3D printed ß-tricalcium phosphate versus synthetic bone mineral scaffolds: A comparative in vitro study of biocompatibility.

BACKGROUND: ß-tricalcium phosphate (ß-TCP) has been successfully utilized as a 3D printed ceramic scaffold in the repair of non-healing bone defects; however, it requires the addition of growth factors to augment its regenerative capacity. Synthetic bone mineral (SBM) is a novel and extrudable carbonate hydroxyapatite with ionic substitutions known to facilitate bone healing. However, its efficacy as a 3D printed scaffold for hard tissue defect repair has not been explored. OBJECTIVE: To evaluate the biocompatibility and cell viability of human osteoprecursor (hOP) cells seeded on 3D printed SBM scaffolds via in vitro analysis. METHODS: SBM and ß-TCP scaffolds were fabricated via 3D printing and sintered at various temperatures. Scaffolds were then subject to qualitative cytotoxicity testing and cell proliferation experiments utilizing (hOP) cells. RESULTS: SBM scaffolds sintered at lower temperatures (600 °C and 700 °C) induced greater levels of acute cellular stress. At higher sintering temperatures (1100 °C), SBM scaffolds showed inferior cellular viability relative to ß-TCP scaffolds sintered to the same temperature (1100 °C). However, qualitative analysis suggested that ß-TCP presented no evidence of morphological change, while SBM 1100 °C showed few instances of acute cellular stress. CONCLUSION: Results demonstrate SBM may be a promising alternative to ß-TCP for potential applications in bone tissue engineering.

Direct inkjet writing of polylactic acid/ß-tricalcium phosphate composites for bone tissue regeneration: A proof-of-concept study.

There is an ever-evolving need of customized, anatomic-specific grafting materials for bone regeneration. More specifically, biocompatible and osteoconductive materials, that may be configured dynamically to fit and fill defects, through the application of an external stimulus. The objective of this study was to establish a basis for the development of direct inkjet writing (DIW)-based shape memory polymer-ceramic composites for bone tissue regeneration applications and to establish material behavior under thermomechanical loading. Polymer-ceramic (polylactic acid [PLA]/ß-tricalcium phosphate [ß-TCP]) colloidal gels were prepared of different w/w ratios (90/10, 80/20, 70/30, 60/40, and 50/50) through polymer dissolution in acetone (15% w/v). Cytocompatibility was analyzed through Presto Blue assays. Rheological properties of the colloidal gels were measured to determine shear-thinning capabilities. Gels were then extruded through a custom-built DIW printer. Space filling constructs of the gels were printed and subjected to thermomechanical characterization to measure shape fixity (Rf) and shape recovery (Rr) ratios through five successive shape memory cycles. The polymer-ceramic composite gels exhibited shear-thinning capabilities for extrusion through a nozzle for DIW. A significant increase in cellular viability was observed with the addition of ß-TCP particles within the polymer matrix relative to pure PLA. Shape memory effect in the printed constructs was repeatable up to 4 cycles followed by permanent deformation. While further research on scaffold macro-/micro-geometries, and engineered porosities are warranted, this proof-of-concept study suggested suitability of this polymer-ceramic material and the DIW 3D printing workflow for the production of customized, patient specific constructs for bone tissue engineering.

Direct inkjet writing type 1 bovine collagen/ß-tricalcium phosphate scaffolds for bone regeneration.

Bone tissue has the capacity to regenerate under healthy conditions, but complex cases like critically sized defects hinder natural bone regeneration, necessitating surgery, and use of a grafting material for rehabilitation. The field of bone tissue engineering (BTE) has pioneered ways to address such issues utilizing different biomaterials to create a platform for cell migration and tissue formation, leading to improved bone reconstruction. One such approach involves 3D-printed patient-specific scaffolds designed to aid in regeneration of boney defects. This study aimed to develop and characterize 3D printed scaffolds composed of type I collagen augmented with ß-tricalcium phosphate (COL/ß-TCP). A custom-built direct inkjet write (DIW) printer was used to fabricate ß-TCP, COL, and COL/ß-TCP scaffolds using synthesized colloidal gels. After chemical crosslinking, the scaffolds were lyophilized and subjected to several characterization techniques, including light microscopy, scanning electron microscopy, and x-ray diffraction to evaluate morphological and chemical properties. In vitro evaluation was performed using human osteoprogenitor cells to assess cytotoxicity and proliferative capacity of the different scaffold types. Characterization results confirmed the presence of ß-TCP in the 3D printed COL/ß-TCP scaffolds, which exhibited crystals that were attributed to ß-TCP due to the presence of calcium and phosphorus, detected through energy dispersive x-ray spectroscopy. In vitro studies showed that the COL/ß-TCP scaffolds yielded more favorable results in terms of cell viability and proliferation compared to ß-TCP and COL scaffolds. The novel COL/ß-TCP scaffold constructs hold promise for improving BTE applications and may offer a superior environment for bone regeneration compared with conventional COL and ß-TCP scaffolds.

