Marta Kisiel

Improving the osteogenic potential of BMP-2 with hyaluronic acid hydrogel modified with integrin-specific fibronectin fragment

While human bone morphogenetic protein-2 (rhBMP-2) is a promising growth factor for bone regeneration, its clinical efficacy has recently shown to be below expectation. In order to improve the clinical translation of rhBMP-2, there exists strong motivation to engineer better delivery systems. Hyaluronic acid (HA) hydrogel is a suitable carrier for the delivery of rhBMP-2, but a major limitation of this scaffold is its low cell adhesive properties. In this study, we have determined whether covalent grafting of an integrin-specific ligands into HA hydrogel could improve cell attachment and further enhance the osteogenic potential of rhBMP-2. A structurally stabilized fibronectin (FN) fragment containing the major integrin-binding domain of full-length FN (FN III9*-10) was engineered, in order to be incorporated into HA hydrogel. Compared to non-functionalized HA hydrogel, HA-FN hydrogel remarkably improved the capacity of the material to support mesenchymal stem cell attachment and spreading. In an ectopic bone formation model in the rat, delivery of rhBMP-2 with HA-FN hydrogel resulted in the formation of twice as much bone with better organization of collagen fibers compared to delivering the growth factor in non-functionalized HA hydrogel. This engineered hydrogel carrier for rhBMP-2 can be relevant in clinical bone repair.

Complexation and sequestration of BMP-2 from an ECM mimetic hyaluronan gel for improved bone formation

Bone morphogenetic protein-2 (BMP-2) is considered a promising adjuvant for the treatment of skeletal non-union and spinal fusion. However, BMP-2 delivery in a conventional collagen scaffold necessitates a high dose to achieve an efficacious outcome. To lower its effective dose, we precomplexed BMP-2 with the glycosaminoglycans (GAGs) dermatan sulfate (DS) or heparin (HP), prior to loading it into a hyaluronic acid (HA) hydrogel. In vitro release studies showed that BMP-2 precomplexed with DS or HP had a prolonged delivery compared to without GAG. BMP-2-DS complexes achieved a slightly faster release in the first 24 h than HP; however, both delivered BMP-2 for an equal duration. Analysis of the kinetic interaction between BMP-2 and DS or HP showed that HP had approximately 10 times higher affinity for BMP-2 than DS, yet it equally stabilized the protein, as determined by alkaline phosphatase activity. Ectopic bone formation assays at subcutaneous sites in rats demonstrated that HA hydrogel-delivered BMP-2 precomplexed with GAG induced twice the volume of bone compared with BMP-2 delivered uncomplexed to GAG.

Critical assessment of rhBMP-2 mediated bone induction: An in vitro and in vivo evaluation

Understanding the influence of formulation and storage conditions on rhBMP-2 bioactivity is extremely important for its clinical application. Reports in the literature show that different research groups employ different parameters such as formulation conditions, storage, doses for in vivo applications etc. that makes it difficult to correlate results from different experiments. We therefore decided to rationalize these anomalies by performing a basic study on such parameters using two commercially available BMPs. Our in vitro experiments suggest that BMPs from different sources have significant differences in their bioactivity. The clinically approved rhBMP-2 (InductOs®; BMP-P) showed superior stability, compared to rhBMP-2 from R&D Systems (BMP-R) at physiological pH (determined by ALP assay). This BMP-P also showed lower binding to polypropylene Eppendorf tube. The BMP-R almost lost its bioactivity within 30 min at physiological pH and also shows more adhesion to plastic surfaces. This aggregation behavior was unequivocally ascertained by performing light scattering studies of the two BMPs, which revealed linear aggregation with time for BMP-R unlike BMP-P. The in vitro results were also reflected in the in vivo experiments, in a rat ectopic model with injectable hyaluronic acid (HA) hydrogel as BMP carrier. After 7 weeks post-implantation we observed larger bone volume with oriented collagen in the BMP-P group but a smaller bone with disoriented collagen in the BMP-R case. Our results highlight the large difference in activity between seemingly identical substances and also the importance of proper handling of such sensitive proteins.

