CRISPR-GEM: A Novel Machine Learning Model for CRISPR Genetic Target Discovery and Evaluation.

CRISPR gene editing strategies are shaping cell therapies through precise and tunable control over gene expression. However, achieving reliable therapeutic effects with improved safety and efficacy requires informed target gene selection. This depends on a thorough understanding of the involvement of target genes in gene regulatory networks (GRNs) that regulate cell phenotype and function. Machine learning models have been previously used for GRN reconstruction using RNA-seq data, but current techniques are limited to single cell types and focus mainly on transcription factors. This restriction overlooks many potential CRISPR target genes, such as those encoding extracellular matrix components, growth factors, and signaling molecules, thus limiting the applicability of these models for CRISPR strategies. To address these limitations, we have developed CRISPR-GEM, a multi-layer perceptron (MLP)-based synthetic GRN constructed to accurately predict the downstream effects of CRISPR gene editing. First, input and output nodes are identified as differentially expressed genes between defined experimental and target cell/tissue types respectively. Then, MLP training learns regulatory relationships in a black-box approach allowing accurate prediction of output gene expression using only input gene expression. Finally, CRISPR-mimetic perturbations are made to each input gene individually and the resulting model predictions are compared to those for the target group to score and assess each input gene as a CRISPR candidate. The top scoring genes provided by CRISPR-GEM therefore best modulate experimental group GRNs to motivate transcriptomic shifts towards a target group phenotype. This machine learning model is the first of its kind for predicting optimal CRISPR target genes and serves as a powerful tool for enhanced CRISPR strategies across a range of cell therapies.

Neutrophil Granulopoiesis Optimized Through Ex Vivo Expansion of Hematopoietic Progenitors in Engineered 3D Gelatin Methacrylate Hydrogels.

Neutrophils are the first line of defense of the innate immune system. In response to methicillin-resistant Staphylococcus aureus infection in the skin, hematopoietic stem, and progenitor cells (HSPCs) traffic to wounds and undergo extramedullary granulopoiesis, producing neutrophils necessary to resolve the infection. This prompted the engineering of a gelatin methacrylate (GelMA) hydrogel that encapsulates HSPCs within a matrix amenable to subcutaneous delivery. The authors study the influence of hydrogel mechanical properties to produce an artificial niche for granulocyte-monocyte progenitors (GMPs) to efficiently expand into functional neutrophils that can populate infected tissue. Lin-cKIT+ HSPCs, harvested from fluorescent neutrophil reporter mice, are encapsulated in GelMA hydrogels of varying polymer concentration and UV-crosslinked to produce HSPC-laden gels of specific stiffness and mesh sizes. Softer 5% GelMA gels yield the most viable progenitors and effective cell-matrix interactions. Compared to suspension culture, 5% GelMA results in a twofold expansion of mature neutrophils that retain antimicrobial functions including degranulation, phagocytosis, and ROS production. When implanted dermally in C57BL/6J mice, luciferase-expressing neutrophils expanded in GelMA hydrogels are visualized at the site of implantation for over 5 days. They demonstrate the potential of GelMA hydrogels for delivering HSPCs directly to the site of skin infection to promote local granulopoiesis.

Solvent-cast 3D printing with molecular weight polymer blends to decouple effects of scaffold architecture and mechanical properties on mesenchymal stromal cell fate.

The biochemical and physical properties of a scaffold can be tailored to elicit specific cellular responses. However, it is challenging to decouple their individual effects on cell-material interactions. Here, we solvent-cast 3D printed different ratios of high and low molecular weight (MW) poly(caprolactone) (PCL) to fabricate scaffolds with significantly different stiffnesses without affecting other properties. Ink viscosity was used to match processing conditions between inks and generate scaffolds with the same surface chemistry, crystallinity, filament diameter, and architecture. Increasing the ratio of low MW PCL resulted in a significant decrease in modulus. Scaffold modulus did not affect human mesenchymal stromal cell (hMSC) differentiation under osteogenic conditions. However, hMSC response was significantly affected by scaffold stiffness in chondrogenic media. Low stiffness promoted more stable chondrogenesis whereas high stiffness drove hMSC progression toward hypertrophy. These data illustrate how this versatile platform can be used to independently modify biochemical and physical cues in a single scaffold to synergistically enhance desired cellular response.

