Background
Cartilage damage is challenging to treat due to its limited self-healing capacity caused by its avascular nature. However, 3D tissue engineering approaches that utilize biomaterials, additives, and cells offer a promising solution to regenerate functional cartilage. These innovative techniques have the potential to restore native cartilage properties, prevent the development of osteoarthritis, and restore joint function. In this study, we investigate a novel alginate-based 3D bioink composed of human articular cartilage for cartilage tissue engineering.

Methods
The bioink was developed by encapsulating C20A4 human articular chondrocytes in a mixture consisting of 20% medium viscosity sodium alginate (Alg), 5% polyvinyl alcohol (PVA), 5% Gum Arabic (GA), and 5% human articular cartilage. The human articular cartilage was obtained from cartilage shavings of distal femurs and ground into a powder for incorporation into the bioink. The bioink was printed into concentric discs using a BIO-X 3D Bioprinter, and the resulting 3D-biofabricated constructs were cross-linked with calcium chloride, rinsed, and cultured in chondrogenic media. Thirty-six total constructs were fabricated. Immediately after 3D printing, a portion of constructs were visualized with SEM (n=1) and underwent compression testing (n=5) to examine the microstructure and mechanical strength respectively. The remaining constructs were placed in culture for 1,7 or 14 days and subsequently analyzed for cellular viability (n=5) and gene expression (n=5) at each time point.

Results
SEM imaging revealed that the 3D-printed constructs exhibited a porous structure. The average elastic modulus of the constructs was 0.48 MPa. Post-printing, the cellular viability was 87.2% which decreased significantly to 76.4% at day 7 (p<0.001) but recovered to 85.9% at day 14 in culture. Furthermore, gene expression analysis showed a significant increase in transcriptional activity of chondrogenic genes Col2a1 (p=0.008) and Sox9 (p=0.021) at day 14 compared to day 1.

Conclusion
This study explored the use of an innovative alginate-based 3D bioink composed of human articular cartilage for cartilage tissue engineering. The bioink successfully formed porous 3D constructs that withstood the printing process. Additionally, the elastic modulus of these engineered constructs approached that of native cartilage (0.45-0.8MPa). Although cellular viability initially decreased, it gradually recovered over a two-week culture period. Moreover, the upregulation of chondrogenic genes indicates the potential of the bioink to promote cartilage regeneration. These findings suggest this bioink holds promise as an effective approach for cartilage tissue engineering, offering possibilities for future therapeutic interventions in cartilage repair and regeneration.

Novel 3D Extrusion Bioink Containing Processed Human Articular Cartilage for Application in Cartilage Tissue Engineering

Background: Wound healing is a complex balance of cascading events orchestrated by dynamic autocrine/paracrine signaling of heterogenous factors. An imbalanced composition of bioactive compounds results in a cycle of sustained, inappropriate cellular signaling, and progression towards chronic wounds. The diverse and complex nature of chronic wounds has resulted in a lack of development of focused and effective therapeutic interventions. Therefore, there remains a critical need for a system that can generate customizable therapies for chronic wounds that can aid in reestablishing proper balance in wound healing signaling. Recent investigations have demonstrated that the diverse milieu of secreted factors from Adipose-derived Mesenchymal Stem Cells (ASCs) could offer a unique approach to treating chronic wounds that circumvents the limitations of other autologous cell therapies. Objective: Investigate the ability to tailor ASC biologics with “wound stimuli” priming.

Methods: A novel 3D-printed tissue-mimetic hydrogel system was utilized as a customizable in vitro bioreactor system to generate ASC-derived acellular biologics. ASCs were exposed to a variety of different native “wound stimuli”, including exposure to collagen peptides and fibrin(ogen). The “wound stimuli” were assessed for their ability to modulate the secretory composition of the ASC secretome and to enhance the wound healing activity of keratinocytes, fibroblasts, and endothelial cells.

Results: Our novel 3D system enhanced the regenerative activity of ASCs. Proteomic evaluation demonstrated ~15% increase in protein secretion, including a number of pro-wound healing growth factors and immunomodulatory compounds. Similarly, ASCs exhibited enhanced production of antioxidants (~25%) and EV/Exosomes (~4-5x). Treatment of keratinocytes, fibroblasts, and endothelial cells with the ASC-derived biologics resulted in enhanced wound healing activity, including increases in metabolic, proliferative, and migratory activity. Furthermore, introduction of “wound stimuli” to the 3D system resulted in modulation of the ASC secretome composition and further enhancement of its wound healing capacity. 

Conclusion: Together, this data demonstrates the potential of a novel 3D system to enhance the regenerative capacity of ASCs and permit the priming of ASCs to tailor secreted biologics. Thus, these studies provide insight towards advancing the development of tailored acellular biologics for future regenerative therapeutics for a variety of soft tissue application, including wound healing.

Tailoring Stem Cell Biologics to Enhance Regenerative Wound Healing

Background: At present, damage to nervous tissue is difficult to treat, often irreversible, and furthermore nerve growth to new sites is very limited. I hypothesize that teeth have a special capability to induce nerves and that we can isolate tissues and factors that could be used in a clinical setting to completely regenerate nerves. In human clinical studies where developing third molar teeth have been transplanted to donor sites, the teeth have continued to grow but more astoundingly have gained the ability for nociception in response to cold stimuli. This finding implies that developing teeth transplanted to a bony defect induce de-novo nerve growth at the recipient site. In this working study I utilize the rat model to demonstrate de-novo nerve growth by transplanting whole teeth to iatrogenic bony-defects created in autologous tibia and performing histological and biochemical analysis. Demonstrating innervation in transplanted teeth of post-natal rats would indicate that dental tissues are indeed capable of nerve growth and would greatly enhance our understanding of nerve induction.

