Chiara E. Ghezzi

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.

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.

Programmable 3D silk bone marrow niche for platelet generation ex vivo and modeling of megakaryopoiesis pathologies

We present a programmable bioengineered 3-dimensional silk-based bone marrow niche tissue system that successfully mimics the physiology of human bone marrow environment allowing us to manufacture functional human platelets ex vivo. Using stem/progenitor cells, megakaryocyte function and platelet generation were recorded in response to variations in extracellular matrix components, surface topography, stiffness, coculture with endothelial cells, and shear forces. Millions of human platelets were produced and showed to be functional based on multiple activation tests. Using adult hematopoietic progenitor cells our system demonstrated the ability to reproduce key steps of thrombopoiesis, including alterations observed in diseased states. A critical feature of the system is the use of natural silk protein biomaterial allowing us to leverage its biocompatibility, nonthrombogenic features, programmable mechanical properties, and surface binding of cytokines, extracellular matrix components, and endothelial-derived proteins. This in turn offers new opportunities for the study of blood component production ex vivo and provides a superior tissue system for the study of pathologic mechanisms of human platelet production.

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.

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.

Clinical Applications of Naturally Derived Biopolymer-Based Scaffolds for Regenerative Medicine

Naturally derived polymeric biomaterials, such as collagens, silks, elastins, alginates, and fibrins are utilized in tissue engineering due to their biocompatibility, bioactivity, and tunable mechanical and degradation kinetics. The use of these natural biopolymers in biomedical applications is advantageous because they do not release cytotoxic degradation products, are often processed using environmentally-friendly aqueous-based methods, and their degradation rates within biological systems can be manipulated by modifying the starting formulation or processing conditions. For these reasons, many recent in vivo investigations and FDA-approval of new biomaterials for clinical use have utilized natural biopolymers as matrices for cell delivery and as scaffolds for cell-free support of native tissues. This review highlights biopolymer-based scaffolds used in clinical applications for the regeneration and repair of native tissues, with a focus on bone, skeletal muscle, peripheral nerve, cardiac muscle, and cornea substitutes.

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.

Real time responses of fibroblasts to plastically compressed fibrillar collagen hydrogels.

In vitro reconstituted type I collagen hydrogels are widely utilized for tissue engineering studies. However, highly hydrated collagen (HHC) gels exhibit insufficient mechanical strength and unstable geometrical properties, thereby limiting their therapeutic application. Plastic compression (PC) is a simple and reproducible approach for the immediate production of dense fibrillar collagen (DC) scaffolds which demonstrate multiple improvements for tissue engineered constructs including extracellular matrix (ECM)-like meso scale characteristics, increased mechanical properties (modulus and strength), enhanced cell growth and differentiation, and reduced long-term scaffold deformation. In order to determine at which stage these benefits become apparent, and the underlying mechanisms involved, the immediate response of NIH/3T3 fibroblasts to PC as well as longer-term cell growth within DC scaffolds were examined herein. The real time three-dimensional (3D) distribution of fluorescently labelled cells during PC was sequentially monitored using confocal laser scanning microscopy (CLSM), observing excellent cell retention and negligible numbers of expelled cells. Relative to cells grown in HHC gels, a significant improvement in cell survival within DC scaffolds was evident as early as day 1. Cell growth and metabolic activity within DC gels were significantly increased over the course of one week. While cells within DC scaffolds reached confluency, an inhomogeneous distribution of cells was present in HHC gels, as detected using x-ray computed micro-tomography analysis of phosphotungstic acid labelled cells and CLSM, which both showed a significant cell loss within the HHC core. Therefore, PC generates a DC gel scaffold without detrimental effects towards seeded cells, surpassing HHC gels as a 3D scaffold for tissue engineering.

Enzymatically crosslinked silk-hyaluronic acid hydrogels

In this study, silk fibroin and hyaluronic acid (HA) were enzymatically crosslinked to form biocompatible composite hydrogels with tunable mechanical properties similar to that of native tissues. The formation of di-tyrosine crosslinks between silk fibroin proteins via horseradish peroxidase has resulted in a highly elastic hydrogel but exhibits time-dependent stiffening related to silk self-assembly and crystallization. Utilizing the same method of crosslinking, tyramine-substituted HA forms hydrophilic and bioactive hydrogels that tend to have limited mechanics and degrade rapidly. To address the limitations of these singular component scaffolds, HA was covalently crosslinked with silk, forming a composite hydrogel that exhibited both mechanical integrity and hydrophilicity. The composite hydrogels were assessed using unconfined compression and infrared spectroscopy to reveal of the physical properties over time in relation to polymer concentration. In addition, the hydrogels were characterized by enzymatic degradation and for cytotoxicity. Results showed that increasing HA concentration, decreased gelation time, increased degradation rate, and reduced changes that were observed over time in mechanics, water retention, and crystallization. These hydrogel composites provide a biologically relevant system with controllable temporal stiffening and elasticity, thus offering enhanced tunable scaffolds for short or long term applications in tissue engineering.

Mesenchymal stem cell-seeded multilayered dense collagen-silk fibroin hybrid for tissue engineering applications

Tissue engineering of multilayered constructs that model complex tissues poses a significant challenge for regenerative medicine. In this study, a three-layered scaffold consisting of an electrospun silk fibroin (SF) mat sandwiched between two dense collagen (DC) layers was designed and characterized. It was hypothesized that the SF layer would endow the DC-SF-DC construct with enhanced mechanical properties (e.g., apparent modulus, tensile strength, and toughness), while the surrounding DC layers provide an extracellular matrix-like environment for mesenchymal stem cell (MSC) growth. MSC-seeded DC-SF-DC hybrids were produced using the plastic compression technique and characterized morphologically, chemically, and mechanically. Moreover, MSC viability was assessed for up to 1 wk in culture. Scaffold analyses confirmed compaction and integration of the meso-scaled multilayered DC-SF-DC hybrid, which was reflected in a significantly higher toughness value when compared to DC and SF alone. MSCs directly incorporated into the DC layers remained viable for up to day 7. The ease of multilayered construct fabrication, enhanced biomechanical properties, along with uniformity of cell distribution confirmed the possibility for the incorporation and segregation of different cell types within distinct layers for the regeneration of complex tissues, such as skin, or central nervous system dura mater.