BMP2 gene transfer induces pericardial effusion and inflammatory response in the ischemic porcine myocardium.

Pro-angiogenic gene therapy is being developed to treat coronary artery disease (CAD). We recently showed that bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor-A synergistically regulate endothelial cell sprouting in vitro. BMP2 was also shown to induce endocardial angiogenesis in neonatal mice post-myocardial infarction. In this study, we investigated the potential of BMP2 gene transfer to improve cardiomyocyte function and neovessel formation in a pig chronic myocardial infarction model. Ischemia was induced in domestic pigs by placing a bottleneck stent in the proximal part of the left anterior descending artery 14 days before gene transfer. Intramyocardial gene transfers with adenovirus vectors (1 × 1012 viral particles/pig) containing either human BMP2 (AdBMP2) or beta-galactosidase (AdLacZ) control gene were performed using a needle injection catheter. BMP2 transgene expression in the myocardium was detected with immunofluorescence staining in the gene transfer area 6 days after AdBMP2 administration. BMP2 gene transfer did not induce angiogenesis or cardiomyocyte proliferation in the ischemic pig myocardium as determined by the quantitations of CD31 or Ki-67 stainings, respectively. Accordingly, no changes in heart contractility were detected in left ventricular ejection fraction and strain measurements. However, BMP2 gene transfer induced pericardial effusion (AdBMP2: 9.41 ± 3.17 mm; AdLacZ: 3.07 ± 1.33 mm) that was measured by echocardiography. Furthermore, an increase in the number of immune cells and CD3+ T cells was found in the BMP2 gene transfer area. No changes were detected in the clinical chemistry analysis of pig serum or histology of the major organs, implicating that the gene transfer did not induce general toxicity, myocardial injury, or off-target effects. Finally, the levels of fibrosis and cardiomyocyte apoptosis detected by Sirius red or caspase 3 stainings, respectively, remained unaltered between the groups. Our results demonstrate that BMP2 gene transfer causes inflammatory changes and pericardial effusion in the adult ischemic myocardium, which thus does not support its therapeutic use in chronic CAD.

SproutAngio: an open-source bioimage informatics tool for quantitative analysis of sprouting angiogenesis and lumen space.

Three-dimensional image analyses are required to improve the understanding of the regulation of blood vessel formation and heterogeneity. Currently, quantitation of 3D endothelial structures or vessel branches is often based on 2D projections of the images losing their volumetric information. Here, we developed SproutAngio, a Python-based open-source tool, for fully automated 3D segmentation and analysis of endothelial lumen space and sprout morphology. To test the SproutAngio, we produced a publicly available in vitro fibrin bead assay dataset with a gradually increasing VEGF-A concentration ( https://doi.org/10.5281/zenodo.7240927 ). We demonstrate that our automated segmentation and sprout morphology analysis, including sprout number, length, and nuclei number, outperform the widely used ImageJ plugin. We also show that SproutAngio allows a more detailed and automated analysis of the mouse retinal vasculature in comparison to the commonly used radial expansion measurement. In addition, we provide two novel methods for automated analysis of endothelial lumen space: (1) width measurement from tip, stalk and root segments of the sprouts and (2) paired nuclei distance analysis. We show that these automated methods provided important additional information on the endothelial cell organization in the sprouts. The pipelines and source code of SproutAngio are publicly available ( https://doi.org/10.5281/zenodo.7381732 ).

BMP6/TAZ-Hippo signaling modulates angiogenesis and endothelial cell response to VEGF.