Immunomodulatory Strategies for Cartilage Regeneration in Osteoarthritis.

Osteoarthritis (OA) is the most prevalent musculoskeletal disorder and a leading cause of disability globally. Although many efforts have been made to treat this condition, current tissue engineering (TE) and regenerative medicine strategies fail to address the inflammatory tissue environment that leads to the rapid progression of the disease and prevents cartilage tissue formation. First, this review addresses in detail the current anti-inflammatory therapies for OA with a special emphasis on pharmacological approaches, gene therapy, and mesenchymal stromal cell (MSC) intra-articular administration, and discusses the reasons behind the limited clinical success of these approaches at enabling cartilage regeneration. Then, we analyze the state-of-the-art TE strategies and how they can be improved by incorporating immunomodulatory capabilities such as the optimization of biomaterial composition, porosity and geometry, and the loading of anti-inflammatory molecules within an engineered structure. Finally, the review discusses the future directions for the new generation of TE strategies for OA treatment, specifically focusing on the spatiotemporal modulation of anti-inflammatory agent presentation to allow for tailored patient-specific therapies. Impact statement Osteoarthritis (OA) is a prevalent and debilitating musculoskeletal disorder affecting millions worldwide. Despite significant advancements in regenerative medicine and tissue engineering (TE), mitigating inflammation while simultaneously promoting cartilage tissue regeneration in OA remains elusive. In this review article, we discuss current anti-inflammatory therapies and explore their potential synergy with cutting-edge cartilage TE strategies, with a special focus on novel spatiotemporal and patient-specific anti-inflammatory strategies.

Engineered Cell-Secreted Extracellular Matrix Modulates Cell Spheroid Mechanosensing and Amplifies Their Response to Inductive Cues for the Formation of Mineralized Tissues.

The clinical translation of mesenchymal stromal cell (MSC)-based therapies remains challenging due to rapid cell death and poor control over cell behavior. Compared to monodisperse cells, the aggregation of MSCs into spheroids increases their tissue-forming potential by promoting cell-cell interactions. However, MSCs initially lack engagement with an endogenous extracellular matrix (ECM) when formed into spheroids. Previously the instructive nature of an engineered, cell-secreted ECM is demonstrated to promote survival and differentiation of adherent MSCs. Herein, it is hypothesized that the incorporation of this cell-secreted ECM during spheroid aggregation would enhance MSC osteogenic potential by promoting cell-matrix and cell-cell interactions. ECM-loaded spheroids contained higher collagen and glycosaminoglycan content, and MSCs exhibited increased mechanosensitivity to ECM through Yes-associated protein (YAP) activation via integrin α2ß1 binding. ECM-loaded spheroids sustained greater MSC viability and proliferation and are more responsive to soluble cues for lineage-specific differentiation than spheroids without ECM or loaded with collagen. The encapsulation of ECM-loaded spheroids in instructive alginate gels resulted in spheroid fusion and enhanced osteogenic differentiation. These results highlight the clinical potential of ECM-loaded spheroids as building blocks for the repair of musculoskeletal tissues.

Alginate-Based Bioinks for 3D Bioprinting and Fabrication of Anatomically Accurate Bone Grafts.

