of operation and cell delivery into the desired area. With regard to these issues, agarose-hydrogel was analyzed as a cell carrier matrix of equine and canine ASCs in vitro, which can be used for minimally invasive application. Isolated ASCs were expanded and 2.5 × 10(6) cells were combined with agarose-hydrogel to build a 0.4% hydrogel-cell solution which was stored at two temperatures (room temperature (RT) vs. 37 °C). Cell viability was investigated (live-dead assay) at different time points (0, 1, 6 and 24 h) in order to determine i) the effect of different temperatures on the cell survival as well as ii) the maximum possible time span before implantation. CFU-assay and WST-1 assay were performed after 24 h incubation in agarose-hydrogel and the cells were induced into adipogenic and osteogenic differentiation to analyze the effects of the incubation on the cell behaviour. No negative effect of the agarose-hydrogel incubation was determined on the different species' cell behaviour at either RT or 37 °C with any of the assays used. We can recommend agarose-hydrogel as a cell carrier for cell implantation with a storage period of up to 24 h at room temperature or at 37 °C prior to implantation.

Cyanoacrylate glue for intra-abdominal mesh fixation of polypropylene-polyvinylidene fluoride meshes in a rabbit model.

BACKGROUND: Cyanoacrylate glues are tissue adhesive with high adherent and hemostatic properties. The aim of this study was to evaluate the efficacy of cyanoacrylates glue for polypropylene-polyvinylidene fluoride (PP-PVDF) intraperitoneal onlay mesh (IPOM) fixation in a rabbit model. MATERIALS AND METHODS: In 40 New Zealand white rabbits, three pieces (3×3cm) of a PP-PVDF mesh (n=120) were fixed in IPOM technique on both sides of a midline laparotomy. For mesh fixation we used spiral tacks, nonabsorbable sutures, or cyanoacrylate glue in a randomized manner. All animals were killed after 12 wk. The prosthetic materials were excised en bloc with the anterior abdominal wall for evaluation of the tensile strength and histologic analysis. Results are presented as mean and standard deviation. RESULTS: Meshes fixed with glue showed a significantly higher tenacity of adhesions (2.75±0.97) compared with those with tacks (2.44±0.97 sutures versus 1.91±0.92 tacks). The percentage of adhesions in the glue group was comparable to the suture group (36.50% ± 27.60% glue, 37.62% ± 27.36% suture). The tensile strength of stapled and sutured meshes was significantly higher than the tensile strength glued mesh (14.15±0.97N suture versus 14.84±0.74 stapler versus 9.64±0.78N glue). Mesh shrinkage was irrespective of the fixation technique. The inflammation reaction was more pronounced in the glue group. CONCLUSIONS: Although cyanoacrylate glue showed a considerable cellular ingrowth in this rabbit model, sutures and tacks proved to be superior for IPOM fixation of PP-PVDF meshes in terms of tensile strength.

Impact of indium-111 oxine labelling on viability of human mesenchymal stem cells in vitro, and 3D cell-tracking using SPECT/CT in vivo.

PURPOSE: This study investigates the effects of (111)In-oxine incorporation on human mesenchymal stem cells' (hMSC) biology and viability, and the applicability of (111)In-oxine for single-photon emission computed tomography/X-ray computed tomography (SPECT/CT) monitoring of hMSC in vivo. PROCEDURES: HMSC were labelled with 10 Bq/cell. Cellular retention of radioactivity, cell survival, and migration were evaluated over 48 h. Metabolic activity was assessed over 14 days and the hMSC's stem cell character was evaluated. Serial SPECT/CT was performed after intra-osseous injection to athymic rats over 48 h. RESULTS: Labelling efficiency was 25%, with 61% of incorporated (111)In remaining in the hMSC at 48 h. The radiolabelling was without effect on cell viability, stem cell character, and plasticity, whereas metabolic activity and migration were significantly reduced. Grafted cells could be imaged in situ with SPECT/CT. CONCLUSIONS: (111)In-oxine labelling moderately impaired hMSC's functional integrity while preserving their stem cell character. Combined SPECT/CT imaging of (111)In-oxine-labelled hMSC opens the possibility for non-invasive sequential monitoring of therapeutic stem cells.

Osteonecrosis of the jaw: effect of bisphosphonate type, local concentration, and acidic milieu on the pathomechanism.