The BMP/TGFß-Smad, Notch and VEGF signaling guides formation of endothelial tip and stalk cells. However, the crosstalk of bone morphogenetic proteins (BMPs) and vascular endothelial growth factor receptor 2 (VEGFR2) signaling has remained largely unknown. We demonstrate that BMP family members regulate VEGFR2 and Notch signaling, and act via TAZ-Hippo signaling pathway. BMPs were found to be regulated after VEGF gene transfer in C57/Bl6 mice and in a porcine myocardial ischemia model. BMPs 2/4/6 were identified as endothelium-specific targets of VEGF. BMP2 modulated VEGF-mediated endothelial sprouting via Delta like Canonical Notch Ligand 4 (DLL4). BMP6 modulated VEGF signaling by regulating VEGFR2 expression and acted via Hippo signaling effector TAZ, known to regulate cell survival/proliferation, and to be dysregulated in cancer. In a matrigel plug assay in nude mice BMP6 was further demonstrated to induce angiogenesis. BMP6 is the first member of BMP family found to directly regulate both Hippo signaling and neovessel formation. It may thus serve as a target in pro/anti-angiogenic therapies.

Cluster analysis of infrared spectra can differentiate intact and repaired articular cartilage.

OBJECTIVE: Successful repair of articular cartilage (AC) defects would be a major advantage due to the low ability of AC to heal spontaneously. Sensitive methods to determine changes in AC composition and structure are required to monitor the success of repair. This study evaluates the ability of unsupervised cluster analysis applied to Fourier transform infrared (FTIR) microspectroscopy to discriminate between healthy and repaired AC. METHODS: Osteochondral lesions (3 mm in depth) were surgically created in patellar grooves of rabbit femurs and were either left to heal spontaneously (n = 6) or surgically repaired with autologous chondrocytes in type II collagen gel (n = 6). After 6 months, tissues were harvested, FTIR microspectroscopy was conducted and Fuzzy c-means (FCM) cluster analysis applied to spectra of pairs of intact and repaired AC samples from each rabbit. Two spectral regions [amide I and carbohydrate (CHO)] were analyzed and the results from the two types of repair were compared. RESULTS: Two separate regions of repair were detected with FCM. The estimated proteoglycan content (from CHO region) in the repaired AC was significantly lower than that in intact AC. The spontaneously repaired AC was better distinguished from the intact AC than the collagen II gel repaired AC. The most distinct clustering was observed for spontaneously repaired samples using CHO region. CONCLUSIONS: This study revealed that unsupervised cluster analysis applied to FTIR microspectroscopy can detect subtle differences in infrared spectra between normal and repaired AC. The method may help in evaluation and optimization of future AC repair strategies.

Repair of osteochondral defects with recombinant human type II collagen gel and autologous chondrocytes in rabbit.

OBJECTIVE: Recombinant human type II collagen (rhCII) gels combined with autologous chondrocytes were tested as a scaffold for cartilage repair in rabbits in vivo. METHOD: Autologous chondrocytes were harvested, expanded and combined with rhCII-gel and further pre-cultivated for 2 weeks prior to transplantation into a 4 mm diameter lesion created into the rabbit's femoral trochlea (n = 8). Rabbits with similar untreated lesions (n = 7) served as a control group. RESULTS: Six months after the transplantation the repair tissue in both groups filled the lesion site, but in the rhCII-repair the filling was more complete. Both repair groups also had high proteoglycan and type II collagen contents, except in the fibrous superficial layer. However, the integration to the adjacent cartilage was incomplete. The O'Driscoll grading showed no significant differences between the rhCII-repair and spontaneous repair, both representing lower quality than intact cartilage. In the repair tissues the collagen fibers were abnormally organized and oriented. No dramatic changes were detected in the subchondral bone structure. The repair cartilage was mechanically softer than the intact tissue. Spontaneously repaired tissue showed lower values of equilibrium and dynamic modulus than the rhCII-repair. However, the differences in the mechanical properties between all three groups were insignificant. CONCLUSION: When rhCII was used to repair cartilage defects, the repair quality was histologically incomplete, but still the rhCII-repairs showed moderate mechanical characteristics and a slight improvement over those in spontaneous repair. Therefore, further studies using rhCII for cartilage repair with emphasis on improving integration and surface protection are required.

Comparison of ultrasound and optical coherence tomography techniques for evaluation of integrity of spontaneously repaired horse cartilage.