Methods: Post-natal rats (Sprague-Dawley) aged 28-30 days were anesthetized and intact tooth germs with fully developed crowns undergoing root development were isolated. A bony defect was then created in the ipsilateral anterior tibia and the extracted tooth was transplanted to the defect. The wound was closed and hemostasis established. Hematoxylin and Eosin staining, and fluorescent immunohistochemistry were performed for nerve specific markers including anti-acetylated tubulin III antibodies. 

Results: Histology reveals a tooth well integrated into the tibia and a significant reduction in inflammation and bony remodeling, with dental specific enamel and dentin present in the leg. Moreover, the transplanted tooth is vascularized and early immunohistochemistry indicates evidence of innervation of transplanted teeth in line with the hypothesis that teeth have nerve inductive capacity postnatally.

Conclusion: Teeth transplanted autologously to tibia can integrate into the leg, survive as independent organs with their own blood supply, and early histology indicates innervation of the transplanted tooth. These data are applicable to patients’ needing re-innervation of eye, spine, extremity, teeth and many other organs. The implications are far-reaching and may lead to novel nerve regeneration therapeutics.




De-Novo Innnervation of a Transplant Organ

Computational methods for characterizing spatial gene expression heterogeneity in tissues

Abstract Body: INTRODUCTION: Graphene – an atomically thin layer of carbon atoms arranged in a hexagonal lattice – has gained interest as a bioscaffold for tissue engineering for its exceptional mechanical, electrical, and thermal properties1-5. Graphene’s structure and properties– tightly coupled to synthesis and processing conditions4– are thought to influence biomolecular interactions at the graphene-cell interfaces. In this study, C2C12 cells, a multipotent mouse myoblast cell line, were grown on graphene bioscaffolds with specific structure–property–processing–performance (SP3) correlations. We found that SP3 correlations significantly influenced C2C12 differentiation, myotube formation, and gene expression, suggesting that graphene–cell interfaces can be engineered to control biomolecule structure and function in adherent cells. The experiments performed are the first to make a direct comparison on scaffold SP3 correlations and their impact on genetic expression of multipotent myoblast cells. METHODS: Chemical vapor deposition (CVD) grown graphene film, transferred to quartz glass slides, resulted with surface roughness (Ra) of 2.37 nm and graphene ink– produced by solvent assisted exfoliation– was printed using a Dimatix inkjet printer onto glass coverslips (Ra of 10.0 nm). Epitaxial graphene on SiC wafers, produced by Si sublimation (Istituto Italiano de Tecnologia, Pisa, Italy) (Ra of 2.24 nm). Prior to cell integration, cell attachment was facilitated with agarose gel applied to the edges of all graphene bioscaffolds and corresponding control bioscaffolds (quartz glass, glass coverslips and SiC wafers). C2C12 cells, maintained in a humidified incubator at 37°C and 5% CO2, were seeded onto the scaffolds at 1.3 x 104 cells/cm2 in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). Cell growth and proliferation were monitored by transmitted light microscopy with images captured to study cell morphology. Gene expression patterns for each bioscaffold were analyzed for muscle myogenesis via qRT-PCR using a PCR array for muscle myogenesis and myopathy. RESULTS: Preliminary results show differences in cell morphology varied in a manner dependent upon the specific bioscaffold used. Myotube-like structures were evident in the control, CVD and epitaxial graphene (Fig.1A). Each of the three graphene bioscaffolds resulted in a unique overall genetic profile related to muscle myogenesis. However, four up-regulated genes were common to all biosaffolds when compared to their control. These genes are as follows: dystrophin (Dmd), myostatin (Mstn), leptin (Lep), pyruvate dehydrogenase kinase isoenzyme 4 (Pdk4) and Utrophin (Utm) (Fig. 1B). DISCUSSION: This preliminary work successfully shows that epitaxial, CVD, and printed graphene bioscaffold’s SP3 correlations impact the gene expression of C2C12 cells in culture. Each graphene bioscaffold presented with different and unique muscle gene expression profiles when compared to the control. The different morphologies of the cells as viewed in the transmitted light microscopy images confirms that the C2C12 cells grown on each bioscaffold, are uniquely different than the control. The following biomarkers: dystrophin, myostatin, leptin, pyruvate dehydrogenase kinase isoenzyme 4, and Utrophin were all upregulated in each of the graphene bioscaffolds. Epitaxial graphene shows the most genes upregulated that correlate to the muscle genotype followed by printed graphene and CVD graphene which showed the least number of upregulated genes relating to muscle (Fig. 1C). REFERENCES: 1. Nayak et al., ACS Nano., 2011. 2. Li et al., Sci Rep., 2013. 3. Krueger et al., ACS Biomaterials Science & Engineering, 2016. 4. Novoselov et al., Nature, (2012): 192-200. 5. Nieto et al., Advanced Functional Materials, 2015. ACKNOWLEDGEMENTS: Support kindly provided by NIGMS under P20GM103408 and P20GM109095 and NSF under 0619793 and 0923535

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Novel 3D Extrusion Bioink Containing Processed Human Articular Cartilage for Application in Cartilage Tissue Engineering

Tailoring Stem Cell Biologics to Enhance Regenerative Wound Healing

De-Novo Innnervation of a Transplant Organ

Computational methods for characterizing spatial gene expression heterogeneity in tissues

Differential Gene Expression in C2C12 Cells Due to Scaffold Structure-Property-Processing-Performance Correlations

Blueprints for Artificial Organs - Tissue Clearing and Beyond

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