Minimally invasive mandibular bone augmentation using injectable hydrogels

Hyaluronic acid‐based hydrogels are proven biocompatible materials and excellent carriers of bone morphogenetic protein‐2 (BMP‐2) that have been successfully tested for bone generation in vivo. Different formulations, with or without nanohydroxyapatite, have shown promise for craniofacial applications. In this study, 28 rats were used to investigate whether it is possible to achieve mandibular bone augmentation upon injection of novel hyaluronic acid‐based hydrogels containing nanohydroxyapatite and different concentrations of BMP‐2 (0, 5 and 150 µg/ml). The biomaterials were injected subperiosteally through fine needles into the innate mandibular diastema, imitating a clinical procedure for resorbed mandibles. No incisions, flaps or sutures were necessary. After 8 weeks the mandibles were evaluated by peripheral quantitative computed tomography (pQCT), micro‐computed tomography (μCT), histology, immunohistochemistry and fluorochrome labelling. As a result, engineered bone was observed in all treated mandibles, with a statistically significant increase in mandibular bone volume correlated with the amount of BMP‐2 loaded in the hydrogel formula. We therefore demonstrated that minimally invasive mandibular bone augmentation is possible upon injection in rats, when using the appropriate injectable scaffolds. This represents an attractive clinical alternative for oral implantology patients. Copyright

Synthesis of guanidinium-modified hyaluronic Acid hydrogel.

In this report, a new guanidinylating reagent is presented, which was developed without any protection/deprotection strategy and was successfully employed for linking to hyaluronan in aqueous solution. The dually functionalised HA biopolymer bearing guanidinium and hydrazide groups was synthesised to form hydrogel in less than a minute when mixed with aldehyde-modified HA. This hydrogel exhibited higher storage modulus with enhanced stability in PBS when compared to the non-guanidine-containing gel. The gel shift assay showed that this biopolymer formed a stable complex with DNA as well as efficient gene transfection to cells that express HA-receptor CD44. The toxicity studies of this polymer with fibroblast cells revealed that the cells were almost 80% viable after 4 d of incubation at high HA concentration (2.5 × 10(-3)  M).

Evaluation of injectable constructs for bone repair with a subperiosteal cranial model in the rat

While testing regenerative medicine strategies, the use of animal models that match the research questions and that are related to clinical translation is crucial. During the initial stage of evaluating new strategies for bone repair, the main goal is to state whether the strategies efficiently induce the formation of new bone tissue at an orthotopic site. Here, we present a subperiosteal model in rat calvaria that allow the evaluation of a broad range of approaches including bone augmentation, replacement and regeneration. The model is a fast to perform, minimally invasive, and has clearly defined control groups. The procedure enables to evaluate the outcomes quantitatively using micro-computed tomography and qualitatively by histology and immunohistochemistry. We established this new model, using bone morphogenetic protein-2 as an osteoinductive factor and hyaluronic acid hydrogel as injectable biomaterial. We showed that this subperiosteal cranial model offers a minimally invasive and promising solution for a rapid initial evaluation of injectables for bone repair. We believe that this approach could be a powerful platform for orthopedic research and regenerative medicine.

Bone Engineering by Biomimetic Injectable Hydrogel

Osteoporosis is a multifactorial bone disease characterized by low bone mineral density (BMD) and deterioration of micro-architecture of cancellous bone leading to bone fragility and risk of fractures. In the current work, a novel tissue engineering strategy was experimented to enhance bone architecture in the risk areas via local injection of a biomimetic/osteoinductive injectable hyaluronan based hydrogel loaded with nano-hydroxyapatite crystals (Hya/HA) with/without bone morphogenetic protein (BMP-2), in distal femur of normal and ovariectomized New Zealand white rabbits. Our results revealed the osteoinductive effect of the Hya/HA composite that enhanced bone density and architecture of the rabbit distal femur.

Smart design of stable hydrazone crosslinked extracellular matrix mimetic hydrogel for tissue engineering application

