Efficacy of Lysostaphin functionalized silicon catheter for the prevention of Staphylococcus aureus biofilm.

Staphylococcus aureus readily forms biofilms on tissue and indwelling catheter surfaces. These biofilms are resistant to antibiotics. Consequently, effective prevention and treatment strategies against staphylococcal biofilms are actively being pursued over the past two decades. One of the proposed strategies involve the incorporation of antibiotics and antiseptics into catheters, however, a persistent concern regarding the possible emergence of antimicrobial resistance is associated with these medical devices. In this study, we developed two types of silicone catheters: one with Lysostaphin (Lst) adsorbed onto the surface, and the other with Lst functionalized on the surface. To confirm the presence of Lst protein on the catheter surface, we conducted FTIR-ATR and SEM-EDS analysis. Both catheters exhibited hemocompatibility, biocompatibility, and demonstrated antimicrobial and biofilm prevention activities against both methicillin-sensitive and resistant strains of S. aureus. Furthermore, the silicone catheters that were surface-functionalized with Lst showed substantially better and more persistent anti-biofilm effects when compared to the catheters where Lst was surface-adsorbed, both under in vitro static and flow conditions, as well as in vivo in BALB/c mice. These results indicate that surface-functionalized Lst catheters have the potential to serve as a promising new medical device for preventing S. aureus biofilm infections in humans.

Smart Design for Hybrid Bioprinting of Scalable and Viable Tissue Constructs.

Hybrid bioprinting uses sequential printing of melt-extruded biodegradable thermoplastic polymer and cell-encapsulated bioink in a predesigned manner using high- and low-temperature print heads for the fabrication of robust three-dimensional (3D) biological constructs. However, the high-temperature print head and melt-extruded polymer cause irreversible thermal damage to the bioprinted cells, and it affects viability and functionality of 3D bioprinted biological constructs. Thus, there is an urgent need to develop innovative approaches to protect the bioprinted cells, coming into contact or at close proximities to the melt-extruded thermoplastic polymer and the high-temperature print head during hybrid bioprinting. Therefore, this study investigated the potential of iterating the structural architecture pattern (SAP) of melt-printed thermoplastic layers and the cell printing pattern (CPP) to protect the cells from temperature-associated damage during hybrid bioprinting. A novel SAP for printing the thermoplastic polymer and an associated CPP for minimizing thermal damage to the 3D bioprinted construct have been developed. The newly developed SAP- and CPP-based hybrid bioprinted biological constructs showed significantly low thermal damage compared to conventionally hybrid bioprinted biological constructs. The results from this study suggest that the newly developed SAP and CPP can be an improved hybrid bioprinting strategy for developing living constructs at the human scale.

Development and characterisation of suitably bioengineered microfibrillar matrix-based 3D prostate cancer model forin vitrodrug testing.

Bioengineered 3D models that can mimic patient-specific pathologiesin vitroare valuable tools for developing and validating anticancer therapeutics. In this study, microfibrillar matrices with unique structural and functional properties were fabricated as 3D spherical and disc-shaped scaffolds with highly interconnected pores and the potential of the newly developed scaffolds for developing prostate cancer model has been investigated. The newly developed scaffolds showed improved cell retention upon seeding with cancer cells compared to conventional electrospun scaffolds. They facilitated rapid growth and deposition of cancer-specific extracellular matrix through-the-thickness of the scaffold. Compared to the prostate cancer cells grown in 2D culture, the newly developed prostate cancer model showed increased resistance to the chemodrug Docetaxel regardless of the drug concentration or the treatment frequency. A significant reduction in the cell number was observed within one week after the drug treatment in the 2D culture for both PC3 and patient-derived cells. Interestingly, almost 20%-30% of the cancer cells in the newly developed 3D model survived the drug treatment, and the patient-derived cells were more resistant than the tested cell line PC3. The results from this study indicate the potential of the newly developed prostate cancer model forin vitrodrug testing.