The aim of this study was to compare sensitivity of ultrasound and optical coherence tomography (OCT) techniques for the evaluation of the integrity of spontaneously repaired horse cartilage. Articular surfaces of horse intercarpal joints, featuring both intact tissue and spontaneously healed chondral or osteochondral defects, were imaged ex vivo with arthroscopic ultrasound and laboratory OCT devices. Quantitative ultrasound (integrated reflection coefficient (IRC), apparent integrated backscattering coefficient (AIB) and ultrasound roughness index (URI)) and optical parameters (optical reflection coefficient (ORC), optical roughness index (ORI) and optical backscattering (OBS)) were determined and compared with histological integrity and mechanical properties of the tissue. Spontaneously healed tissue could be quantitatively discerned from the intact tissue with ultrasound and OCT techniques. Furthermore, several significant correlations (p < 0.05) were detected between ultrasound and OCT parameters. Superior resolution of OCT provided a more accurate measurement of cartilage surface roughness, while the ultrasound backscattering from the inner structures of the cartilage matched better with the histological findings. Since the techniques were found to be complementary to each other, dual modality imaging techniques could provide a useful tool for the arthroscopic evaluation of the integrity of articular cartilage.

Contrast-Enhanced Micro-Computed Tomography in Evaluation of Spontaneous Repair of Equine Cartilage.

OBJECTIVE: Contrast-enhanced computed tomography (CECT) has been introduced for the evaluation of cartilage integrity. Furthermore, CECT enables imaging of the structure and density of subchondral bone. In this laboratory study, we investigate the potential of microCECT to simultaneously image cartilage and subchondral bone for the evaluation of tissue healing. DESIGN: Osteochondral lesions (Ø = 6 mm) were surgically created in equine intercarpal joints (n = 7). After spontaneous healing for 12 months, the horses were sacrificed and osteochondral plugs (Ø = 14 mm), including the repair cartilage and adjacent intact tissue, were harvested. The nonfibrillar and fibrillar moduli and the permeability of cartilage were determined using indentation testing. Contrast agent diffusion into the samples was imaged for 36 hours using high-resolution CT. Results from CECT, mechanical testing, and microscopic analyses were compared and correlated. RESULTS: The contrast agent diffusion coefficient showed a significant (P < 0.05) difference between the repair and adjacent intact tissue. MicroCECT revealed altered (P < 0.05) bone volume fraction, mineral density, and microstructure of subchondral bone at the repair site. The contrast agent diffusion coefficient correlated with the moduli of the nonfibrillar matrix (R = -0.662, P = 0.010), collagen fibril parallelism index (R = -0.588, P = 0.035), and glycosaminoglycan content (R = -0.503, P = 0.067). The repair cartilage was mechanically and structurally different from adjacent intact tissue (P < 0.05). CONCLUSIONS: MicroCECT enabled simultaneous quantitative evaluation of subchondral bone and monitoring of cartilage repair, distinguishing quantitatively the repair site from the adjacent intact tissue. As the only technique able to simultaneously image cartilage and determine subchondral bone mineral density and microstructure, CECT has potential clinical value.

Engineering of cartilage in recombinant human type II collagen gel in nude mouse model in vivo.

OBJECTIVE: Our goal was to test the recombinant human type II collagen (rhCII) material as a gel-like scaffold for chondrocytes in a nude mouse model in vivo. DESIGN: Isolated bovine chondrocytes (6x10(6)) were seeded into rhCII gels (rhCII-cell) and injected subcutaneously into the backs of nude mice. For comparison, chondrocytes (6x10(6)) in culture medium (Med-cell) and cell-free rhCII gels (rhCII-gel) were similarly injected (n=24 animals, total of three injections/animal). After 6 weeks, the tissue constructs were harvested and analyzed. RESULTS: Chondrocytes with or without rhCII-gel produced white resilient tissue, which in histological sections had chondrocytes in lacunae-like structures. Extracellular matrix stained heavily with toluidine blue stain and had strongly positive collagen type II immunostaining. The tissue did not show any evidence of vascular invasion or mineralization. The cell-free rhCII-gel constructs showed no signs of cartilage tissue formation. Cartilage tissue produced by Med-cell was thin and macroscopically uneven, while the rhCII-cell construct was smooth and rounded piece of neotissue. RhCII-cell constructs were statistically thicker than Med-cell ones. However, no statistical differences were found between the groups in terms of glycosaminoglycan (GAG) content or biomechanical properties. CONCLUSIONS: These results show that rhCII-gel provides good expansion and mechanical support for the formation of cartilage neotissue. RhCII material may allow favorable conditions in the repair of chondral lesions.