To realize the promise of three-dimensional (3D) bioprinting, it is imperative to develop bioinks that possess the necessary biological and rheological characteristics for printing cell-laden tissue grafts. Alginate is widely used as a bioink because its rheological properties can be modified through precrosslinking or the addition of thickening agents to increase printing resolution. However, modification of alginate's physiochemical characteristics using common crosslinking agents can affect its cytocompatibility. Therefore, we evaluated the printability, physicochemical properties, and osteogenic potential of four common alginate bioinks: alginate-CaCl2 (alg-CaCl2), alginate-CaSO4 (alg-CaSO4), alginate-gelatin (alg-gel), and alginate-nanocellulose (alg-ncel) for the 3D bioprinting of anatomically accurate osteogenic grafts. While all bioinks possessed similar viscosity, printing fidelity was lower in the precrosslinked bioinks. When used to print geometrically defined constructs, alg-CaSO4 and alg-ncel exhibited higher mechanical properties and lower mesh size than those printed with alg-CaCl2 or alg-gel. The physical properties of these constructs affected the biological performance of encapsulated bone marrow-derived mesenchymal stromal cells (MSCs). Cell-laden constructs printed using alg-CaSO4 and alg-ncel exhibited greater cell apoptosis and contained fewer living cells 7 days postprinting. In addition, effective cell-matrix interactions were only observed in alg-CaCl2 printed constructs. When cultured in osteogenic media, MSCs in alg-CaCl2 constructs exhibited increased osteogenic differentiation compared to the other three bioinks. This bioink was then used to 3D print anatomically accurate cell-laden scaphoid bones that were capable of partial mineralization after 14 days of in vitro culture. These results highlight the importance of bioink properties to modulate cell behavior and the biofabrication of clinically relevant bone tissues. Impact statement Alginate-based bioinks are widely used for three-dimensional (3D) bioprinting of bone tissues. However, a direct systematic comparison between alginate-based bioinks is needed to assess the optimal bioink properties for mesenchymal stromal cell survival and osteogenesis. This study evaluates the printability, physical properties, biocompatibility, and osteogenic potential of four commonly used alginate-based bioinks and establishes the importance of bioink properties for advancing toward the clinical translation of 3D bioprinted bone grafts.

Three-Dimensional Printed Stamps for the Fabrication of Patterned Microwells and High-Throughput Production of Homogeneous Cell Spheroids.

Aggregation of cells into spheroids and organoids is a promising tool for regenerative medicine, cancer and cell biology, and drug discovery due to their recapitulation of the cell-cell and cell-matrix interactions found in vivo. Traditional approaches for the production of spheroids, such as the hanging drop method, are limited by the lack of reproducibility and the use of labor-intensive and time-consuming techniques. The need for high-throughput approaches allowing for the quick and reproducible formation of cell aggregates has driven the development of soft lithography techniques based on the patterning of microwells into nonadherent hydrogels. However, these methods are also limited by costly, labor-intensive, and multistep protocols that could impact the sterility of the process and efficiency of spheroid formation. In this study, we describe a one-step method for the fabrication of patterned nonadherent microwells into tissue culture plates using three-dimensional (3D) printed stamps and evaluate the production of cell spheroids of different sizes and cell sources. The generation of bone marrow-derived mesenchymal stromal cell and endothelial cell spheroids by the use of 3D printed stamps was superior in comparison with a widely used multistep mold technique, yielding spheroids of larger sizes and higher DNA content. The 3D stamps produced spheroids of more consistent diameter and DNA content when compared with other commercially available methods. These 3D printed stamps offer a tunable, simple, fast, and cost-effective approach for the production of reproducible spheroids and organoids for a wide range of applications.

Multi-peptide presentation and hydrogel mechanics jointly enhance therapeutic duo-potential of entrapped stromal cells.

The native extracellular matrix (ECM) contains a host of matricellular proteins and bioactive factors that regulate cell behavior, and many ECM components have been leveraged to guide cell fate. However, the large size and chemical characteristics of these constituents complicate their incorporation into biomaterials without interfering with material properties, motivating the need for alternative approaches to regulate cellular responses. Mesenchymal stromal cells (MSCs) can promote osseous regeneration in vivo directly or indirectly through multiple means including (1) secretion of proangiogenic and mitogenic factors to initiate formation of a vascular template and recruit host cells into the tissue site or (2) direct differentiation into osteoblasts. As MSC behavior is influenced by the properties of engineered hydrogels, we hypothesized that the biochemical and biophysical properties of alginate could be manipulated to promote the dual contributions of encapsulated MSCs toward bone formation. We functionalized alginate with QK peptide to enhance proangiogenic factor secretion and RGD to promote adhesion, while biomechanical-mediated osteogenic cues were controlled by modulating viscoelastic properties of the alginate substrate. A 1:1 ratio of QK:RGD resulted in the highest levels of both proangiogenic factor secretion and mineralization in vitro. Viscoelastic alginate outperformed purely elastic gels in both categories, and this effect was enhanced by stiffness up to 20 kPa. Furthermore, viscoelastic constructs promoted vessel infiltration and bone regeneration in a rat calvarial defect over 12 weeks. These data suggest that modulating viscoelastic properties of biomaterials, in conjunction with dual peptide functionalization, can simultaneously enhance multiple aspects of MSC regenerative potential and improve neovascularization of engineered tissues.