Human Adipose-Derived Mesenchymal Stem Cell-Secreted Extracellular Matrix Coating on a Woven Nanotextile Vascular Patch for Improved Endothelial Cell Response.

Biomedical implants possessing the structural and functional characteristics of extracellular matrix (ECM) are pivotal for vascular applications. This study investigated the potential of recreating a natural ECM-like structural and functional environment on the surface of biodegradable polymeric nanotextiles for vascular implants. Human adipose-derived mesenchymal stem cells (MSCs) were grown on a suitably engineered polycaprolactone (PCL) nanofibrous textile and were allowed to modify its surface through the deposition of MSC-specific ECM. This surface-modified nanotextile showed mechanical characteristics and functionality appropriate for vascular patch material. The uniformity of ECM coating significantly improved the viability, proliferation, and migration of human endothelial cells compared to bare and xenogeneic collagen-coated PCL nanotextile patches. Thus, a polymeric nanotextile, which is surface modified using MSC-driven ECM, provided a rapid and improved endothelialization, thereby suggesting its potential for vascular patch applications.

Bioengineered 3D microfibrous-matrix modulates osteopontin release from MSCs and facilitates the expansion of hematopoietic stem cells.

The osteopontin (OPN) released from mesenchymal stem cells (MSCs) undergoing lineage differentiation can negatively influence the expansion of hematopoietic stem cells (HSCs) in coculture systems developed for expanding HSCs. Therefore, minimizing the amount of OPN in the coculture system is important for the successful ex vivo expansion of HSCs. Toward this goal, a bioengineered three dimensional (3D) microfibrous-matrix that can maintain MSCs in less OPN-releasing conditions has been developed, and its influence on the expansion of HSCs has been studied. The newly developed 3D matrix significantly decreased the release of OPN, depending on the MSC culture conditions used during the priming period before HSC seeding. The culture system with the lowest amount of OPN facilitated a more than 24-fold increase in HSC number in 1 week time period. Interestingly, the viability of expanded cells and the CD34+   pure population of HSCs were found to be the highest in the low OPN-containing system. Therefore, bioengineered microfibrous 3D matrices seeded with MSCs, primed under suitable culture conditions, can be an improved ex vivo expansion system for HSC culture.

Soft Hydrogel Environments that Facilitate Cell Spreading and Aggregation Preferentially Support Chondrogenesis of Adult Stem Cells.

Mesenchymal stem/stromal cells (MSCs) represent a promising cell type for treating damaged synovial joints. The therapeutic potential of MSCs will be facilitated by the engineering of biomaterial environments capable of directing their fate. Here the interplay between matrix elasticity and cell morphology in regulating the chondrogenic differentiation of MSCs when seeded onto or encapsulated within hydrogels made of interpenetrating networks (IPN) of alginate and collagen type I is explored. This IPN system enables the independent control of substrate stiffness (in 2D and in 3D) and cell morphology (3D only). The expression of chondrogenic markers SOX9, ACAN, and COL2 increases when MSCs are cultured onto the soft substrate, which correlates with increased SMAD2/3 nuclear localization, enhanced MSCs condensation, and the formation of larger cellular aggregates. The encapsulation of spread MSCs within a soft IPN increases the expression of cartilage-specific genes, which is linked to cellular condensation and nuclear SMAD2/3 localization. Surprisingly, cells forced to adopt a more rounded morphology within the same soft IPNs expressed higher levels of the osteogenic markers RUNX2 and COL1. The insight provided by this study suggests that a mechanobiology informed approach to biomaterial development will be integral to the development of successful cartilage tissue engineering strategies.

Improving the Intercellular Uptake and Osteogenic Potency of Calcium Phosphate via Nanocomplexation with the RALA Peptide.

