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.

Histomorphometric Analysis of Callus Formation Stimulated by Axial Dynamisation in a Standardised Ovine Osteotomy Model.

The cyclic axial dynamisation of a stabilised fracture is intended to promote callus formation and bone healing. Most studies focused on biomechanical properties or the quantity of new bone formation. Far less is known about the quality of newly formed callus tissues, such as tissue distribution and arrangement within the callus. The aim of this current study was to investigate the effect of cyclic, axial dynamisation on the quantity and quality of callus in an established delayed fracture healing model. In 41 sheep transverse osteotomies with a gap size of 3 mm were stabilised with a unilateral external fixator. In 32 of these, fracture ends were axially stimulated with displacement amplitudes of 0.8 mm, 0.4 mm, 0.2 mm, or 0.0 mm, respectively, for six weeks. In the remaining 9 sheep of the control group, an additional external fixator was mounted to achieve almost total rigidity. Animal material originating from a past animal experiment was reanalysed in this study. Histological thin-ground sections were histomorphometrically analysed regarding the histological structure and composition of the defect region. A slight tendency towards an increase in size of total callus area, area of new bone (nB.Ar), and cartilage (Cg.Ar) was detected with increasing displacement amplitudes compared to the control group. At the anterior callus side nB.Ar and Cg.Ar were significantly larger than at the posterior side in all groups independent of treatment. Regarding the quality of callus, areas of very compact bone were predominant in the treatment groups whereas in the control group a slight shift to more porous bone was observed. No difference of callus compactness was observed between the anterior and the posterior side. The established method to assess the local compactness of callus areas is a useful tool to quantitatively determine the spatial distribution of new bone tissue within the callus. The application of this method in combination with biomechanical testing might reveal interesting relations between tissue distribution and bone strength that, with traditional histomorphometry, cannot be identified.

Adipose-tissue-derived therapeutic cells in their natural environment as an autologous cell therapy strategy: the microtissue-stromal vascular fraction.

The prerequisite for a successful clinical use of autologous adipose-tissue-derived cells is the highest possible regenerative potential of the applied cell population, the stromal vascular fraction (SVF). Current isolation methods depend on high enzyme concentration, lysis buffer, long incubation steps and mechanical stress, resulting in single cell dissociation. The aim of the study was to limit cell manipulation and obtain a derivative comprising therapeutic cells (microtissue-SVF) without dissociation from their natural extracellular matrix, by employing a gentle good manufacturing practice (GMP)-grade isolation. The microtissue-SVF yielded larger numbers of viable cells as compared to the improved standard-SVF, both with low enzyme concentration and minimal dead cell content. It comprised stromal tissue compounds (collagen, glycosaminoglycans, fibroblasts), capillaries and vessel structures (CD31+, smooth muscle actin+). A broad range of cell types was identified by surface-marker characterisation, including mesenchymal, haematopoietic, pericytic, blood and lymphatic vascular and epithelial cells. Subpopulations such as supra-adventitial adipose-derived stromal/stem cells and endothelial progenitor cells were significantly more abundant in the microtissue-SVF, corroborated by significantly higher potency for angiogenic tube-like structure formation in vitro. The microtissue-SVF showed the characteristic phenotype and tri-lineage mesenchymal differentiation potential in vitro and an immunomodulatory and pro-angiogenic secretome. In vivo implantation of the microtissue-SVF combined with fat demonstrated successful graft integration in nude mice. The present study demonstrated a fast and gentle isolation by minor manipulation of liposuction material, achieving a therapeutically relevant cell population with high vascularisation potential and immunomodulatory properties still embedded in a fraction of its original matrix.

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.

A critical review of the in vitro and in vivo models for the evaluation of anti-infective meshes.

BACKGROUND: Infectious complications following mesh implantation for abdominal wall repair appear in 0.7 up to 26.6% of hernia repairs and can have a detrimental impact for the patient. To prevent or to treat mesh-related infection, the scientific community is currently developing a veritable arsenal of antibacterial meshes. The numerous and increasing reports published every year describing new technologies indicate a clear clinical need, and an academic interest in solving this problem. Nevertheless, to really appreciate, to challenge, to compare and to optimize the antibacterial properties of next generation meshes, it is important to know which models are available and to understand them. PURPOSE: We proposed for the first time, a complete overview focusing only on the in vitro and in vivo models which have been employed specifically in the field of antibacterial meshes for hernia repair. RESULTS AND CONCLUSION: From this investigation, it is clear that there has been vast progress and breadth in new technologies and models to test them. However, it also shows that standardization or adoption of a more restricted number of models would improve comparability and be a benefit to the field of study.

Photobiomodulation of freshly isolated human adipose tissue-derived stromal vascular fraction cells by pulsed light-emitting diodes for direct clinical application.

A highly interesting source for adult stem cells is adipose tissue, from which the stromal vascular fraction (SVF)-a heterogeneous cell population including the adipose-derived stromal/stem cells-can be obtained. To enhance the regenerative potential of freshly isolated SVF cells, low-level light therapy (LLLT) was used. The effects of pulsed blue (475 nm), green (516 nm), and red (635 nm) light from light-emitting diodes applied on freshly isolated SVF were analysed regarding cell phenotype, cell number, viability, adenosine triphosphate content, cytotoxicity, and proliferation but also osteogenic, adipogenic, and proangiogenic differentiation potential. The colony-forming unit fibroblast assay revealed a significantly increased colony size after LLLT with red light compared with untreated cells, whereas the frequency of colony-forming cells was not affected. LLLT with green and red light resulted in a stronger capacity to form vascular tubes by SVF when cultured within 3D fibrin matrices compared with untreated cells, which was corroborated by increased number and length of the single tubes and a significantly higher concentration of vascular endothelial growth factor. Our study showed beneficial effects after LLLT on the vascularization potential and proliferation capacity of SVF cells. Therefore, LLLT using pulsed light-emitting diode light might represent a new approach for activation of freshly isolated SVF cells for direct clinical application.

Infections associated with mesh repairs of abdominal wall hernias: Are antimicrobial biomaterials the longed-for solution?

The incidence of mesh-related infection after abdominal wall hernia repair is low, generally between 1 and 4%; however, worldwide, this corresponds to tens of thousands of difficult cases to treat annually. Adopting best practices in prevention is one of the keys to reduce the incidence of mesh-related infection. Once the infection is established, however, only a limited number of options are available that provides an efficient and successful treatment outcome. Over the past few years, there has been a tremendous amount of research dedicated to the functionalization of prosthetic meshes with antimicrobial properties, with some receiving regulatory approval and are currently available for clinical use. In this context, it is important to review the clinical importance of mesh infection, its risk factors, prophylaxis and pathogenicity. In addition, we give an overview of the main functionalization approaches that have been applied on meshes to confer anti-bacterial protection, the respective benefits and limitations, and finally some relevant future directions.

Fibrin-based delivery strategies for acute and chronic wound healing.

Fibrin, a natural hydrogel, is the end product of the physiological blood coagulation cascade and naturally involved in wound healing. Beyond its role in hemostasis, it acts as a local reservoir for growth factors and as a provisional matrix for invading cells that drive the regenerative process. Its unique intrinsic features do not only promote wound healing directly via modulation of cell behavior but it can also be fine-tuned to evolve into a delivery system for sustained release of therapeutic biomolecules, cells and gene vectors. To further augment tissue regeneration potential, current strategies exploit and modify the chemical and physical characteristics of fibrin to employ combined incorporation of several factors and their timed release. In this work we show advanced therapeutic approaches employing fibrin matrices in wound healing and cover the many possibilities fibrin offers to the field of regenerative medicine.