Synthetic bone mimetic matrix-mediated in situ bone tissue formation through host cell recruitment.

Advances in tissue engineering have offered new opportunities to restore anatomically and functionally compromised tissues. Although traditional tissue engineering approaches that utilize biomaterials and cells to create tissue constructs for implantation or biomaterials as a scaffold to deliver cells are promising, strategies that can activate endogenous cells to promote tissue repair are more clinically attractive. Here, we demonstrate that an engineered injectable matrix mimicking a calcium phosphate (CaP)-rich bone-specific microenvironment can recruit endogenous cells to form bone tissues in vivo. Comparison of matrix alone with that of bone marrow-soaked or bFGF-soaked matrix demonstrates similar extent of neo-bone formation and bridging of decorticated transverse processes in a posterolateral lumbar fusion rat model. Synthetic biomaterials that stimulate endogenous cells without the need for biologics to assist tissue repair could circumvent limitations associated with conventional tissue engineering approaches, including ex vivo cell processing and laborious efforts, thereby accelerating the translational aspects of regenerative medicine.

Calcium phosphate-bearing matrices induce osteogenic differentiation of stem cells through adenosine signaling.

Synthetic matrices emulating the physicochemical properties of tissue-specific ECMs are being developed at a rapid pace to regulate stem cell fate. Biomaterials containing calcium phosphate (CaP) moieties have been shown to support osteogenic differentiation of stem and progenitor cells and bone tissue formation. By using a mineralized synthetic matrix mimicking a CaP-rich bone microenvironment, we examine a molecular mechanism through which CaP minerals induce osteogenesis of human mesenchymal stem cells with an emphasis on phosphate metabolism. Our studies show that extracellular phosphate uptake through solute carrier family 20 (phosphate transporter), member 1 (SLC20a1) supports osteogenic differentiation of human mesenchymal stem cells via adenosine, an ATP metabolite, which acts as an autocrine/paracrine signaling molecule through A2b adenosine receptor. Perturbation of SLC20a1 abrogates osteogenic differentiation by decreasing intramitochondrial phosphate and ATP synthesis. Collectively, this study offers the demonstration of a previously unknown mechanism for the beneficial role of CaP biomaterials in bone repair and the role of phosphate ions in bone physiology and regeneration. These findings also begin to shed light on the role of ATP metabolism in bone homeostasis, which may be exploited to treat bone metabolic diseases.

Effect of scaffold microarchitecture on osteogenic differentiation of human mesenchymal stem cells.

Design of macroporous synthetic grafts that can promote infiltration of cells, their differentiation, and synthesis of bone-specific extracellular matrix is a key determinant for in vivo bone tissue regeneration and repair. In this study, we investigated the effect of the microarchitecture of the scaffold on osteogenic differentiation of human mesenchymal stem cells (hMSCs). Poly(ethylene glycol) diacrylate-co-N-acryloyl 6-aminocaproic acid cryogels were fabricated to have either a pore network consisting of cellular, randomly oriented pores (termed 'spongy') or a pore network consisting of lamellar columns (termed 'columnar'), with both cryogel types showing a similar porosity. Both spongy and columnar cryogels supported comparable levels of cell viability and proliferation of hMSCs in vitro. However, spongy cryogels promoted osteogenic differentiation to a greater extent than their columnar counterparts, as evidenced by increased alkaline phosphatase activity and osteoblastic gene expression over 21 days post culture. Leveraging upon our previous work, we further evaluated the ability of these synthetic scaffolds in conjunction with mineralisation to promote ectopic bone formation upon subcutaneous implantation in nude rats. Mineralised spongy and columnar cryogels, both in the presence and absence of exogenous hMSCs, promoted ectopic bone formation in vivo. No such bone formation was observed in acellular cryogels devoid of mineralisation, with extensive host cell infiltration and vascularisation in columnar cryogels, and negligible infiltration into spongy cryogels. Our results thus present a novel method to tune the microarchitecture of porous polymeric scaffolds, in addition to suggesting their efficacy as synthetic bone grafts.

Mineralized synthetic matrices as an instructive microenvironment for osteogenic differentiation of human mesenchymal stem cells.