3D Printing Type 1 Bovine Collagen Scaffolds for Tissue Engineering Applications-Physicochemical Characterization and In Vitro Evaluation.

Collagen, an abundant extracellular matrix protein, has shown hemostatic, chemotactic, and cell adhesive characteristics, making it an attractive choice for the fabrication of tissue engineering scaffolds. The aim of this study was to synthesize a fibrillar colloidal gel from Type 1 bovine collagen, as well as three dimensionally (3D) print scaffolds with engineered pore architectures. 3D-printed scaffolds were also subjected to post-processing through chemical crosslinking (in N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide) and lyophilization. The scaffolds were physicochemically characterized through Fourier Transform Infrared Spectroscopy (FTIR), Thermogravimetric Analysis, Differential Scanning Calorimetry, and mechanical (tensile) testing. In vitro experiments using Presto Blue and Alkaline Phosphatase assays were conducted to assess cellular viability and the scaffolds' ability to promote cellular proliferation and differentiation. Rheological analysis indicated shear thinning capabilities in the collagen gels. Crosslinked and lyophilized 3D-printed scaffolds were thermally stable at 37 °C and did not show signs of denaturation, although crosslinking resulted in poor mechanical strength. PB and ALP assays showed no signs of cytotoxicity as a result of crosslinking. Fibrillar collagen was successfully formulated into a colloidal gel for extrusion through a direct inkjet writing printer. 3D-printed scaffolds promoted cellular attachment and proliferation, making them a promising material for customized, patient-specific tissue regenerative applications.

Custom 3D Printable Silicones with Tunable Stiffness.

Silicone elastomers have broad versatility within a variety of potential advanced materials applications, such as soft robotics, biomedical devices, and metamaterials. A series of custom 3D printable silicone inks with tunable stiffness is developed, formulated, and characterized. The silicone inks exhibit excellent rheological behavior for 3D printing, as observed from the printing of porous structures with controlled architectures. Herein, the capability to tune the stiffness of printable silicone materials via careful control over the chemistry, network formation, and crosslink density of the ink formulations in order to overcome the challenging interplay between ink development, post-processing, material properties, and performance is demonstrated.

Development of a 3D printable maxillofacial silicone: Part II. Optimization of moderator and thixotropic agent.

STATEMENT OF PROBLEM: Conventionally, maxillofacial prostheses are fabricated by hand carving the missing anatomic defect in wax and creating a mold into which pigmented silicone elastomer is placed. Digital technologies such as computer numerical control milling and 3-dimensional (3D) printing have been used to prepare molds, directly or indirectly, into which a biocompatible pigmented silicone elastomer can be placed. PURPOSE: The purpose of this in vitro study was to develop a silicone elastomer that could be 3D printed directly without a mold to create facial or body prostheses by varying its composition. MATERIAL AND METHODS: The room temperature vulcanizing silicone composition was divided into 2 components which were mixed 1:1 to initiate polymerization in the printer before printing began. Different types of moderators and thixotropic agents were used, and the base composition was varied to obtain 11 formulations. The specimens were printed and polymerized from these formulations and tested for tear and tensile strength and hardness. Ten readings of the specimens were recorded for tear and tensile strength and 6 for hardness. Results were analyzed using ANOVA (α=.05). Visual assessment of uncured printed specimens was undertaken for 5 formulations to assess any differences in their ability to hold their shape after printing. RESULTS: The tear and tensile strength of the 11 formulations with varying moderators, thixotropic agents, and base compositions were statistically similar to each other (P>.05). Five of 11 formulations were chosen for the visual assessment as they had sufficient thixotropic agent to avoid slumping while printing. The specimens showed varied slumping behavior until they polymerized. The filler content was increased in the selected formulation, and the tear and tensile strength of the formulation was increased to 6.138 kNm-1 and 3.836 MPa; these increases were comparable to those of commercial silicones currently used for the fabrication of facial prostheses. CONCLUSIONS: The optimum combination of mechanical properties implies the use of one of the formulations as a suitable material for the 3D printing of facial prostheses.

3D-Printed Transparent Glass.

Silica inks are developed, which may be 3D printed and thermally processed to produce optically transparent glass structures with sub-millimeter features in forms ranging from scaffolds to monoliths. The inks are composed of silica powder suspended in a liquid and are printed using direct ink writing. The printed structures are then dried and sintered at temperatures well below the silica melting point to form amorphous, solid, transparent glass structures. This technique enables the mold-free formation of transparent glass structures previously inaccessible using conventional glass fabrication processes.