Tunable fibrin-alginate interpenetrating network hydrogels to support cell spreading and network formation.

Hydrogels are effective platforms for use as artificial extracellular matrices, cell carriers, and to present bioactive cues. Two common natural polymers, fibrin and alginate, are broadly used to form hydrogels and have numerous advantages over synthetic materials. Fibrin is a provisional matrix containing native adhesion motifs for cell engagement, yet the interplay between mechanical properties, degradation, and gelation rate is difficult to decouple. Conversely, alginate is highly tunable yet bioinert and requires modification to present necessary adhesion ligands. To address these challenges, we developed a fibrin-alginate interpenetrating network (IPN) hydrogel to combine the desirable adhesion and stimulatory characteristics of fibrin with the tunable mechanical properties of alginate. We tested its efficacy by examining capillary network formation with entrapped co-cultures of mesenchymal stromal cells (MSCs) and endothelial cells (ECs). We manipulated thrombin concentration and alginate crosslinking density independently to modulate the fibrin structure, mesh size, degradation, and biomechanical properties of these constructs. In IPNs of lower stiffness, we observed a significant increase in total cell area (1.7 × 105 ± 7.9 × 104 µm2) and decrease in circularity (0.56 ± 0.03) compared to cells encapsulated in stiffer IPNs (4.0 × 104 ± 1.5 × 104 µm2 and 0.77 ± 0.09, respectively). Fibrinogen content did not influence capillary network formation. However, higher fibrinogen content led to greater retention of these networks confirmed via increased spreading and presence of F-actin at 7 days. This is an elegant platform to decouple cell adhesion and hydrogel bulk stiffness that will be broadly useful for cell instruction and delivery. STATEMENT OF SIGNIFICANCE: Hydrogels are widely used as drug and cell delivery vehicles and as artificial extracellular matrices to study cellular responses. However, there are limited opportunities to simultaneously control mechanical properties and degradation while mimicking the complex native adhesion motifs and ligands known to encourage cell engagement with the hydrogel. In this study, we describe a fibrin-alginate interpenetrating network (IPN) hydrogel designed to balance the compliance and provisional qualities of fibrin with the mechanical stability and tunability of alginate to interrogate these contributions on cell response. We used clinically relevant cell sources, a co-culture of endothelial cells and mesenchymal stromal cells, to test its efficacy in supporting capillary formation in vitro. These data demonstrate the promise of this IPN for use in tissue engineering.

Nano-particle mediated M2 macrophage polarization enhances bone formation and MSC osteogenesis in an IL-10 dependent manner.

Engineering a pro-regenerative immune response following scaffold implantation is integral to functional tissue regeneration. The immune response to implanted biomaterials is determined by multiple factors, including biophysical cues such as material stiffness, topography and particle size. In this study we developed an immune modulating scaffold for bone defect healing containing bone mimetic nano hydroxyapatite particles (BMnP). We first demonstrate that, in contrast to commercially available micron-sized hydroxyapatite particles, in-house generated BMnP preferentially polarize human macrophages towards an M2 phenotype, activate the transcription factor cMaf and specifically enhance production of the anti-inflammatory cytokine, IL-10. Furthermore, nano-particle treated macrophages enhance mesenchymal stem cell (MSC) osteogenesis in vitro and this occurs in an IL-10 dependent manner, demonstrating a direct pro-osteogenic role for this cytokine. BMnPs were also capable of driving pro-angiogenic responses in human macrophages and HUVECs. Characterization of immune cell subsets following incorporation of functionalized scaffolds into a rat femoral defect model revealed a similar profile, with micron-sized hydroxyapatite functionalized scaffolds eliciting pro-inflammatory responses characterized by infiltrating T cells and elevated expression of M1 macrophages markers compared to BMnP functionalized scaffolds which promoted M2 macrophage polarization, tissue vascularization and increased bone volume. Taken together these results demonstrate that nano-sized Hydroxyapatite has immunomodulatory potential and is capable of directing anti-inflammatory innate immune-mediated responses that are associated with tissue repair and regeneration.