PURPOSE: Osteonecrosis of the jaw has been reported in patients receiving high doses of intravenous nitrogen-containing bisphosphonates (N-BPs) because of malignant disease. The exact pathomechanisms have been elusive and questions of paramount importance remain unanswered. Recent studies have indicated toxic effects of bisphosphonates on different cell types, apart from osteoclast inhibition. Multipotent stem cells play an important role in the processes of wound healing and bone regeneration, which seem to be especially impaired in the jaws of patients receiving high doses of N-BPs. Therefore, the aim of the present study was to investigate the effects of different bisphosphonate derivatives and dose levels combined with varying pH levels on the mesenchymal stem cells in vitro. MATERIALS AND METHODS: The effect of 2 N-BPs (zoledronate and ibandronate) and 1 non-N-BP (clodronate) on immortalized mesenchymal stem cells was tested at different concentrations, reflecting 1, 3, and 6 months and 1, 3, 5, and 10 years of exposure to standard oncology doses of the 2 N-BPs and equimolar concentrations of clodronate at different pH values (7.4, 7.0, 6.7, and 6.3). Cell viability and activity were analyzed using a WST assay. Cell motility was investigated using scratch wound assays and visualized using time-lapse microscopy. RESULTS: Both types of bisphosphonates revealed remarkable differences. Zoledronate and ibandronate showed a dose- and pH-dependent cellular toxicity. Increasing concentrations of both N-BPs and an acidic milieu led to a significant decrease in cell viability and activity (P < .01), with more pronounced effects for zoledronate. Equimolar concentrations of clodronate did not affect the cell survival or activity significantly, apart from the effect of pH reduction itself, which was also detectable in the patients in the control group who did not receive bisphosphonates. CONCLUSIONS: Our results have shown that high concentrations of N-BPs and a local acidic milieu, which is commonly present in infections of the jaw, might play a key role in the pathogenesis of osteonecrosis of the jaw in patients receiving high doses of N-BPs for malignant diseases. Also the potency of N-BPs might be different, suggesting a greater risk of osteonecrosis of the jaw with zoledronate.

Quantification of fluorescence intensity of labeled human mesenchymal stem cells and cell counting of unlabeled cells in phase-contrast imaging: an open-source-based algorithm.

Assessment of cell fate is indispensable to evaluate cell-based therapies in regenerative medicine. Therefore, a widely used technique is fluorescence labeling. A major problem still is the standardized, noninvasive, and reliable quantification of fluorescence intensity of adherent cell populations on single-cell level, since total fluorescence intensity must be correlated to the cell number. Consequently, the aim of the present study was to produce and validate an open-source-based algorithm, capable of measuring the total fluorescence intensity of cell populations and assessing the total cell number in phase-contrast images. To verify the algorithms' capacity to assess fluorescence intensity, human mesenchymal stem cells were transduced to stably express enhanced green fluorescent protein and results produced by the algorithm were compared to flow cytometry analysis. No significant differences could be observed at any time (p ≥ 0.443). For validation of the algorithm for cell counting in phase-contrast images, adherent human mesenchymal stem cells were manually counted and compared to results produced by the algorithm (correlation coefficient [CC] r = 0.975), nuclei staining (CC r = 0.997), and hemocytometer (CC r = 0.629). We conclude that applying the developed algorithm in routine practice allows robust, fast, and reproducible assessment of fluorescence intensity and cell numbers in simple large-scale microscopy. The method is easy to perform and open source based.

Hypoxic preconditioning of human mesenchymal stem cells overcomes hypoxia-induced inhibition of osteogenic differentiation.