Calcium phosphate-base materials (e.g., alpha tri-calcium phosphate (α-TCP)) have been shown to promote osteogenic differentiation of stem/progenitor cells, enhance osteoblast osteogenic activity and mediate in vivo bone tissue formation. However, variable particle size and hydrophilicity of the calcium phosphate result in an extremely low bioavailability. Therefore, an effective delivery system is required that can encapsulate the calcium phosphate, improve cellular entry and, consequently, elicit a potent osteogenic response in osteoblasts. In this study, collagenous matrix deposition and extracellular matrix mineralization of osteoblast lineage cells were assessed to investigate osteogenesis following intracellular delivery of α-TCP nanoparticles. The nanoparticles were formed via condensation with a novel, cationic 30 mer amphipathic peptide (RALA). Nanoparticles prepared at a mass ratio of 5:1 demonstrated an average particle size of 43 nm with a zeta potential of +26 mV. The average particle size and zeta potential remained stable for up to 28 days at room temperature and across a range of temperatures (4-37 °C). Cell viability decreased 24 h post-transfection following RALA/α-TCP nanoparticle treatment; however, recovery ensued by Day 7. Immunocytochemistry staining for Type I collagen up to Day 21 post-transfection with RALA/α-TCP nanoparticles (NPs) in MG-63 cells exhibited a significant enhancement in collagen expression and deposition compared to an untreated control. Furthermore, in porcine mesenchymal stem cells (pMSCs), there was enhanced mineralization compared to α-TCP alone. Taken together these data demonstrate that internalization of RALA/α-TCP NPs elicits a potent osteogenic response in both MG-63 and pMSCs.

Spatial Presentation of Tissue-Specific Extracellular Matrix Components along Electrospun Scaffolds for Tissue Engineering the Bone-Ligament Interface.

The bone-ligament interface transitions from a highly organized type I collagen rich matrix to a nonmineralized fibrocartilage region and finally to a mineralized fibrocartilage region that interfaces with the bone. Therefore, engineering the bone-ligament interface requires a biomaterial substrate capable of maintaining or directing the spatially defined differentiation of multiple cell phenotypes. To date the appropriate combination of biophysical and biochemical factors that can be used to engineer such a biomaterial substrate remain unknown. Here we show that microfiber scaffolds functionalized with tissue-specific extracellular matrix (ECM) components can direct the differentiation of MSCs toward the phenotypes seen at the bone-ligament interface. Ligament-ECM (L-ECM) promoted the expression of the ligament-marker gene tenomodulin (TNMD) and higher levels of type I and III collagen expression compared to functionalization with commercially available type I collagen. Functionalization of microfiber scaffolds with cartilage-ECM (C-ECM) promoted chondrogenesis of MSCs, as evidenced by adoption of a round cell morphology and increased SRY-box 9 (SOX9) expression in the absence of exogenous growth factors. Next, we fabricated a multiphasic scaffold by controlling the spatial presentation of L-ECM and C-ECM along the length of a single electrospun microfiber construct, with the distal region of the C-ECM coated fibers additionally functionalized with an apatite layer (using simulated body fluid) to promote endochondral ossification. These ECM functionalized scaffolds promoted spatially defined differentiation of MSCs, with higher expression of TNMD observed in the region functionalized with L-ECM, and higher expression of type X collagen and osteopontin (markers of endochondral ossification) observed at the end of the scaffold functionalized with C-ECM and the apatite coating. Our results demonstrate the utility of tissue-specific ECM derived components as a cue for directing MSC differentiation when engineering complex multiphasic interfaces such as the bone-ligament enthesis.

Electrospinning of highly porous yet mechanically functional microfibrillar scaffolds at the human scale for ligament and tendon tissue engineering.

