Evaluation of BMP2/miRNA co-expression systems for potent therapeutic efficacy in bone-tissue regeneration.

Reconstruction of bone defects and compensation of deficient repair mechanisms represent important goals within the field of regenerative medicine and require novel safe strategies for translation into the clinic. A non-viral osteogenic gene therapeutic vector system ('hybrid vectors') was generated, combining an improved bone morphogenetic protein 2 (BMP2) gene cassette and single pro-osteogenic microRNAs (miR-148b-3p, miR-20-5p, miR-590b-5p), driven by the U6 promoter. The vectors were tested in vitro for their osteogenic differentiation potential in C2C12 and C3H/10T1/2 cell lines, using BMP2 alone as control. After confirming BMP2 expression and miRNA transcription, increased osteogenic differentiation was observed by all hybrid vectors, but most consistently by BMP2/miR-590-5p, using alkaline phosphatase enzyme activity assays and osteogenic marker mRNA quantitation, including runt-related transcription factor 2 (Runx2), collagen type 1 (Col1a1) and osteocalcin. To visualise target mRNAs of the respective miRNAs, next generation sequencing was performed, confirming down-regulation of mRNA targets of the hybrid vectors. Since the hybrid vector consisting of BMP2 and miR-590-5p showed the largest increase in osteogenic differentiation in vitro, this was tested in a mouse ectopic-bone model. Mineralisation was more than with BMP2 alone. The present study showed hybrid vectors as a novel non-viral gene therapeutic plasmid system for combining therapeutic effects of recombinant protein expression and miRNA transcription that did not add to the burden of the translation machinery, while improving the therapeutic efficacies. In vivo proof-of-principle in the context of bone regeneration suggested that such hybrid vectors will be applicable in a wide array of gene therapeutic strategies.

Repopulation of decellularised articular cartilage by laser-based matrix engraving.

BACKGROUND: In spite of advances in the treatment of cartilage defects using cell and scaffold-based therapeutic strategies, the long-term outcome is still not satisfying since clinical scores decline years after treatment. Scaffold materials currently used in clinical settings have shown limitations in providing suitable biomechanical properties and an authentic and protective environment for regenerative cells. To tackle this problem, we developed a scaffold material based on decellularised human articular cartilage. METHODS: Human articular cartilage matrix was engraved using a CO2 laser and treated for decellularisation and glycosaminoglycan removal. Characterisation of the resulting scaffold was performed via mechanical testing, DNA and GAG quantification and in vitro cultivation with adipose-derived stromal cells (ASC). Cell vitality, adhesion and chondrogenic differentiation were assessed. An ectopic, unloaded mouse model was used for the assessment of the in vivo performance of the scaffold in combination with ASC and human as well as bovine chondrocytes. The novel scaffold was compared to a commercial collagen type I/III scaffold. FINDINGS: Crossed line engravings of the matrix allowed for a most regular and ubiquitous distribution of cells and chemical as well as enzymatic matrix treatment was performed to increase cell adhesion. The biomechanical characteristics of this novel scaffold that we term CartiScaff were found to be superior to those of commercially available materials. Neo-tissue was integrated excellently into the scaffold matrix and new collagen fibres were guided by the laser incisions towards a vertical alignment, a typical feature of native cartilage important for nutrition and biomechanics. In an ectopic, unloaded in vivo model, chondrocytes and mesenchymal stromal cells differentiated within the incisions despite the lack of growth factors and load, indicating a strong chondrogenic microenvironment within the scaffold incisions. Cells, most noticeably bone marrow-derived cells, were able to repopulate the empty chondrocyte lacunae inside the scaffold matrix. INTERPRETATION: Due to the better load-bearing, its chondrogenic effect and the ability to guide matrix-deposition, CartiScaff is a promising biomaterial to accelerate rehabilitation and to improve long term clinical success of cartilage defect treatment. FUNDING: Austrian Research Promotion Agency FFG ("CartiScaff" #842455), Lorenz Böhler Fonds (16/13), City of Vienna Competence Team Project Signaltissue (MA23, #18-08).