The effect of substrate-mediated signals on osteogenic differentiation of hMSCs is studied using a synthetic bone-like material comprising both organic and inorganic components that supports adhesion, spreading, and proliferation of hMSCs. hMSCs undergo osteogenic differentiation even in the absence of osteogenesis-inducing supplements. They exhibit higher expressions of Runx2, BSP, and OCN compared to their matrix-rigidity-matched, non-mineralized hydrogel counterparts. The mineralized-hydrogel-assisted osteogenic differentiation of hMSCs could be attributed to their exposure to high local concentrations of calcium and phosphate ions in conjunction with chemical and topological cues arising from the hydrogel-bound calcium phosphate mineral layer.

Spatial tuning of negative and positive Poisson's ratio in a multi-layer scaffold.

While elastic modulus is tunable in tissue engineering scaffolds, it is substantially more challenging to tune the Poisson's ratio of scaffolds. In certain biological applications, scaffolds with a tunable Poisson's ratio may be more suitable for emulating the behavior of native tissue mechanics. Here, we design and fabricate a scaffold, which exhibits simultaneous negative and positive Poisson's ratio behavior. Custom-made digital micro-mirror device stereolithography was used to fabricate single- and multiple-layer scaffolds using polyethylene glycol-based biomaterial. These scaffolds are composed of pore structures having special geometries, and deformation mechanisms, which can be tuned to exhibit both negative Poisson's ratio (NPR) and positive Poisson's ratio (PPR) behavior in a side-to-side or top-to-bottom configuration. Strain measurement results demonstrate that analytical deformation models and simulations accurately predict the Poisson's ratios of both the NPR and PPR regions. This hybrid Poisson's ratio property can be imparted to any photocurable material, and potentially be applicable in a variety of biomedical applications.

Rapid self-healing hydrogels.

Synthetic materials that are capable of autonomous healing upon damage are being developed at a rapid pace because of their many potential applications. Despite these advancements, achieving self-healing in permanently cross-linked hydrogels has remained elusive because of the presence of water and irreversible cross-links. Here, we demonstrate that permanently cross-linked hydrogels can be engineered to exhibit self-healing in an aqueous environment. We achieve this feature by arming the hydrogel network with flexible-pendant side chains carrying an optimal balance of hydrophilic and hydrophobic moieties that allows the side chains to mediate hydrogen bonds across the hydrogel interfaces with minimal steric hindrance and hydrophobic collapse. The self-healing reported here is rapid, occurring within seconds of the insertion of a crack into the hydrogel or juxtaposition of two separate hydrogel pieces. The healing is reversible and can be switched on and off via changes in pH, allowing external control over the healing process. Moreover, the hydrogels can sustain multiple cycles of healing and separation without compromising their mechanical properties and healing kinetics. Beyond revealing how secondary interactions could be harnessed to introduce new functions to chemically cross-linked polymeric systems, we also demonstrate various potential applications of such easy-to-synthesize, smart, self-healing hydrogels.

A Three-dimensional Polymer Scaffolding Material Exhibiting a Zero Poisson's Ratio.

Poisson's ratio describes the degree to which a material contracts (expands) transversally when axially strained. A material with a zero Poisson's ratio does not transversally deform in response to an axial strain (stretching). In tissue engineering applications, scaffolding having a zero Poisson's ratio (ZPR) may be more suitable for emulating the behavior of native tissues and accommodating and transmitting forces to the host tissue site during wound healing (or tissue regrowth). For example, scaffolding with a zero Poisson's ratio may be beneficial in the engineering of cartilage, ligament, corneal, and brain tissues, which are known to possess Poisson's ratios of nearly zero. Here, we report a 3D biomaterial constructed from polyethylene glycol (PEG) exhibiting in-plane Poisson's ratios of zero for large values of axial strain. We use digital micro-mirror device projection printing (DMD-PP) to create single- and double-layer scaffolds composed of semi re-entrant pores whose arrangement and deformation mechanisms contribute the zero Poisson's ratio. Strain experiments prove the zero Poisson's behavior of the scaffolds and that the addition of layers does not change the Poisson's ratio. Human mesenchymal stem cells (hMSCs) cultured on biomaterials with zero Poisson's ratio demonstrate the feasibility of utilizing these novel materials for biological applications which require little to no transverse deformations resulting from axial strains. Techniques used in this work allow Poisson's ratio to be both scale-independent and independent of the choice of strut material for strains in the elastic regime, and therefore ZPR behavior can be imparted to a variety of photocurable biomaterial.