Electrospun fibers offer tremendous potential for tendon and ligament tissue engineering, yet developing porous scaffolds mimicking the size, stiffness and strength of human tissues remains a challenge. Previous studies have rolled, braided, or stacked electrospun sheets to produce three-dimensional (3D) scaffolds with tailored sizes and mechanical properties. A common limitation with such approaches is the development of low porosity scaffolds that impede cellular infiltration into the body of the implant, thereby limiting their regenerative potential. Here, we demonstrate how varying the rotational speed of the collecting mandrel during the electrospinning of poly(ε-caprolactone) (PCL) can be used to limit inter-fiber fusion (or fiber welding). Increasing the fraction of unfused fibers reduced the flexural rigidity of the electrospun sheets, which in turn allowed us to bundle the fibers into 3D scaffolds with similar dimensions to the human anterior cruciate ligament (ACL). These unfused fibers allowed for higher levels of porosity (up to 95%) that facilitated the rapid migration of mesenchymal stem cells (MSCs) into the body of the scaffolds. Mechanical testing demonstrated that the fiber-bundles possessed a Young's modulus approaching that of the native human ACL. The scaffolds were also capable of supporting the differentiation of MSCs towards either the fibrocartilage or ligament/tendon lineage. This novel electrospinning strategy could be used to produce mechanically functional, yet porous, scaffolds for a wide range of biomedical applications.

Hypoxia mimicking hydrogels to regulate the fate of transplanted stem cells.

Controlling the phenotype of transplanted stem cells is integral to ensuring their therapeutic efficacy. Hypoxia is a known regulator of stem cell fate, the effects of which can be mimicked using hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitors such as dimethyloxalylglycine (DMOG). By releasing DMOG from mesenchymal stem cell (MSC) laden alginate hydrogels, it is possible to stabilize HIF-1α and enhance its nuclear localization. This correlated with enhanced chondrogenesis and a reduction in the expression of markers associated with chondrocyte hypertrophy, as well as increased SMAD 2/3 nuclear localization in the encapsulated MSCs. In vivo, DMOG delivery significantly reduced mineralisation of the proteoglycan-rich cartilaginous tissue generated by MSCs within alginate hydrogels loaded with TGF-ß3 and BMP-2. Together these findings point to the potential of hypoxia mimicking hydrogels to control the fate of stem cells following their implantation into the body. STATEMENT OF SIGNIFICANCE: There are relatively few examples where in vivo delivery of adult stem cells has demonstrated a true therapeutic benefit. This may be attributed, at least in part, to a failure to control the fate of transplanted stem cells in vivo. In this paper we describe the development of hydrogels that mimic the effects of hypoxia on encapsulated stem cells. In vitro, these hydrogels enhance chondrogenesis of MSCs and suppress markers associated with chondrocyte hypertrophy. In an in vivo environment that otherwise supports progression along an endochondral pathway, we show that these hydrogels will instead direct mesenchymal stem cells (MSCs) to produce a more stable, cartilage-like tissue. In addition, we explore potential molecular mechanisms responsible for these phenotypic changes in MSCs.

nCP:Fe-A Biomineral Magnetic Nanocontrast Agent for Tracking Implanted Stem Cells in Brain Using MRI.

In vivo tracking of transplanted stem cells to monitor their migration, biodistribution, and engraftment in the host tissue is important for assessing the efficacy of stem cell therapeutics. Here, we report a biomineral nanocontrast agent, iron doped calcium phosphate nanoparticles (nCP:Fe), for the in vivo tracking of stem cells in brain using magnetic resonance imaging (MRI). We have synthesized ∼100 nm sized nCP nanoparticles doped with 9.81 wt % Fe3+. In vitro studies using mesenchymal stem cells (MSCs) showed excellent biocompatibility for nCP:Fe with ∼87% labeling efficiency under optimized conditions (100 µg/mL, 6 h). Most importantly, the labeling was not found to affect the neurogenic differentiation potential of MSCs. MRI of labeled cells (∼22.34 pg Fe/cell) showed significant reduction in T2 relaxation time from 195 to 89 ms, rendering dark contrast. In vivo transplantation of labeled cells (1 × 106 cells) in external capsule of healthy rat brain showed a clearly distinguishable hypointense (dark) region in T2 weighted MR images, which remained visible up to 30 days. Subsequently, MRI tracking of labeled MSCs transplanted intracerebrally, 3 mm near to the LPS induced inflammatory site in brain, showed successful migration of labeled MSCs toward the site of inflammation. The cell migration was confirmed ex vivo by Prussian-blue (Fe3+) and Alizarin-red (Ca2+) staining of tissue sections, where individual cells were found migrated to the site of inflammation over a period of 30 days. In summary, our results clearly show that, as a biocompatible mineral composition, nCP:Fe is a promising magnetic nanocontrast agent for MRI based cell tracking in vivo.

