Benedetto Marelli

Accelerated mineralization of dense collagen-nano bioactive glass hybrid gels increases scaffold stiffness and regulates osteoblastic function

Plastically compressed dense collagen (DC) gels mimic the microstructural, mechanical, and biological properties of native osteoid. This study investigated the effect of hybridizing DC with osteoinductive nano-sized bioactive glass (nBG) particles in order to potentially produce readily implantable, and mineralizable, cell seeded hydrogel scaffolds for bone tissue engineering. Due to the high surface area of nBG and increased reactivity, calcium phosphate formation was immediately detected within as processed DC-nGB hybrid gel scaffolds. By day 3 in simulated body fluid, accelerated mineralization was confirmed through the homogeneous growth of carbonated hydroxylapatite on the nanofibrillar collagen framework. At day 7, there was a 13 fold increase in the hybrid gel scaffold compressive modulus. MC3T3-E1 pre-osteoblasts, three-dimensionally seeded at the point of nanocomposite self-assembly, were viable up to day 28 in culture. In the absence of osteogenic supplements, MC3T3-E1 metabolic activity and alkaline phosphatase production were affected by the presence of nBG, indicating accelerated osteogenic differentiation. Additionally, no cell-induced contraction of DC-nBG gel scaffolds was detected. The accelerated mineralization of rapidly produced DC-nBG hybrid gels indicates their potential suitability as osteoinductive cell delivery scaffolds for bone regenerative therapy.

Compliant electrospun silk fibroin tubes for small vessel bypass grafting.

Processing silk fibroin (SF) by electrospinning offers a very attractive opportunity for producing three-dimensional nanofibrillar matrices in tubular form, which may be useful for a biomimetic approach to small calibre vessel regeneration. Bypass grafting of small calibre vessels, with a diameter less than 6mm, is performed mainly using autografts, like the saphenous vein or internal mammary artery. At present no polymeric grafts made of SF are commercially available, mainly due to inadequate properties (low compliance and lack of endothelium cells). The aim of this work was to electrospin SF into tubular structures (Ø=6mm) for small calibre vessel grafting, characterize the morphological, chemico-physical and mechanical properties of the electrospun SF structures and to validate their potential to interact with cells. The morphological properties of electrospun SF nanofibres were investigated by scanning electron microscopy. Chemico-physical analyses revealed an increase in the crystallinity of the structure of SF nanofibres on methanol treatment. Mechanical tests, i.e. compliance and burst pressure measurements, of the electrospun SF tubes showed that the inner pressure to radial deformation ratio was linear for elongation up to 15% and pressure up to 400 mm Hg. The mean compliance value between 80 and 120 mm Hg was higher than the values reported for both Goretex(R) and Dacron(R) grafts and for bovine heterografts, but still slightly lower than those of saphenous and umbilical vein, which nowadays represent the gold standard for the replacement of small calibre arteries. The electrospun tubes resisted up to 575+/-17 mmHg, which is more than four times the upper physiological pressure of 120 mmHg and more than twice the pathological upper pressures (range 180-220 mmHg). The in vitro tests showed a good cytocompatibility of the electrospun SF tubes. Therefore, the electrospun SF tubes developed within this work represent a suitable candidate for small calibre blood vessel replacement.

Silk-based resorbable electronic devices for remotely controlled therapy and in vivo infection abatement

Significance We present the demonstration of in vivo operation of a subcutaneously implanted, resorbable electronic device. The remotely controlled device was wirelessly activated after implantation, successfully eliminating infection, and subsequently dissolving in the surrounding tissue. This approach is a first step for the development of a class of implantable, technological, biomedical devices that resorb harmlessly, eliminating the need for retrieval after use. A paradigm shift for implantable medical devices lies at the confluence between regenerative medicine, where materials remodel and integrate in the biological milieu, and technology, through the use of recently developed material platforms based on biomaterials and bioresorbable technologies such as optics and electronics. The union of materials and technology in this context enables a class of biomedical devices that can be optically or electronically functional and yet harmlessly degrade once their use is complete. We present here a fully degradable, remotely controlled, implantable therapeutic device operating in vivo to counter a Staphylococcus aureus infection that disappears once its function is complete. This class of device provides fully resorbable packaging and electronics that can be turned on remotely, after implantation, to provide the necessary thermal therapy or trigger drug delivery. Such externally controllable, resorbable devices not only obviate the need for secondary surgeries and retrieval, but also have extended utility as therapeutic devices that can be left behind at a surgical or suturing site, following intervention, and can be externally controlled to allow for infection management by either thermal treatment or by remote triggering of drug release when there is retardation of antibiotic diffusion, deep infections are present, or when systemic antibiotic treatment alone is insufficient due to the emergence of antibiotic-resistant strains. After completion of function, the device is safely resorbed into the body, within a programmable period.