Stromal vascular fraction cells as biologic coating of mesh for hernia repair.

BACKGROUND: The interest in non-manipulated cells originating from adipose tissue has raised tremendously in the field of tissue engineering and regenerative medicine. The resulting stromal vascular fraction (SVF) cells have been successfully used in numerous clinical applications. The aim of this experimental work is, first to combine a macroporous synthetic mesh with SVF isolated using a mechanical disruption process, and to assess the effect of those cells on the early healing phase of hernia. METHODS: Human SVF cells combined with fibrin were used to coat commercial titanized polypropylene meshes. In vitro, viability and growth of the SVF cells were assessed using live/dead staining and scanning electron microscopy. The influence of SVF cells on abdominal wall hernia healing was conducted on immunodeficient rats, with a focus on short-term vascularization and fibrogenesis. RESULTS: Macroporous meshes were easily coated with SVF using a fibrin gel as temporary carrier. The in vitro experiments showed that the whole process including the isolation of human SVF cells and their coating on PP meshes did not impact on the SVF cells' viability and on their capacity to attach and to proliferate. In vivo, the SVF cells were well tolerated by the animals, and coating mesh with SVF resulted in a decrease degree of vascularity compared to control group at day 21. CONCLUSIONS: The utilization of SVF-coated mesh influences the level of angiogenesis during the early onset of tissue healing. Further long-term animal experiments are needed to confirm that this effect correlates with a more robust mesh integration compared to non-SVF-coated mesh.

Repopulation of an auricular cartilage scaffold, AuriScaff, perforated with an enzyme combination.

Biomaterials currently in use for articular cartilage regeneration do not mimic the composition or architecture of hyaline cartilage, leading to the formation of repair tissue with inferior characteristics. In this study we demonstrate the use of "AuriScaff", an enzymatically perforated bovine auricular cartilage scaffold, as a novel biomaterial for repopulation with regenerative cells and for the formation of high-quality hyaline cartilage. AuriScaff features a traversing channel network, generated by selective depletion of elastic fibers, enabling uniform repopulation with therapeutic cells. The complex collagen type II matrix is left intact, as observed by immunohistochemistry, SEM and TEM. The compressive modulus is diminished, but three times higher than in the clinically used collagen type I/III scaffold that served as control. Seeding tests with human articular chondrocytes (hAC) alone and in co-culture with human adipose-derived stromal/stem cells (ASC) confirmed that the network enabled cell migration throughout the scaffold. It also guides collagen alignment along the channels and, due to the generally traverse channel alignment, newly deposited cartilage matrix corresponds with the orientation of collagen within articular cartilage. In an osteochondral plug model, AuriScaff filled the complete defect with compact collagen type II matrix and enabled chondrogenic differentiation inside the channels. Using adult articular chondrocytes from bovine origin (bAC), filling of even deep defects with high-quality hyaline-like cartilage was achieved after 6 weeks in vivo. With its composition and spatial organization, AuriScaff provides an optimal chondrogenic environment for therapeutic cells to treat cartilage defects and is expected to improve long-term outcome by channel-guided repopulation followed by matrix deposition and alignment. STATEMENT OF SIGNIFICANCE: After two decades of tissue engineering for cartilage regeneration, there is still no optimal strategy available to overcome problems such as inconsistent clinical outcome, early and late graft failures. Especially large defects are dependent on biomaterials and their scaffolding, guiding and protective function. Considering the currently used biomaterials, structure and mechanical properties appear to be insufficient to fulfill this task. The novel scaffold developed within this study is the first approach enabling the use of dense cartilage matrix, repopulate it via channels and provide the cells with a compact collagen type II environment. Due to its density, it also provides better mechanical properties than materials currently used in clinics. We therefore think, that the auricular cartilage scaffold (AuriScaff) has a high potential to improve future cartilage regeneration approaches.