Engineering large cartilage tissues using dynamic bioreactor culture at defined oxygen conditions.

Mesenchymal stem cells maintained in appropriate culture conditions are capable of producing robust cartilage tissue. However, gradients in nutrient availability that arise during three-dimensional culture can result in the development of spatially inhomogeneous cartilage tissues with core regions devoid of matrix. Previous attempts at developing dynamic culture systems to overcome these limitations have reported suppression of mesenchymal stem cell chondrogenesis compared to static conditions. We hypothesize that by modulating oxygen availability during bioreactor culture, it is possible to engineer cartilage tissues of scale. The objective of this study was to determine whether dynamic bioreactor culture, at defined oxygen conditions, could facilitate the development of large, spatially homogeneous cartilage tissues using mesenchymal stem cell laden hydrogels. A dynamic culture regime was directly compared to static conditions for its capacity to support chondrogenesis of mesenchymal stem cells in both small and large alginate hydrogels. The influence of external oxygen tension on the response to the dynamic culture conditions was explored by performing the experiment at 20% O2 and 3% O2. At 20% O2, dynamic culture significantly suppressed chondrogenesis in engineered tissues of all sizes. In contrast, at 3% O2 dynamic culture significantly enhanced the distribution and amount of cartilage matrix components (sulphated glycosaminoglycan and collagen II) in larger constructs compared to static conditions. Taken together, these results demonstrate that dynamic culture regimes that provide adequate nutrient availability and a low oxygen environment can be employed to engineer large homogeneous cartilage tissues. Such culture systems could facilitate the scaling up of cartilage tissue engineering strategies towards clinically relevant dimensions.

Modulating microfibrillar alignment and growth factor stimulation to regulate mesenchymal stem cell differentiation.

The ideal tissue engineering (TE) strategy for ligament regeneration should recapitulate the bone - calcified cartilage - fibrocartilage - soft tissue interface. Aligned electrospun-fibers have been shown to guide the deposition of a highly organized extracellular matrix (ECM) necessary for ligament TE. However, recapitulating the different tissues observed in the bone-ligament interface using such constructs remains a challenge. This study aimed to explore how fiber alignment and growth factor stimulation interact to regulate the chondrogenic and ligamentous differentiation of mesenchymal stem cells (MSCs). To this end aligned and randomly-aligned electrospun microfibrillar scaffolds were seeded with bone marrow derived MSCs and stimulated with transforming growth factor ß3 (TGFß3) or connective tissue growth factor (CTGF), either individually or sequentially. Without growth factor stimulation, MSCs on aligned-microfibers showed higher levels of tenomodulin (TNMD) and aggrecan gene expression compared to MSCs on randomly-oriented fibers. MSCs on aligned-microfibers stimulated with TGFß3 formed cellular aggregates and underwent robust chondrogenesis, evidenced by increased type II collagen expression and sulphated glycosaminoglycans (sGAG) synthesis compared to MSCs on randomly-oriented scaffolds. Bone morphogenetic protein 2 (BMP2) and type I collagen gene expression were higher on randomly-oriented scaffolds stimulated with TGFß3, suggesting this substrate was more supportive of an endochondral phenotype. In the presence of CTGF, MSCs underwent ligamentous differentiation, with increased TNMD expression on aligned compared to randomly aligned scaffolds. Upon sequential growth factor stimulation, MSCs expressed types I and II collagen and deposited higher overall levels of collagen compared to scaffolds stimulated with either growth factor in isolation. These findings demonstrate that modulating the alignment of microfibrillar scaffolds can be used to promote either an endochondral, chondrogenic, fibrochondrogenic or ligamentous MSC phenotype upon presentation of appropriate biochemical cues. STATEMENT OF SIGNIFICANCE: Polymeric electrospun fibers can be tuned to match the fibrillar size and anisotropy of collagen fibers in ligaments, and can be mechanically competent. Therefore, their use is attractive when attempting to tissue engineer the bone-ligament interface. A central challenge in this field is recapitulating the cellular phenotypes observed across the bone-ligament interface. Here we demonstrated that it is possible to direct MSCs seeded onto aligned electrospun fibres towards either a ligamentogenic, chondrogenic or fibrochondrogenic phenotype upon presentation of appropriate biochemical cues. This opens the possibility of using aligned microfibrillar scaffolds that are spatially functionalized with specific growth factors to direct MSC differentiation for engineering the bone-ligament interface.