Bony engineering using time-release porous scaffolds to provide sustained growth factor delivery.

Microporous scaffolds designed to improve bony repair have had limited success; therefore, we sought to evaluate whether time-released porous scaffolds with or without recombinant bone morphogenetic protein 2 (rhBMP-2) could enhance stem cell osteoinduction. Custom-made 15/85 hydroxyapatite/ß-tricalcium phosphate scaffolds were left empty (E) or filled with rhBMP-2 (E+), calcium sulfate (CS), or CS and rhBMP-2 (CS+). All scaffolds were placed in media and weighed daily. Conditioned supernatant was analyzed for rhBMP-2 and then used to feed human adipose-derived mesenchymal stem cells (ASCs). Adipose-derived mesenchymal stem cell ALP activity, OSTERIX expression, and bone nodule formation were determined. E scaffolds retained 97% (SD, 2%) of the initial weight, whereas CS scaffolds had a near-linear 30% (SD, 3%) decrease over 60 days. E+ scaffolds released 155 (SD, 5) ng of rhBMP-2 (77%) by day 2. In contrast, CS+ scaffolds released only 30 (SD, 2) ng (10%) by day 2, and the remaining rhBMP-2 was released over 20 days. Conditioned media from E+ scaffolds stimulated the highest ALP activity and OSTERIX expression in ACSs on day 2. However, after day 6, media from CS+ scaffolds stimulated the highest ALP activity and OSTERIX expression in ASCs. Adipose-derived mesenchymal stem cells exposed to day 8 CS+-conditioned media produced significantly more bone nodules (10.1 [SD, 1.7] nodules per high-power field) than all other scaffolds. Interestingly, day 8 conditioned media from CS scaffolds simulated significantly more bone nodules than either E or E+ scaffold (P < 0.05 for both). Time-released hydroxyapatite/ß-tricalcium phosphate porosity provides sustained growth factor release, enhances ASC osteoinduction, and may result in better in vivo bone formation.

Three-dimensional printing of bone repair and replacement materials: impact on craniofacial surgery.

Solid freeform fabrication techniques such as direct write technology can be used to fabricate tissue-engineering scaffolds in 3 dimensions with high levels of reproducibility and precision. These can comprise complex structures made of osteoconductive, remodelable lattices to conduct bone ingrowth and solid barriers to prevent soft tissue invasion. As such, they act as a combination of bone graft and barrier membrane. Results from animal studies have shown that these structures fill rapidly with healing bone and can conduct bone across critical-size defects to fill large defects in rabbit skull. Results indicate that this technology can be used to produce both off-the-shelf and custom-fabricated bone graft substitutes. These may initially be used to restore alveolar ridge defects, but could also be used, in the future, to repair or replace complex craniofacial bone defects such as cleft palate defects. In the more distant future, these technologies could be combined with controlled-release bioactive substances such as growth factors and pharmaceuticals to regenerate complex structures comprising multiple tissue types.

In vivo bone response to 3D periodic hydroxyapatite scaffolds assembled by direct ink writing.

The in vivo bone response of 3D periodic hydroxyapatite (HA) scaffolds is investigated. Two groups of HA scaffolds (11 mm diameter x 3.5 mm thick) are fabricated by direct-write assembly of a concentrated HA ink. The scaffolds consist of cylindrical rods periodically arranged into four quadrants with varying separation distances between rods. In the first group, HA rods (250 microm in diameter) are patterned to create pore channels, whose areal dimensions are 250 x 250 microm(2) in quadrant 1, 250 x 500 microm(2) in quadrants 2 and 4, and 500 x 500 microm(2) in quadrant 3. In the second group, HA rods (400 microm in diameter) are patterned to create pore channels, whose areal dimensions of 500 x 500 microm(2) in quadrant 1, 500 x 750 microm(2) in quadrants 2 and 4, and 750 x 750 microm(2) in quadrant 3. Each group of scaffolds is partially densified by sintering at 1200 degrees C prior to being implanted bilaterally in trephine defects of skeletally mature New Zealand White rabbits. Their tissue response is evaluated at 8 and 16 weeks using micro-computed tomography, histology, and scanning electron microscopy. New trabecular bone is conducted rapidly and efficiently across substantial distances within these patterned 3D HA scaffolds. Our observations suggest that HA rods are first coated with a layer of new bone followed by subsequent scaffold infilling via outward and inward radial growth of the coated regions. Direct-write assembly of 3D periodic scaffolds composed of micro-porous HA rods arrayed to produce macro-pores that are size-matched to trabecular bone may represent an optimal strategy for bone repair and replacement structures.