Bio-instructive materials for musculoskeletal regeneration.

The prevalence and cost of disorders affecting the musculoskeletal system are predicted to rise significantly in the coming years due to the aging global population and the increase of associated risk factors. Despite being the second largest cause of disability, the clinical options for therapeutic intervention remain limited. The clinical translation of cell-based therapies for the treatment of musculoskeletal disorders faces many challenges including maintenance of cell survival in the harsh in vivo environment and the lack of control over regulating cell phenotype upon implantation. In order to address these challenges, the development of bio-instructive materials to modulate cell behavior has taken center stage as a strategy to increase the therapeutic potential of various cell populations. However, the determination of the necessary cues for a specific application and how these signals should be presented from a biomaterial remains elusive. This review highlights recent biochemical and physical strategies used to engineer bio-instructive materials for the repair of musculoskeletal tissues. There is a particular emphasis on emerging efforts such as the engineering of immunomodulatory and antibacterial materials, as well as the incorporation of these strategies into biofabrication and organ-on-a-chip approaches. STATEMENT OF SIGNIFICANCE: Disorders affecting the musculoskeletal system affect individuals across the lifespan and have a profound effect on mobility and quality of life. While small defects in many tissues can heal successfully, larger defects are often unable to heal or instead heal with inferior quality fibrous tissue and require clinical intervention. Cell-based therapies are a promising option for clinical translation, yet challenges related to maintaining cell survival and instructing cell phenotype upon implantation have limited the success of this approach. Bio-instructive materials provide an exciting opportunity to modulate cell behavior and enhance the efficacy of cell-based approaches for musculoskeletal repair. However, the identification of critical instructive cues and how to present these stimuli is a focus of intense investigation. This review highlights recent biochemical and physical strategies used to engineer bio-instructive materials for the repair of musculoskeletal tissues, while also considering exciting progress in the engineering of immunomodulatory and antibacterial materials.

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.

Defining hydrogel properties to instruct lineage- and cell-specific mesenchymal differentiation.

The maintenance and direction of stem cell lineage after implantation remains challenging for clinical translation. Aggregation and encapsulation into instructive biomaterials after preconditioning can bolster retention of differentiated phenotypes. Since these procedures do not depend on cell type or lineage, we hypothesized we could use a common, tunable platform to engineer formulations that retain and enhance multiple lineages from different cell populations. To test this, we varied alginate stiffness and adhesive ligand content, then encapsulated spheroids of varying cellularity. We used Design-of-Experiments to determine the effect of these parameters and their interactions on phenotype retention. The combination of parameters leading to maximal differentiation varied with lineage and cell type, inducing a 2-4-fold increase over non-optimized levels. Phenotype was also retained for 4 weeks in a murine subcutaneous model. This widely applicable approach can facilitate translation of cell-based therapies by instructing phenotype in situ without prolonged induction or costly growth factors.

Dual non-viral gene delivery from microparticles within 3D high-density stem cell constructs for enhanced bone tissue engineering.

High-density mesenchymal stem cell (MSC) aggregates can be guided to form bone-like tissue via endochondral ossification in vitro when culture media is supplemented with proteins, such as growth factors (GFs), to first guide the formation of a cartilage template, followed by culture with hypertrophic factors. Recent reports have recapitulated these results through the controlled spatiotemporal delivery of chondrogenic transforming growth factor-ß1 (TGF-ß1) and chondrogenic and osteogenic bone morphogenetic protein-2 (BMP-2) from microparticles embedded within human MSC aggregates to avoid diffusion limitations and the lengthy, costly in vitro culture necessary with repeat exogenous supplementation. However, since GFs have limited stability, localized gene delivery is a promising alternative to the use of proteins. Here, mineral-coated hydroxyapatite microparticles (MCM) capable of localized delivery of Lipofectamine-plasmid DNA (pDNA) nanocomplexes encoding for TGF-ß1 (pTGF-ß1) and BMP-2 (pBMP-2) were incorporated, alone or in combination, within MSC aggregates from three healthy porcine donors to induce sustained production of these transgenes. Three donor populations were investigated in this work due to the noted MSC donor-to-donor variability in differentiation capacity documented in the literature. Delivery of pBMP-2 within Donor 1 aggregates promoted chondrogenesis at week 2, followed by an enhanced osteogenic phenotype at week 4. Donor 2 and 3 aggregates did not promote robust glycosaminoglycan (GAG) production at week 2, but by week 4, Donor 2 aggregates with pTGF-ß1/pBMP-2 and Donor 3 aggregates with both unloaded MCM and pBMP-2 enhanced osteogenesis compared to controls. These results demonstrate the ability to promote osteogenesis in stem cell aggregates through controlled, non-viral gene delivery within the cell masses. These findings also indicate the need to screen donor MSC regenerative potential in response to gene transfer prior to clinical application. Taken together, this work demonstrates a promising gene therapy approach to control stem cell fate in biomimetic 3D condensations for treatment of bone defects.