Recombinant human type II collagen as a material for cartilage tissue engineering.

PURPOSE: Collagen type II is the major component of cartilage and would be an optimal scaffold material for reconstruction of injured cartilage tissue. In this study, the feasibility of recombinant human type II collagen gel as a 3-dimensional culture system for bovine chondrocytes was evaluated in vitro. METHODS: Bovine chondrocytes (4x106 cells) were seeded within collagen gels and cultivated for up to 4 weeks. The gels were investigated with confocal microscopy, histology, and biochemical assays. RESULTS: Confocal microscopy revealed that the cells maintained their viability during the entire cultivation period. The chondrocytes were evenly distributed inside the gels, and the number of cells and the amount of the extracellular matrix increased during cultivation. The chondrocytes maintained their round phenotype during the 4-week cultivation period. The glycosaminoglycan levels of the tissue increased during the experiment. The relative levels of aggrecan and type II collagen mRNA measured with realtime polymerase chain reaction (PCR) showed an increase at 1 week. CONCLUSION: Our results imply that recombinant human type II collagen is a promising biomaterial for cartilage tissue engineering, allowing homogeneous distribution in the gel and biosynthesis of extracellular matrix components.

Bioreactor improves the growth and viability of chondrocytes in the knitted poly-L,D-lactide scaffold.

In the present study bovine chondrocytes were cultured in two different environments (static flasks and bioreactor) in knitted poly-L,D-lactide (PLDLA) scaffolds up to 4 weeks. Chondrocyte viability was assessed by employing cell viability fluorescence markers. The cells were visualized using confocal laser scanning microscopy and scanning electron microscopy. The mechanical properties and uronic acid contents of the scaffolds were tested. Our results showed that cultivation in a bioreactor improved the growth and viability of the chondrocytes in the PLDLA scaffolds. Cells were observed both on and in between the fibrils of scaffold. Furthermore, chondrocytes cultured in the bioreactor, regained their original round phenotypes, whereas those in the static flask culture were flattened in shape. Confocal microscopy revealed that chondrocytes from the bioreactor were attached on both sides of the scaffold and sustained viability better during the culture period. Uronic acid contents of the scaffolds, cultured in bioreactor, were significantly higher than in those cultured in static flasks for 4 weeks. In summary, our data suggests that the bioreactor is superior over the static flask culture when culturing chondrocytes in knitted PLDLA scaffold.

Brief intervention for male heavy drinkers in routine general practice: a three-year randomized controlled study.

The aim of this research was to evaluate the effectiveness of long-term brief intervention in routine general practice. In five primary care out-patient clinics in a Finnish town, 296 male early-phase heavy drinkers consulting a general practitioner (GP) for various reasons were identified. Control group C (n = 88) was informed of the risks of drinking after the screening and were advised at the subsequent feedback about 2 weeks later to reduce their drinking. Groups A (n = 109) and B (n = 99) were offered in addition seven and three brief intervention sessions, respectively. All GPs took part, whether or not they indicated a special interest. The main outcome measures were differences between beginning and end-point at 3 years in self-reported alcohol consumption, mean corpuscular volume (MCV), and serum carbohydrate-deficient transferrin, aspartate aminotransferase, alanine aminotransferase and gamma-glutamyltransferase. There were no statistically significant differences between study groups A, B and C in mean changes in outcome measures. Within all the groups, MCV decreased. Depending on the outcome measure used and the study group analysed, clinically significant reduction of drinking was found in 25-53% of the subjects. In routine general practice, giving additional sessions of brief intervention may not be as effective as in special research conditions. Factors reducing the effectiveness of brief intervention programmes should be investigated, so that primary health care staff can be better supported in their efforts.