Three-dimensional mineralization of dense nanofibrillar collagen-bioglass hybrid scaffolds.

Scaffolds for bone tissue engineering must meet a number of requirements such as biocompatibility, osteoconductivity, osteoinductivity, biodegradability, and appropriate biomechanical properties. A combination of type I collagen and 45S5 Bioglass may meet these requirements, however, little has been demonstrated on the effect of Bioglass on the potential of the collagen nanofibrillar three-dimensional mineralization and its influence on the structural and mechanical properties of the scaffolds. In this work, rapidly fabricated dense collagen-Bioglass hybrid scaffolds were assessed for their potential for immediate implantation. Hybrid scaffolds were conditioned, in vitro, in simulated body fluid (SBF) for up to 14 days and assessed in terms of changes in structural, chemical, and mechanical properties. MicroCT and SEM analyses showed a homogeneous distribution of Bioglass particles in the as-made hybrids. Mineralization was detected at day 1 in SBF, while ATR-FTIR microscopy and XRD revealed the presence of hydroxyl-carbonated apatite on the surface and within the two hybrid scaffolds at days 7 and 14. FTIR and SEM confirmed that the triple helical structure and typical banding pattern of fibrillar collagen was maintained as a function of time in SBF. Principal component analysis executed on ATR-FTIR microscopy revealed that the mineralization extent was a function of both Bioglass content and conditioning time in SBF. Tensile mechanical analysis showed an increase in the elastic modulus and a corresponding decrease in strain at ultimate tensile strength (UTS) as imparted by mineralization of scaffolds as a function of time in SBF and Bioglass content. Change in UTS was affected by Bioglass content. These results suggested the achievement of a hybrid matrix potentially suitable for bone tissue engineering.

Inkjet Printing of Regenerated Silk Fibroin: From Printable Forms to Printable Functions.

A formulation of regenerated silk fibroin solution that can be easily functionalized and inkjet printed on numerous surfaces is developed. As an example, the inks can be printed on laboratory gloves that change color when exposed to bacteria.

Silk fibroin derived polypeptide-induced biomineralization of collagen.

Silk fibroin (SF) is extensively investigated in osteoregenerative therapy as it combines extraordinary mechanical properties and directs calcium-phosphate formation. However, the role of the peptidic fractions in inducing the protein mineralization has not been previously decoded. In this study, we investigated the mineralization of fibroin-derived polypeptides (FDPs), which were obtained through the chymotryptic separation of the hydrophobic crystalline (Cp) fractions and of the hydrophilic electronegative amorphous (Cs) fractions. When immersed in simulated body fluid (SBF), only Cs fragments demonstrated the formation of carbonated apatite, providing experimental evidence that the mineralization of SF is dictated exclusively by its electronegative amino-acidic sequences. The potential of Cs to conceptually mimic the role of anionic non-collagenous proteins in biomineralization processes was investigated via their incorporation (up to 10% by weight) in bulk osteoid-like dense collagen (DC) gels. Within 6 h in SBF, apatite was formed in DC-Cs hybrid gels, and by day 7, carbonated hydroxylapatite crystals were extensively formed. This accelerated 3-D mineralization resulted in a nine-fold increase in the compressive modulus of the hydrogel. The tailoring of the mineralization and mechanical properties of hydrogels through hybridization with FDPs could potentially have a significant impact on cell delivery and bone regenerative medicine.

Laser-based three-dimensional multiscale micropatterning of biocompatible hydrogels for customized tissue engineering scaffolds

Significance In this paper we present results on 3D, multiscale laser machining of soft, transparent biomaterials suited for cellular growth and/or implantation. We use an ultrafast laser to generate high-resolution, 3D structures within the bulk of a transparent soft-biomaterial formulation that can support cell growth and allow cells to penetrate deep within the material. The structure is created by multiphoton absorption which, thanks to the clarity of the silk gels, is possible nearly 1 cm below the surface of the material. This depth represents an ∼10× improvement over other materials. The ability to create micrometer-scale voids over such a large volume has promising applications in the biomedical field and its efficacy was demonstrated both in vitro and in vivo. Light-induced material phase transitions enable the formation of shapes and patterns from the nano- to the macroscale. From lithographic techniques that enable high-density silicon circuit integration, to laser cutting and welding, light–matter interactions are pervasive in everyday materials fabrication and transformation. These noncontact patterning techniques are ideally suited to reshape soft materials of biological relevance. We present here the use of relatively low-energy (< 2 nJ) ultrafast laser pulses to generate 2D and 3D multiscale patterns in soft silk protein hydrogels without exogenous or chemical cross-linkers. We find that high-resolution features can be generated within bulk hydrogels through nearly 1 cm of material, which is 1.5 orders of magnitude deeper than other biocompatible materials. Examples illustrating the materials, results, and the performance of the machined geometries in vitro and in vivo are presented to demonstrate the versatility of the approach.

