3D-Printed Demineralized Bone Matrix-Based Conductive Scaffolds Combined with Electrical Stimulation for Bone Tissue Engineering Applications.

Bone is remodeled through a dynamic process facilitated by biophysical cues that support cellular signaling. In healthy bone, signaling pathways are regulated by cells and the extracellular matrix and transmitted via electrical synapses. To this end, combining electrical stimulation (ES) with conductive scaffolding is a promising approach for repairing damaged bone tissue. Therefore, "smart" biomaterials that can provide multifunctionality and facilitate the transfer of electrical cues directly to cells have become increasingly more studied in bone tissue engineering. Herein, 3D-printed electrically conductive composite scaffolds consisting of demineralized bone matrix (DBM) and polycaprolactone (PCL), in combination with ES, for bone regeneration were evaluated for the first time. The conductive composite scaffolds were fabricated and characterized by evaluating mechanical, surface, and electrical properties. The DBM/PCL composites exhibited a higher compressive modulus (107.2 MPa) than that of pristine PCL (62.02 MPa), as well as improved surface properties (i.e., roughness). Scaffold electrical properties were also tuned, with sheet resistance values as low as 4.77 × 105 Ω/sq for our experimental coating of the highest dilution (i.e., 20%). Furthermore, the biocompatibility and osteogenic potential of the conductive composite scaffolds were tested using human mesenchymal stromal cells (hMSCs) both with and without exogenous ES (100 mV/mm for 5 min/day four times/week). In conjunction with ES, the osteogenic differentiation of hMSCs grown on conductive DBM/PCL composite scaffolds was significantly enhanced when compared to those cultured on PCL-only and nonconductive DBM/PCL control scaffolds, as determined through xylenol orange mineral staining and osteogenic protein analysis. Overall, these promising results suggest the potential of this approach for the development of biomimetic hybrid scaffolds for bone tissue engineering applications.

3D-Printed conductive polymeric scaffolds with direct current electrical stimulation for enhanced bone regeneration.

Various methods have been used to treat bone defects caused by genetic disorders, injury, or disease. Yet, there is still great need to develop alternative approaches to repair damaged bone tissue. Bones naturally exhibit piezoelectric potential, or the ability to convert mechanical stresses into electrical impulses. This phenomenon has been utilized clinically to enhance bone regeneration in conjunction with electrical stimulation (ES) therapies; however, oftentimes with critical-sized bone defects, the bioelectric potential at the site of injury is compromised, resulting in less desirable outcomes. In the present study, the potential of a 3D-printed conductive polymer blend to enhance bone formation through restoration of the bioelectrical microenvironment was evaluated. A commercially available 3D printer was used to create circular, thin-film scaffolds consisting of either polylactide (PLA) or a conductive PLA (CPLA) composite. Preosteoblast cells were seeded onto the scaffolds and subjected to direct current ES via a purpose-built cell culture chamber. It was found that CPLA scaffolds had no adverse effects on cell viability, proliferation or differentiation when compared with control scaffolds. The addition of ES, however, resulted in a significant increase in the expression of osteocalcin, a protein indicative of osteoblast maturation, after 14 days of culture. Furthermore, xylenol orange staining also showed the presence of increased mineralized calcium nodules in cultures undergoing stimulation. This study demonstrates the potential for low-cost, conductive scaffolding materials to support cell viability and enhance in vitro mineralization in conjunction with ES.

Conductive Scaffolds for Bone Tissue Engineering: Current State and Future Outlook.

Bone tissue engineering strategies attempt to regenerate bone tissue lost due to injury or disease. Three-dimensional (3D) scaffolds maintain structural integrity and provide support, while improving tissue regeneration through amplified cellular responses between implanted materials and native tissues. Through this, scaffolds that show great osteoinductive abilities as well as desirable mechanical properties have been studied. Recently, scaffolding for engineered bone-like tissues have evolved with the use of conductive materials for increased scaffold bioactivity. These materials make use of several characteristics that have been shown to be useful in tissue engineering applications and combine them in the hope of improved cellular responses through stimulation (i.e., mechanical or electrical). With the addition of conductive materials, these bioactive synthetic bone substitutes could result in improved regeneration outcomes by reducing current factors limiting the effectiveness of existing scaffolding materials. This review seeks to overview the challenges associated with the current state of bone tissue engineering, the need to produce new grafting substitutes, and the promising future that conductive materials present towards alleviating the issues associated with bone repair and regeneration.

Bioprinted Three-Dimensional Cell-Laden Hydrogels to Evaluate Adipocyte-Breast Cancer Cell Interactions.