Adequate cellular in-growth into biomaterials is one of the fundamental requirements in regenerative medicine. Type-I-collagen is the most commonly used material for soft tissue engineering, because it is nonimmunogenic and a highly porous network for cellular support. However, adequate cell in-growth and cell seeding has been suboptimal. Different densities of collagen scaffolds (0.3% to 0.8% (w/v)) with/without polymer knitting (poly-caprolactone (PCL)) were prepared. The structure of collagen scaffolds was characterized using scanning electronic microscopy (SEM) and HE staining. The mechanical strength of hybrid scaffolds was determined using tensile strength analysis. Cellular penetration and interconnectivity were evaluated using fluorescent bead distribution and human bladder smooth muscle cells and urothelium seeding. SEM and HE analysis showed the honeycomb structure and the hybrid scaffolds were adequately connected. The hybrid scaffolds were much stronger than collagen alone. The distribution of the beads and cells were highly dependent on the collagen density: at lower densities the beads and cells were more evenly distributed and penetrated deeper into the scaffold. The lower density collagen scaffolds showed remarkably deeper cellular penetration and by combining it with PCL knitting the tensile strength was enhanced. This study indicated that a 0.4% hybrid scaffold strengthened with knitting achieved the best cellular distribution.Human adult heart harbors a population of resident progenitor cells that can be isolated by Sca-1 antibody and expanded in culture. These cells can differentiate into cardiomyocytes and vascular cells in vitro and contribute to cardiac regeneration in vivo. However, when directly injected as single cell suspension, the survival rate and retention is really poor, less than 1% of injected cells being detectable in the hosttissue within few weeks. The present study aimed at investigating the possibility to produce scaffoldless, thick cardiac progenitor cell-derived cardiac patches by thermo-responsive technology. Human cardiac progenitors obtained from the auricles of patients were cultured as scaffoldless engineered tissues fabricated using temperature-responsive surfaces obtained by poly-N-isopropylacrylamide (PNIPAAm) surface immobilization. In the engineered tissue, progenitor cells established proper three-dimensional intercellular relationships and produced abundant extracellular matrix, while preserving their phenotype and plasticity. Cell phenotype and viability within the 3D construct were followed for 1 week, showing that no significant differentiation or apoptotic events occurred within the construct. After engineered tissues were leant on visceral pericardium, a number of cells migrated into the myocardium and in the vascular walls, where they integrated in the respective textures. The study demonstrates the suitability of such approach to deliver stem cells.Spinal cord injury and repair is one of the important focus areas in tissue regeneration. Mechanical trauma caused due to factors such as contusion, compression or involuntary stretching induce post-traumatic secondary tissue damage in many Spinal Cord Injury (SCI) patients. Therefore, there is a need for scaffolds that provide a conducive threedimensionsal (3D) environment for injured cells to attach and grow. In this study we propose to synthesize 3D polymeric scaffolds in order to study the mechanical and adhesive properties & the nature of the interactions between hyaluronan-based (HY) biomaterials and cells and tissues both in vitroandin vivo. Here we have synthesized 3D HY-based hydrogels with robust mechanical and adhesive properties and demonstrate the use of this material for neuronal-related applications such as the treatment of SCI. Cell culture and survivability studies were done with NSC-34 cells. Live/Dead assay performed on the cells revealed significant differences in the staining of live cells and showed increased viability and proliferation. The number of live cells in the HY-based hydrogels with 0.1% collagen showed higher cell numbers compared with the other hydrogels. In this study we show that Injectable HYbased hydrogels with high elasticity, comparable to the mechanical properties of nervous tissue have been used in this study to study their biocompatibility and neuroprotective properties and they show better affinity for neuronal cells.Calcium phosphates (CaP) obtained by biomineralisation in Simulated Boby Fluid have been used for decades to assess the mineralisation capability of biomaterials. Recently, they have been envisioned as potential agents to promote bone formation. In this study, we have fabricated and coated with calcium phosphate melt electrospun scaffolds whereby macropores permit adequate cell migration and nutrient transfer. We have systematically investigated the effect of coating and osteoinduction onto the response of ovine osteoblasts and we observed that the coating up-regulated alkaline phosphatase activity regardless of the in vitro culture conditions. Micro Computed Tomography revealed that only scaffolds cultured in an osteoinductive cocktail were capable of depositing mineralised matrix, and that CaP coated scaffolds were more efficient at promoting mineralisation. Theses scaffolds were subcutaneously implanted in athymic rats and this demonstrated that the osteoinduction was a pre-requisite for bone formation in this ectopic model. It showed that although the bone formation was not significantly different after 8 weeks, the CaP coated scaffolds were superior at inducing bone formation as evidenced by higher levels of mineralisation at earlier time points. This work demonstrated that CaP coating is not sufficient to induce bone formation; however the combination of osteoinduction and CaP coating resulted in earlier bone formation in an ectopic model.Introduction: Bladder regeneration using minced bladder mucosa is an alternative to costly and time-consuming conventional in vitro culturing of urothelial cells. In this method, the uroepithelium ...