Engineered microenvironments for self-renewal and musculoskeletal differentiation of stem cells.

Stem cells hold great promise for therapies aimed at regenerating damaged tissue, drug screening and studying in vitro models of human disease. However, many challenges remain before these applications can become a reality. One such challenge is developing chemically defined and scalable culture conditions for derivation and expansion of clinically viable human pluripotent stem cells, as well as controlling their differentiation with high specificity. Interaction of stem cells with their extracellular microenvironment plays an important role in determining their differentiation commitment and functions. Regenerative medicine approaches integrating cell-matrix and cell-cell interactions, and soluble factors could lead to development of robust microenvironments to control various cellular responses. Indeed, several of these recent developments have provided significant insight into the design of microenvironments that can elicit the targeted cellular response. In this article, we will focus on some of these developments with an emphasis on matrix-mediated expansion of human pluripotent stem cells while maintaining their pluripotency. We will also discuss the role of matrix-based cues and cell-cell interactions in the form of soluble signals in directing stem cell differentiation into musculoskeletal lineages.

Oligo(trimethylene carbonate)-poly(ethylene glycol)-oligo(trimethylene carbonate) triblock-based hydrogels for cartilage tissue engineering.

A triblock co-polymer of oligo(trimethylene carbonate)-block-poly(ethylene glycol) 20000-block-oligo(trimethylene carbonate) diacrylate (TMC20) was used as a photo-polymerizable precursor for the encapsulation of primary articular chondrocytes. The efficacy of TMC20 as a biodegradable scaffold for cartilage tissue engineering was compared with non-degradable poly(ethylene glycol) 20000 diacrylate (PEG20) hydrogel. Chondrocytes encapsulated in PEG hydrogels containing oligo(trimethylene carbonate) (OTMC) moieties underwent spontaneous aggregation during in vitro culture, which was not observed in the PEG hydrogel counterparts. The aggregation of cells was found to be dependent on the initial cell density, as well as the mesh size of the hydrogels. Similarly, cell aggregation was also found in biodegradable PEG hydrogels containing caprolactone moieties. The aggregation of cells in TMC20 hydrogels resulted in enhanced cartilage matrix production compared with their PEG20 counterparts over 3 weeks of culture. Taken together, these results indicate that PEG hydrogels containing degradable OTMC moieties promote the aggregation and biosynthetic activity of encapsulated chondrocytes, indicating their potential as scaffolds for the repair of cartilage tissue.

Templated mineralization of synthetic hydrogels for bone-like composite materials: role of matrix hydrophobicity.

Bone-mimetic mineral-polymer composite materials have several applications ranging from artificial bone grafts to scaffolds for bone tissue engineering; templated mineralization is an effective approach to fabricate such composites. In this study, we synthesized bone-like composites using synthetic hydrogels having pendant side chains terminating with carboxyl groups as a template for mineralization. The role of matrix hydrophobicity on mineralization was examined using poly(ethylene glycol) hydrogels modified with varying lengths of anionic pendant side chains (CH(2) horizontal lineCHCONH(CH(2))(n)COOH, where n = 1, 3, 5, and 7). The ability of these hydrogels to undergo templated mineralization was found to be strongly dependent upon the length of the pendant side chain as is evident from the extent of calcification and morphology of the minerals. Moreover, mineralized phases formed on the hydrogels were confirmed to resemble apatite-like structures. In addition to demonstrating the importance of material hydrophobicity as a design parameter for the development of bone-like synthetic materials, our study also provides a potential explanation for the in vitro differences between the apatite-nucleating capacity of aspartate-rich osteopontin and glutamate-rich bone sialoprotein.