Osteogenic differentiation of human mesenchymal stem cells (hMSCs) into osteoblasts is a prerequisite for subsequent bone formation. Numerous studies have explored osteogenic differentiation under standard tissue culture conditions, which usually employ 21% of oxygen. However, bone precursor cells such as hMSCs reside in stem cell niches of low-oxygen atmospheres. Furthermore, they are subjected to low oxygen concentrations when cultured on three-dimensional scaffolds in vitro, and even more so after transplantation when vascularization has yet to be established. Similarly, hMSCs are exposed to low oxygen in the fracture microenvironment following bony injury. Recent studies revealed that hypoxic preconditioning improves cellular engraftment and survival in low-oxygen atmospheres. In our study we investigated the osteogenic differentiation potential of hMSCs under 2% O(2) (hypoxia) in comparison to a standard tissue culture oxygen atmosphere of 21% (normoxia). We assessed the osteogenic differentiation of hMSCs following hypoxic preconditioning to address whether this pretreatment is beneficial for subsequent differentiation processes as well. To validate our findings we carefully characterized the extent of hypoxia exerted and its effect on cell survival and proliferation. We found that hMSCs proliferate better if cultured under 2% of oxygen. We confirmed that osteogenic differentiation of hMSCs is indeed inhibited if osteogenic induction is carried out under constant hypoxia. Finally, we showed for the first time that hypoxic preconditioning of hMSCs prior to osteogenic induction restores osteogenic differentiation of hMSCs under hypoxic conditions. Collectively, our results indicate that maintaining constant levels of oxygen improves the osteogenic potential of hMSCs and suggest that low oxygen concentrations may preserve the stemness of hMSCs. In addition, our data support the hypothesis that if low-oxygen atmospheres are expected at the site of implantation, hypoxic pretreatment may be beneficial for the cells' subsequent in vivo performance.

Beta1 integrin deficiency results in multiple abnormalities of the knee joint.

The lack of beta1 integrins on chondrocytes leads to severe chondrodysplasia associated with high mortality rate around birth. To assess the impact of beta1 integrin-mediated cell-matrix interactions on the function of adult knee joints, we conditionally deleted the beta1 integrin gene in early limb mesenchyme using the Prx1-cre transgene. Mutant mice developed short limbed dwarfism and had joint defects due to beta1 integrin deficiency in articular regions. The articular cartilage (AC) was structurally disorganized, accompanied by accelerated terminal differentiation, altered shape, and disrupted actin cytoskeleton of the chondrocytes. Defects in chondrocyte proliferation, cytokinesis, and survival resulted in hypocellularity. However, no significant differences in cartilage erosion, in the expression of matrix-degrading proteases, or in the exposure of aggrecan and collagen II cleavage neoepitopes were observed between control and mutant AC. We found no evidence for disturbed activation of MAPKs (ERK1/2, p38, and JNK) in vivo. Furthermore, fibronectin fragment-stimulated ERK activation and MMP-13 expression were indistinguishable in control and mutant femoral head explants. The mutant synovium was hyperplastic and frequently underwent chondrogenic differentiation. beta1-null synoviocytes showed increased proliferation and phospho-focal adhesion kinase expression. Taken together, deletion of beta1 integrins in the limb bud results in multiple abnormalities of the knee joints; however, it does not accelerate AC destruction, perturb cartilage metabolism, or influence intracellular MAPK signaling pathways.

Tissue engineering for bone defect healing: an update on a multi-component approach.

SUMMARY: The need for an interdisciplinary approach in order to establish new therapeutic strategies for the therapy of bone defects has been acknowledged by the scientific community for many years. This awareness makes itself felt when looking at the multitude of approaches--ranging from cell-based to scaffold-based strategies and also including the use of osteogenic growth factors and genetic engineering--that are currently being combined to assess their potential to develop effective concepts for the treatment of extensive loss of osseous tissue. With a strong focus on the preclinical research in this field, the goal of this review is to give an update on the multi-component approaches that are currently being investigated in tissue engineering of bone.

Hypoxia in static and dynamic 3D culture systems for tissue engineering of bone.

Tissue engineering of sizeable cell-scaffold constructs is limited by gradients in tissue quality from the periphery toward the center. Because homogenous delivery of oxygen to three-dimensional (3D) cell cultures remains an unsolved challenge, we hypothesized that uneven oxygen supply may impede uniform cellular growth on scaffolds. In this study we challenged static and dynamic 3D culture systems designed for bone tissue engineering applications with a well-growing subclone of MC3T3-E1 preosteoblasts and continuously measured the oxygen concentrations in the center of cell-seeded scaffolds and in the surrounding medium. After as little as 5 days in static culture, central oxygen concentrations dropped to 0%. Subsequently, cells died in central regions of the scaffold but not in its periphery, where oxygen levels were approximately 4%. The use of perfusion bioreactors successfully prevented cell death, yet central oxygen concentrations did not rise above 4%. We conclude that 3D culture in vitro is associated with relevant oxygen gradients, which can be the cause of inhomogeneous tissue quality. Perfusion bioreactors prevent cell death but they do not entirely eliminate 3D culture-associated oxygen gradients. Therefore, we advise continuous oxygen monitoring of 3D culture systems to ensure tissue quality throughout engineered constructs.