3D Bioprinting for Cartilage and Osteochondral Tissue Engineering.

Significant progress has been made in the field of cartilage and bone tissue engineering over the last two decades. As a result, there is real promise that strategies to regenerate rather than replace damaged or diseased bones and joints will one day reach the clinic however, a number of major challenges must still be addressed before this becomes a reality. These include vascularization in the context of large bone defect repair, engineering complex gradients for bone-soft tissue interface regeneration and recapitulating the stratified zonal architecture present in many adult tissues such as articular cartilage. Tissue engineered constructs typically lack such spatial complexity in cell types and tissue organization, which may explain their relatively limited success to date. This has led to increased interest in bioprinting technologies in the field of musculoskeletal tissue engineering. The additive, layer by layer nature of such biofabrication strategies makes it possible to generate zonal distributions of cells, matrix and bioactive cues in 3D. The adoption of biofabrication technology in musculoskeletal tissue engineering may therefore make it possible to produce the next generation of biological implants capable of treating a range of conditions. Here, advances in bioprinting for cartilage and osteochondral tissue engineering are reviewed.

* 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.

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.

Gene Delivery of TGF-ß3 and BMP2 in an MSC-Laden Alginate Hydrogel for Articular Cartilage and Endochondral Bone Tissue Engineering.

Incorporating therapeutic genes into three-dimensional biomaterials is a promising strategy for enhancing tissue regeneration. Alginate hydrogels have been extensively investigated for cartilage and bone tissue engineering, including as carriers of transfected cells to sites of injury, making them an ideal gene delivery platform for cartilage and osteochondral tissue engineering. The objective of this study was to develop gene-activated alginate hydrogels capable of supporting nanohydroxyapatite (nHA)-mediated nonviral gene transfer to control the phenotype of mesenchymal stem cells (MSCs) for either cartilage or endochondral bone tissue engineering. To produce these gene-activated constructs, MSCs and nHA complexed with plasmid DNA (pDNA) encoding for transforming growth factor-beta 3 (pTGF-ß3), bone morphogenetic protein 2 (pBMP2), or a combination of both (pTGF-ß3-pBMP2) were encapsulated into alginate hydrogels. Initial analysis using reporter genes showed effective gene delivery and sustained overexpression of the transgenes were achieved. Confocal microscopy demonstrated that complexing the plasmid with nHA before hydrogel encapsulation led to transport of the plasmid into the nucleus of MSCs, which did not happen with naked pDNA. Gene delivery of TGF-ß3 and BMP2 and subsequent cell-mediated expression of these therapeutic genes resulted in a significant increase in sulfated glycosaminoglycan and collagen production, particularly in the pTGF-ß3-pBMP2 codelivery group in comparison to the delivery of either pTGF-ß3 or pBMP2 in isolation. In addition, stronger staining for collagen type II deposition was observed in the pTGF-ß3-pBMP2 codelivery group. In contrast, greater levels of calcium deposition were observed in the pTGF-ß3- and pBMP2-only groups compared to codelivery, with a strong staining for collagen type X deposition, suggesting these constructs were supporting MSC hypertrophy and progression along an endochondral pathway. Together, these results suggest that the developed gene-activated alginate hydrogels were able to support transfection of encapsulated MSCs and directed their phenotype toward either a chondrogenic or an osteogenic phenotype depending on whether TGF-ß3 and BMP2 were delivered in combination or isolation.