* Three-Dimensional Bioprinting of Polycaprolactone Reinforced Gene Activated Bioinks for Bone Tissue Engineering.

Regeneration of complex bone defects remains a significant clinical challenge. Multi-tool biofabrication has permitted the combination of various biomaterials to create multifaceted composites with tailorable mechanical properties and spatially controlled biological function. In this study we sought to use bioprinting to engineer nonviral gene activated constructs reinforced by polymeric micro-filaments. A gene activated bioink was developed using RGD-γ-irradiated alginate and nano-hydroxyapatite (nHA) complexed to plasmid DNA (pDNA). This ink was combined with bone marrow-derived mesenchymal stem cells (MSCs) and then co-printed with a polycaprolactone supporting mesh to provide mechanical stability to the construct. Reporter genes were first used to demonstrate successful cell transfection using this system, with sustained expression of the transgene detected over 14 days postbioprinting. Delivery of a combination of therapeutic genes encoding for bone morphogenic protein and transforming growth factor promoted robust osteogenesis of encapsulated MSCs in vitro, with enhanced levels of matrix deposition and mineralization observed following the incorporation of therapeutic pDNA. Gene activated MSC-laden constructs were then implanted subcutaneously, directly postfabrication, and were found to support superior levels of vascularization and mineralization compared to cell-free controls. These results validate the use of a gene activated bioink to impart biological functionality to three-dimensional bioprinted constructs.

Simple Radical Polymerization of Poly(Alginate-Graft-N-Isopropylacrylamide) Injectable Thermoresponsive Hydrogel with the Potential for Localized and Sustained Delivery of Stem Cells and Bioactive Molecules.

In this study, thermoresponsive copolymers that are fully injectable, biocompatible, and biodegradable and are synthesized via graft copolymerization of poly(N-isopropylacrylamide) onto alginate using a free-radical reaction are presented. This new synthesis method does not involve multisteps or associated toxicity issues, and has the potential to reduce scale-up difficulties. Chemical and physical analyses verify the resultant graft copolymer structure. The lower critical solution temperature, which is a characteristic of sol-gel transition, is observed at 32 °C. The degradation properties indicate suitable degradation kinetics for drug delivery and bone tissue engineering applications. The synthesized P(Alg-g-NIPAAm) hydrogel is noncytotoxic with both human osteosarcoma (MG63) cells and porcine bone marrow derived mesenchymal stem cells (pBMSCs). pBMSCs encapsulated in the P(Alg-g-NIPAAm) hydrogel remain viable, show uniform distribution within the injected hydrogel, and undergo osteogenic and chondrogenic differentiation under appropriate culture conditions. Furthermore, for the first time, this work will explore the influence of alginate viscosity on the viscoelastic properties of the resulting copolymer hydrogels, which influences the rate of medical device formation and subsequent drug release. Together the results of this study indicate that the newly synthesized P(Alg-g-NIPAAm) hydrogel has potential to serve as a versatile and improved injectable platform for drug delivery and bone tissue engineering applications.

* Hierarchically Structured Electrospun Scaffolds with Chemically Conjugated Growth Factor for Ligament Tissue Engineering.