Abdominal wall reinforcement: biologic vs. degradable synthetic devices.

BACKGROUND: New biodegradable synthetic and biologic hernia implants have been promoted for rapid integration and tissue reinforcement in challenging repairs, e.g. at the hiatus or in contaminated wound fields. Interestingly, experimental data to support or falsify this assumption is scarce. METHODS: Synthetic (BioA®) and biologic implants (porcine and bovine collagen matrices Strattice® and Veritas®) have been tested in experimental onlay hernia repair in rats in observation periods of 30 and 60 days. The key outcome parameters were mesh integration and reinforcement of the tissue at the implant site over sutured and sealed defects as well as comparison to native abdominal wall. Macroscopic assessment, biomechanical analysis and histology with haematoxylin/eosin staining, collagen staining and van Willebrand factor staining for detection of neovascularization were performed. RESULTS: BioA® was well integrated. Although the matrices were already fragmented at 60 days follow-up, hernia sites treated with synthetic scaffolds showed a significantly enhanced tissue deflection and resistance to burst force when compared to the native abdominal wall. In porcine and bovine matrices, tissue integration and shrinkage were significantly inferior to BioA®. Histology revealed a lack of fibroblast ingrowth through mesh interstices in biologic samples, whereas BioA® was tightly connected to the underlying tissue by reticular collagen fibres. CONCLUSIONS: Strattice® and Veritas® yielded reduced tissue integration and significant shrinkage, prohibiting further biomechanical tests. The synthetic BioA® provides little inherent strength but reticular collagen remodelling led to an augmentation of the scar due to significantly higher burst force resistance in comparison to native tissue.

Liquid antiadhesive agents for intraperitoneal hernia repair procedures: Artiss® compared to CoSeal® and Adept® in an IPOM rat model.

BACKGROUND: Adhesion formation remains an important issue in hernia surgery. Liquid agents were developed for easy and versatile application, especially in laparoscopy. The aim of this study was to compare the antiadhesive effect of fibrin sealant (FS, Artiss®), Icodextrin (ID, Adept®) and Polyethylene glycol (PEG, CoSeal®) alone and in combination and to evaluate the resulting effect on tissue integration of the mesh. METHODS: A total of 56 Sprague-Dawley rats were operated in open IPOM technique. A middleweight polypropylene mesh of 2 × 2 cm size was implanted and covered with 1: FS, 2: ID, 3: PEG, 4: FS + ID, 5: FS + PEG, 6: PEG + ID, 7: control group, uncovered mesh (n = 8 per treatment/control). Observation period was 30 days. Macroscopic and histological evaluation was performed. RESULTS: Severe adhesions were found in group 2 (ID), group 6 (PEG + ID) and the controls. Best results were achieved with FS alone or FS + ID. Mesh integration in the treatment groups was reduced in comparison with the control group. This is a new finding possibly relevant for the outcome of intraperitoneal mesh repair. Group 6 (PEG + ID) showed an impairment of tissue integration with <50 % of the mesh surface in seven samples. CONCLUSION: FS alone and in combination with ID yielded excellent adhesion prevention. ID alone did not show significant adhesion prevention after 30 days. Tissue integration of FS-covered meshes was superior to ID or PEG alone or combined. PEG did show adhesion prevention comparable to FS but evoked impaired tissue integration. So Artiss® is among the most potent antiadhesive agents in IPOM repair.

Erratum to: The impact of hydrophobic hernia mesh coating by omega fatty acid on atraumatic fibrin sealant fixation.

Erratum to: Hernia DOI 10.1007/s10029-014-1304-y. The authors would like to notify readers that unfortunately, Fig. 1 and associated captions are incorrectly published in the original publication. The correct figure and legend are given here.

The impact of hydrophobic hernia mesh coating by omega fatty acid on atraumatic fibrin sealant fixation.