Validation of a femoral critical size defect model for orthotopic evaluation of bone healing: a biomechanical, veterinary and trauma surgical perspective.

Numerous in vivo studies have been conducted to investigate bone regeneration in orthotopic defect models, but a reliably standardized critical-size defect (CSD) model in small animals is still lacking in tissue-engineering research. Utilizing the expertise of trauma surgeons, veterinary surgeons, and engineers, we evaluated the optimal fixation strategy for in vivo application in terms of surgical suitability and conducted biomechanical studies for 3 fixation devices. Fixation strategies were an external fixation device made of polymethylmethacrylate, widely used in animal care; a self-constructed external clamp-fixation device, designed and manufactured using rapid prototyping techniques; and commercially available 1.2-mm titanium plates used in hand surgery. The CSD was 6 mm in size. Biomechanical testing included compression, 4-point bending, and torsion tests. The surgical procedure was optimized in vitro and validated in a clinical setting in athymic rats in vivo. Despite differences in the results of the biomechanical tests, all fixation devices tested proved suitable for the intended purpose. In conclusion, the evaluated model for stabilizing a CSD in a rat's femur can reliably be used for standardized bone regeneration studies in small animals.

Introducing a single-cell-derived human mesenchymal stem cell line expressing hTERT after lentiviral gene transfer.

Human mesenchymal stem cells (hMSCs) can be readily isolated from bone marrow and differentiate into multiple tissues, making them a promising target for future cell and gene therapy applications. The low frequency of hMSCs in bone marrow necessitates their isolation and expansion in vitro prior to clinical use, but due to senescence-associated growth arrest during culture, limited cell numbers can be generated. The lifespan of hMSCs has been extended by ectopic expression of human telomerase reverse transcriptase (hTERT) using retroviral vectors. Since malignant transformation was observed in hMSCs and retroviral vectors cause insertional mutagenesis, we ectopically expressed hTERT using lentiviral gene transfer. Single-cell-derived hMSC clones expressing hTERT did not show malignant transformation in vitro and in vivo after extended culture periods. There were no changes observed in the expression of tumour suppressor genes and karyotype. Cultured hMSCs lack telomerase activity, but it was significantly increased by ectopic expression of hTERT. HTERT expression prevented hMSC senescence and the cells showed significantly higher and unlimited proliferation capacity. Even after an extended culture period, hMSCs expressing hTERT preserved their stem cells character as shown by osteogenic, adipogenic and chondrogenic differentiation. In summary, extending the lifespan of human mesenchymal stem cells by ectopic expression of hTERT using lentiviral gene transfer may be an attractive and safe way to generate appropriate cell numbers for cell and gene therapy applications.

Influence of in vitro cultivation on the integration of cell-matrix constructs after subcutaneous implantation.

Dynamic cultivation of scaffolds loaded with undifferentiated stem cells can lead toward osteogenic differentiation in vivo. The aim of this study was to examine the influence of different in vitro cultivation setups on the integration of cell-matrix constructs after subcutaneous implantation. Human mesenchymal stem cells (hMSC) were inoculated on clinically approved scaffolds. These cell-matrix constructs were then cultured under static (12 hours or 14 days) or dynamic (14 days) conditions, followed by paravertebral subcutaneous implantation in athymic nude mice. After 2 weeks and 12 weeks the constructs and selected organs were harvested for histological evaluation, and qualitative and quantitative polymerase chain reaction (PCR). Histological analysis showed good integration of cell-matrix constructs independent of culture conditions and a differential effect of static and dynamic in vitro culture on fat cell formation in vivo. Human DNA (hDNA) was detected in explanted cell-matrix constructs at all time points with a significant decrease in human cells on the constructs compared to the initial amount of cells seeded. No hDNA was detected in the explanted organs. In conclusion, we could prove the survival of hMSC on scaffolds after in vitro cultivation and consecutive implantation in vivo. While the amount of adipose tissue increased after static cultivation, we could not achieve osteogenic differentiation.