@article {19833, title = {Novel Enzymatically Cross-linked Silk Fibroin Bioink for Bioprinting of Patient-Specific Memory-Shape Implants for Meniscus Regeneration}, journal = {Society for Biomaterials 2019}, year = {2019}, month = {2019-04-03 00:00:00}, address = {Seattle}, abstract = {

Statement of Purpose: The regeneration of the meniscus tissue using a biomimetic implant is still a big challenge in the orthopedic field. Arthroscopic knee surgery, in part due to meniscus lesions, is the most common orthopedic procedure with an estimative of more than two million surgeries performed each year at a global scale that can cost more than $3billion per year in the US alone [1]. The use of 3D printing combined with the development of novel biomaterials-based bioinks can be the perfect solution to achieve a rapid and successful progress in this field. In this work, a fast setting enzymatic-crosslinked silk fibroin (SF) bioink was developed allowing the production of patient-specific implants with memory-shape properties. This novel bioink can be a step forward in the meniscus regeneration field providing a reproducible and reliable way to produce patient-specific implants with good mechanical properties and suitable biological performance. Bioprinted silk scaffolds were submitted to a battery of in vitro tests, including physico-chemical and biological assays.
Methods: The methodology used to develop this novel SF bioink is based on the hydrogel formation using tyrosine groups present in silk through enzymatic cross-link, by using horseradish peroxidase (HRP) as enzyme and hydrogen peroxide (H2O2) as subtract. The ratio of HRP and H2O2 used underwent an optimization process in order to reach a good printability. Rheological properties of different formulations were tested using Kinexus pro+ rheometer. Silk cube shape scaffolds with 4 layers were printed and freeze-dried, where their chemical structure were posteriorly characterized using Fourier transform infrared (FTIR) spectroscopy, as well as their architecture using scanning electron microscope (SEM) and micro-computed tomography (CT). The mechanical properties were investigated under a uniaxial compression test (Universal Testing Machine) and dynamic compression test (dynamic mechanical analysis (DMA)). Biological performance was evaluated by seeding human adipose-derived stem cells (hASCs) and analyzing cell viability (Live/dead assay), metabolic activity (Alamar Blue) and proliferation (DNA quantification) up to 7 days of culturing.
Results: The novel SF bioink showed good printability (Figure 1A) and allowed the fabrication of structures with good resolution and porosity (Figure 1B and C).The porosity and architecture, as previously mentioned, were assessed by SEM and CT revealing a total porosity of 59.1 {\textpm} 3.4\%, of which 26.1 {\textpm} 3.2\% relates with micro
porosity and 33.1 {\textpm} 6.3\% reports to macro porosity. The mechanical tests results showed a suitable mechanical performance compared with the native tissue. The SF scaffolds revealed also memory-shape properties proved by a similar mechanical behavior over 5 cycles after a cyclic compressive test. hASCs seeded onto SF scaffolds remained viable showing an increase of metabolic activity and a sustained proliferation after 7 days of culturing (Figure 1D). Finally, as proof of concept, a SF patient-specific human meniscus implant was printed using a model previously developed in our group [2].
Figure 1 {\textendash} A) SF structure after printing (conformation before freeze-drying), B) SF structure after freeze-drying, C) SEM image of freeze-dried SF structure, D) SEM image of the bioprinted scaffolds seeded with hASCs after culture for 7 days, E) patient specific memory-shape SF meniscus implant.
Conclusions: In this work, it was demonstrated the development of a fast-setting SF bioink with unique and suitable properties to produce patient-specific implants for meniscus regeneration. In addition, the in vitro biological assays proved the biocompatibility of the SF constructs making them a promising approach in the tissue engineering field. Furthermore, the versatility of this novel bioink can open new horizons giving a great alternative for all types of approaches that require a patient-specific memory-shape implantable construct.

}, keywords = {3D printing, Silk bioink}, author = {Costa, J. B. and Silva-Correia, J. and Reis, R. L. and Oliveira, J. M.} }