BACKGROUND: Fibrin sealant (FS) is a safe and efficient fixation method in open intraperitoneal hernia repair. While favourable results have been achieved with hydrophilic meshes, hydrophobic (such as Omega fatty acid coated) meshes (OFM) have not been specifically assessed so far. Atrium C-qur lite(®) mesh was tested in rats in models of open onlay and intraperitoneal hernia repair. METHODS: 44 meshes (2 × 2 cm) were implanted in 30 male Sprague-Dawley rats in open (n = 2 meshes per animal) and intraperitoneal technique (IPOM; n = 1 mesh per animal). Animals were randomised to four groups: onlay and IPOM sutured vs. sealed. Follow-up was 6 weeks, sutured groups serving as controls. Evaluation criteria were mesh dislocation, adhesions and foreign body reaction. RESULTS: FS provided a reliable fixation in onlay technique, whereas OFM meshes dislocated in the IPOM position when sealed only. CONCLUSION: FS mesh fixation was safe with OFM meshes in open onlay repair. Intraperitoneal placement of hydrophobic meshes requires additional fixation and cannot be achieved with FS alone.

A comparison of Progrip(®) and Adhesix (®) self-adhering hernia meshes in an onlay model in the rat.

BACKGROUND: Atraumatic fixation is a key element of modern hernia repair. Two different concepts of self-adhering meshes were directly compared in this study. Adhesix(®) (AH) is coated with polyethylene glycol (PEG) and polyvinylpyrrolidone (PVP), whereas Parietene Progrip(®) (PP) relies on the mechanical principle of micro grips made of polylactic acid (PLA). These meshes are the main competitors in the field. METHODS: AH and PP were tested in Sprague-Dawley rats at 14 and 90 days. Four groups were operated (n = 8 animals per group). Two meshes were implanted per animal in an operation model of onlay hernia repair. Dislocation, tissue integration and foreign-body reaction were evaluated. RESULTS: AH dislocated significantly more frequently (every second mesh) at both time points of observation than PP. Tissue integration was good with PP and could not be reliably assessed in AH due to frequent dislocation. Histologic examination revealed only a mild foreign body reaction in all groups. CONCLUSIONS: In our hands, PP (mechanical grip fixation) was superior to hydrogel fixation with PEG and PVP in AH in an onlay model. The reason for dislocation of AH requires further clarification as well as the impact of long-term degradation of the PLA grips.

Comparison of Ringer's solution with 0.4% glucose or without in intraoperative infusion regimens for the prevention of hypoglycemia in juvenile pigs.

Juvenile pigs are prone to hypoglycemia, nevertheless they are commonly used in preclinical studies. The optimal perioperative fluid management with crystalloid solutions in such settings has not yet been studied. The aim of the current study was to correlate the perioperative fluid administration of Ringer's solution, with and without the addition of a minimal amount of glucose (0.4% glucose) to the blood glucose, and the incidence of perioperative hypoglycemia in 20 juvenile pigs (31.7 ± 5.2 kg) undergoing surgery for approximately one hour. Infusion of Ringer's solution without glucose (group Ri, n = 10) led to a significant decrease of the serum blood glucose level from 105.7 ± 27.1 mg/dL at baseline compared with 87.8 ± 31.7 mg/dL post surgery. The pigs (50%) in group Ri were observed to be hypoglycemic (<72 mg/dL). The second group received Ringer's solution containing a final concentration of 0.4% glucose (group Ri-Glu, n = 10) and showed a significant increase in blood glucose level from 104.4 ± 15.8 mg/dL at baseline compared with 122.3 ± 14.3 mg/dL post surgery. No animal in group Ri-Glu was observed to be hypoglycemic. In conclusion, we showed that even minor changes in perioperative crystalloid fluid management significantly influence blood glucose levels. In order to avoid hypoglycemia in juvenile pigs we recommend adding a minimal amount of glucose (0.4% final concentration) to the Ringer's solution given perioperatively during surgery of approximately one hour.