In vitro reactivity of Cu doped 45S5 Bioglass® derived scaffolds for bone tissue engineering

Cu-doped 45S5 bioactive glasses with varying Cu contents were fabricated and used to process 3D porous scaffolds via the foam replica technique. Cu-doping results in the weakening of the glass network and a decrease in its glass transition temperature. Acellular in vitro studies revealed very high bioactivity independent of Cu doping as indicated by the fast formation of a carbonated hydroxyapatite layer (CHA) on scaffold surfaces after immersion in simulated body fluid (SBF). The kinetics of the glass-ceramic scaffolds transition to an amorphous calcium phosphate layer (ACP) and the crystallisation of CHA were explored by FT-IR and SEM analyses. The elemental distribution in the scaffold/fluid interface region was monitored by the advanced micro-PIXE-RBS (particle induced X-ray emission/Rutherford backscattering spectrometry) method. Cu-containing glasses showed slower release of Si, Ca and P from the scaffold periphery, whereas traces of Cu were found incorporated in the CaP layer on the scaffold surface. Cu release kinetics from the scaffolds in SBF were found to depend on culturing conditions while highest Cu concentrations of ∼3.1 ppm and ∼4.6 ppm under static and quasi-dynamic conditions, respectively, were observed. Since Cu exhibits potential angiogenic and osteogenic properties, the Cu-containing scaffolds are suggested as promising materials for bone tissue engineering applications.

Immediate production of a tubular dense collagen construct with bioinspired mechanical properties.

The intrinsic complexity of tissues and organs demands tissue engineering approaches that extend beyond planar constructs currently in clinical use. However, the engineering of cylindrical or tubular tissue constructs with a hollow lumen presents significant challenges arising from geometrical and architectural considerations required to tailor biomaterials for tissue and organ repair. Type I collagen is an ideal scaffolding material due to its outstanding biocompatibility and high processability. However, the highly hydrated nature of collagen hydrogels results in their lack of mechanical properties and instability, as well as extensive cell-mediated contraction, which must be overcome to achieve process control. Herein, tubular dense collagen constructs (TDCCs) were produced simply and rapidly (in less than 1h) by circumferentially wrapping plastically compressed dense collagen gel sheets around a cylindrical support. The effects of collagen source, i.e. rat-tail tendon and bovine dermis-derived acid solubilized collagen, and concentration on TDCC properties were investigated through morphological, mechanical and chemical characterizations. Both tensile strength and apparent modulus correlated strongly with physiologically relevant collagen gel fibrillar densities. The clinical potential of TDCC as a tubular tissue substitute was demonstrated mechanically, through circumferential tensile properties, theoretical burst pressure, which ranged from 1225 to 1574 mm Hg, compliance values of between 8.3% to 14.2% per 100mm Hg and suture retention strength in the range of 116-151 grams-force, which were compatible with surgical procedures. Moreover, NIH/3T3 fibroblast viability and uniform distribution within the construct wall were confirmed up to day 7 in culture. TDCCs with fibrillar densities equivalent to native tissues can be readily engineered in various dimensions with tunable morphological and mechanical properties, which can be easily handled for use as tissue models and adapted to clinical needs.

Osteoid-Mimicking Dense Collagen/Chitosan Hybrid Gels

Bone extracellular matrix (ECM) is a 3D network, composed of collagen type I and a number of other macromolecules, including glycosaminoglycans (GAGs), which stimulate signaling pathways that regulate osteoblast growth and differentiation. To model the ECM of bone for tissue regenerative approaches, dense collagen/chitosan (Coll/CTS) hybrid hydrogels were developed using different proportions of CTS to mimic GAG components of the ECM. MC3T3-E1 mouse calvaria preosteoblasts were seeded within plastically compressed Coll/CTS hydrogels with solid content approaching that of native bone osteoid. Dense, cellular Coll/CTS hybrids were maintained for up to 8 weeks under either basal or osteogenic conditions. Higher CTS content significantly increased gel resistance to collagenase degradation. The incorporation of CTS to collagen gels decreased the apparent tensile modulus from 1.82 to 0.33 MPa. In contrast, the compressive modulus of Coll/CTS hybrids increased in direct proportion to CTS content exhibiting an increase from 23.50 to 55.25 kPa. CTS incorporation also led to an increase in scaffold resistance to cell-induced contraction. MC3T3-E1 viability, proliferation, and matrix remodeling capability (via matrix metalloproteinase expression) were maintained. Alkaline phosphatase activity was increased up to two-fold, and quantification of phosphate mineral deposition was significantly increased with CTS incorporation. Thus, dense Coll/CTS scaffolds provide osteoid-like models for the study of osteoblast differentiation and bone tissue engineering.