Three-dimensional (3D) bioprinting, although still in its infancy as a fabrication tool, has the potential to effectively mimic many biological environments. Cell-laden 3D printed structures have demonstrated to be an improvement from the widely used monolayer platforms, largely because of recapitulation of native tissue architecture with the 3D structures. Thus, 3D in vitro models have been increasingly investigated for improved modeling of cell and disease systems, such as for breast cancer. In the present work, multicellular cell-laden hydrogels comprised of adipocytes and breast cancer cells were bioprinted and evaluated. An ideal bioink of 3:2 5% alginate was determined to mimic the tissue stiffness observed in a physiological breast cancer tumor environment. Rheological characterization and degradation studies were performed to verify the stability of the artificial breast hydrogel environment. It was found that both the breast cancer cells and adipocytes remained viable directly after printing and throughout the 10-day culture period within the printed hydrogels. Direct printing of the cells in co-culture resulted in morphology changes and variations in cell localization within printed structures. Overall, the feasibility of efficiently fabricating multicellular cell-laden bioprinted models of the breast tumor microenvironment was established.

4D Biofabrication Using Shape-Morphing Hydrogels.

Despite the tremendous potential of bioprinting techniques toward the fabrication of highly complex biological structures and the flourishing progress in 3D bioprinting, the most critical challenge of the current approaches is the printing of hollow tubular structures. In this work, an advanced 4D biofabrication approach, based on printing of shape-morphing biopolymer hydrogels, is developed for the fabrication of hollow self-folding tubes with unprecedented control over their diameters and architectures at high resolution. The versatility of the approach is demonstrated by employing two different biopolymers (alginate and hyaluronic acid) and mouse bone marrow stromal cells. Harnessing the printing and postprinting parameters allows attaining average internal tube diameters as low as 20 µm, which is not yet achievable by other existing bioprinting/biofabrication approaches and is comparable to the diameters of the smallest blood vessels. The proposed 4D biofabrication process does not pose any negative effect on the viability of the printed cells, and the self-folded hydrogel-based tubes support cell survival for at least 7 d without any decrease in cell viability. Consequently, the presented 4D biofabrication strategy allows the production of dynamically reconfigurable architectures with tunable functionality and responsiveness, governed by the selection of suitable materials and cells.

Lithium-end-capped polylactide thin films influence osteoblast progenitor cell differentiation and mineralization.

End-capping by covalently binding functional groups to the ends of polymer chains offers potential advantages for tissue engineering scaffolds, but the ability of such polymers to influence cell behavior has not been studied. As a demonstration, polylactide (PLA) was end-capped with lithium carboxylate ionic groups (hPLA13kLi) and evaluated. Thin films of the hPLA13kLi and PLA homopolymer were prepared with and without surface texturing. Murine osteoblast progenitor cells from collagen 1α1 transgenic reporter mice were used to assess cell attachment, proliferation, differentiation, and mineralization. Measurement of green fluorescent protein expressed by these cells and xylenol orange staining for mineral allowed quantitative analysis. The hPLA13kLi was biologically active, increasing initial cell attachment and enhancing differentiation, while reducing proliferation and strongly suppressing mineralization, relative to PLA. These effects of bound lithium ions (Li(+) ) had not been previously reported, and were generally consistent with the literature on soluble additions of lithium. The surface texturing generated here did not influence cell behavior. These results demonstrate that end-capping could be a useful approach in scaffold design, where a wide range of biologically active groups could be employed, while likely retaining the desirable characteristics associated with the unaltered homopolymer backbone.

Characterization of the differentiation and leptin secretion profile of adult stem cells on patterned polylactide films.

Several issues need to be better understood before breast tissue engineering becomes a clinically viable option. One of the most important aspects is the interaction between cells and the microtopography of the implant surface. The aim of this study was to evaluate the efficacy of D1 cells, multipotent mouse bone marrow stromal precursors, in differentiating to adipocytes and to characterize their metabolic activity (lactic acid released and glucose consumed), leptin secretion and lipid production when cultured on patterned poly(L-lactide) (PLLA) films. It was determined that, by appropriate stimulation, the D1 cells displayed morphological characteristics of adipocytes and produced lipid. The results showed that a patterned surface did affect the rate of lipid production. Polynomial models were proposed to predict the amount of leptin secreted by the cells over a period of time.

Stem cells and adipose tissue engineering.

A large proportion of the plastic and reconstructive surgical procedures performed each year are to repair soft tissue defects that result from traumatic injury, tumor resection, and congenital defects. These defects typically result from the loss of a large volume of adipose tissue. To date, no ideal filler material which is successful in all cases has been developed. Additionally, the success of using autologous fat tissue grafts to repair soft tissue defects has been limited. Researchers are thus investigating strategies to engineer volumes of adipose tissue that may be used in these cases. A necessary component for engineering a viable tissue construct is an appropriate cell source. Attempts to engineer adipose tissue have involved the use of preadipocytes and adipocytes as the base cell source. Increased interest surrounding the research and development of stem cells as a source of cells for tissue engineering has, however, led to a new path of investigation for developing adipose tissue-engineering strategies. This manuscript serves as a review of the current state of adipose tissue-engineering methods and describes the shift toward tissue-engineering strategies using stem cells.