The anterior cruciate ligament (ACL) of the knee is vital for proper joint function and is commonly ruptured during sports injuries or car accidents. Due to a lack of intrinsic healing capacity and drawbacks with allografts and autografts, there is a need for a tissue-engineered ACL replacement. Our group has previously used aligned sheets of electrospun polycaprolactone nanofibers to develop solid cylindrical bundles of longitudinally aligned nanofibers. We have shown that these nanofiber bundles support cell proliferation and elongation and the hierarchical structure and material properties are similar to the native human ACL. It is possible to combine multiple nanofiber bundles to create a scaffold that attempts to mimic the macroscale structure of the ACL. The goal of this work was to develop a hierarchical bioactive scaffold for ligament tissue engineering using connective tissue growth factor (CTGF)-conjugated nanofiber bundles and evaluate the behavior of mesenchymal stem cells (MSCs) on these scaffolds in vitro and in vivo. CTGF was immobilized onto the surface of individual nanofiber bundles or scaffolds consisting of multiple nanofiber bundles. The conjugation efficiency and the release of conjugated CTGF were assessed using X-ray photoelectron spectroscopy, assays, and immunofluorescence staining. Scaffolds were seeded with MSCs and maintained in vitro for 7 days (individual nanofiber bundles), in vitro for 21 days (scaled-up scaffolds of 20 nanofiber bundles), or in vivo for 6 weeks (small scaffolds of 4 nanofiber bundles), and ligament-specific tissue formation was assessed in comparison to non-CTGF-conjugated control scaffolds. Results showed that CTGF conjugation encouraged cell proliferation and ligament-specific tissue formation in vitro and in vivo. The results suggest that hierarchical electrospun nanofiber bundles conjugated with CTGF are a scalable and bioactive scaffold for ACL tissue engineering.

RALA complexed α-TCP nanoparticle delivery to mesenchymal stem cells induces bone formation in tissue engineered constructs in vitro and in vivo.

A range of bone regeneration strategies, from growth factor delivery and/or mesenchymal stem cell (MSC) transplantation to endochondral tissue engineering, have been developed in recent years. Despite their tremendous promise, the clinical translation and future use of many of these strategies is being hampered by concerns such as off target effects associated with growth factor delivery. Therefore the overall objective of this study was to investigate the influence of alpha-tricalcium phosphate (α-TCP) nanoparticle delivery into MSCs using an amphipathic cell penetrating peptide RALA, on osteogenesis in vitro and both intramembranous and endochondral bone formation in vivo. RALA complexed α-TCP nanoparticle delivery to MSCs resulted in an increased expression of bone morphogenetic protein-2 (BMP-2) and an upregulation in a number of key osteogenic genes. When α-TCP stimulated MSCs were encapsulated into alginate hydrogels, enhanced mineralization of the engineered construct was observed over a 28 day culture period. Furthermore, the in vivo bone forming potential of RALA complexed α-TCP nanoparticle delivery to MSCs was found to be comparable to growth factor delivery. Recognizing the potential and limitations associated with endochondral bone tissue engineering strategies, we then sought to explore how α-TCP nanoparticle delivery to MSCs influences early mineralization of engineered cartilage templates in vitro and their subsequent ossification in vivo. Despite accelerating mineralization of engineered cartilage templates in vitro, RALA complexed α-TCP nanoparticle delivery did not enhance endochondral bone formation in vivo. Therefore the potential of RALA complexed α-TCP nanoparticle delivery appears to be as an alternative to growth factor delivery as a single stage strategy for promoting bone generation.

Anisotropic Shape-Memory Alginate Scaffolds Functionalized with Either Type I or Type II Collagen for Cartilage Tissue Engineering.

Regenerating articular cartilage and fibrocartilaginous tissue such as the meniscus is still a challenge in orthopedic medicine. While a range of different scaffolds have been developed for joint repair, none have facilitated the development of a tissue that mimics the complexity of soft tissues such as articular cartilage. Furthermore, many of these scaffolds are not designed to function in mechanically challenging joint environments. The overall goal of this study was to develop a porous, biomimetic, shape-memory alginate scaffold for directing cartilage regeneration. To this end, a scaffold was designed with architectural cues to guide cellular and neo-tissue alignment, which was additionally functionalized with a range of extracellular matrix cues to direct stem cell differentiation toward the chondrogenic lineage. Shape-memory properties were introduced by covalent cross-linking alginate using carbodiimide chemistry, while the architecture of the scaffold was modified using a directional freezing technique. Introducing such an aligned pore structure was found to improve the mechanical properties of the scaffold, and promoted higher levels of sulfated glycosaminoglycans (sGAG) and collagen deposition compared to an isotropic (nonaligned) pore geometry when seeded with adult human stem cells. Functionalization with collagen improved stem cell recruitment into the scaffold and facilitated more homogenous cartilage tissue deposition throughout the construct. Incorporating type II collagen into the scaffolds led to greater cell proliferation, higher sGAG and collagen accumulation, and the development of a stiffer tissue compared to scaffolds functionalized with type I collagen. The results of this study demonstrate how both scaffold architecture and composition can be tailored in a shape-memory alginate scaffold to direct stem cell differentiation and support the development of complex cartilaginous tissues.

3D Bioprinting of Developmentally Inspired Templates for Whole Bone Organ Engineering.

The ability to print defined patterns of cells and extracellular-matrix components in three dimensions has enabled the engineering of simple biological tissues; however, bioprinting functional solid organs is beyond the capabilities of current biofabrication technologies. An alternative approach would be to bioprint the developmental precursor to an adult organ, using this engineered rudiment as a template for subsequent organogenesis in vivo. This study demonstrates that developmentally inspired hypertrophic cartilage templates can be engineered in vitro using stem cells within a supporting gamma-irradiated alginate bioink incorporating Arg-Gly-Asp adhesion peptides. Furthermore, these soft tissue templates can be reinforced with a network of printed polycaprolactone fibers, resulting in a ≈350 fold increase in construct compressive modulus providing the necessary stiffness to implant such immature cartilaginous rudiments into load bearing locations. As a proof-of-principal, multiple-tool biofabrication is used to engineer a mechanically reinforced cartilaginous template mimicking the geometry of a vertebral body, which in vivo supported the development of a vascularized bone organ containing trabecular-like endochondral bone with a supporting marrow structure. Such developmental engineering approaches could be applied to the biofabrication of other solid organs by bioprinting precursors that have the capacity to mature into their adult counterparts over time in vivo.

Bone Tissue Engineering with Multilayered Scaffolds-Part II: Combining Vascularization with Bone Formation in Critical-Sized Bone Defect.

Our previous in vivo study showed that multilayered scaffolds made of an angiogenic layer embedded between an osteogenic layer and an osteoconductive layer, with layer thickness in the 100-400 µm range, resulted in through-the-thickness vascularization of the construct even in the absence of exogenous endothelial cells. The angiogenic layer was a collagen-fibronectin gel, and the osteogenic layer was made from nanofibrous polycaprolactone while the osteoconductive layer was made either from microporous hydroxyapatite or microfibrous polycaprolactone. In this follow-up study, we implanted these acellular and cellular multilayered constructs in critical-sized rat calvarial defects and evaluated their vascularization and bone formation potential. Vascularization and bone formation at the defect were evaluated and quantified using microcomputed tomography (microCT) followed by perfusion of the animals with the radio opaque contrast agent, MICROFIL. The extent of bony bridging and union within the critical-sized defect was evaluated using a previously established scoring system from the microCT data set. Similarly the new bone formation in the defect was quantified from the microCT data set as previously reported. Histological evaluation at 4 and 12 weeks validated the microCT findings. Our experimental results showed that acellular multilayered scaffolds with microscale-thick nanofibers and porous ceramic discs with angiogenic zone at their interface can regenerate functional vasculature and bone similar to that of cellular constructs in critical-sized calvarial defects. This result suggests that suitably bioengineered acellular multilayered constructs can be an improved and more translational approach in functional in vivo bone regeneration.