Core-shell silk hydrogels with spatially tuned conformations as drug delivery system

Hydrogels of spatially controlled physicochemical properties are appealing platforms for tissue engineering and drug delivery. In this study, core-shell silk fibroin (SF) hydrogels of spatially controlled conformation were developed. The core-shell structure in the hydrogels was formed by means of soaking the preformed (enzymatically crosslinked) random coil SF hydrogels in methanol. When increasing the methanol treatment time from 1 to 10 minutes, the thickness of the shell layer can be tuned from about 200 to around 850 µm as measured in wet status. After lyophilization of the rehydrated core-shell hydrogels, the shell layer displayed compact morphology and the core layer presented porous structure, when observed by scanning electron microscopy. The conformation of the hydrogels was evaluated by Fourier transform infrared spectroscopy in wet status. The results revealed that the shell layer possessed dominant β-sheet conformation and the core layer maintained mainly random coil conformation. Enzymatic degradation data showed that the shell layers presented superior stability to the core layer. The mechanical analysis displayed that the compressive modulus of the core-shell hydrogels ranged from around 25 kPa to about 1.1 MPa by increasing the immersion time in methanol. When incorporated with albumin, the core-shell SF hydrogels demonstrated slower and more controllable release profiles compared with the non-treated hydrogel. These core-shell SF hydrogels of highly tuned properties are useful systems as drug delivery system and may be applied as cartilage substitute.This study was funded by the Portuguese Foundation for Science and Technology (FCT) projects Tissue2Tissue (PTDC/CTM/105703/2008) and OsteoCart (PTDC/CTM-BPC/115977/2009), as well as the European Union’s FP7 Programme under grant agreement no. REGPOT-CT2012-316331-POLARIS. Le-Ping Yan was awarded a FCT PhD scholarship (SFRH/BD/ 64717/2009). The FCT distinctions attributed to J.M. Oliveira and A.L. Oliveira under the Investigador FCT program (IF/ 00423/2012) and (IF/00411/2013) are also greatly acknowledged, respectively

Current concepts and challenges in osteochondral tissue engineering and regenerative medicine

"Publication Date (Web): February 20, 2015"In the last few years, great progress has been made to validate tissue engineering strategies in preclinical studies and clinical trials on the regeneration of osteochondral defects. In the preclinical studies, one of the dominant strategies comprises the development of biomimetic/bioactive scaffolds, which are used alone or incorporated with growth factors and/or stem cells. Many new trends are emerging for modulation of stem cell fate towards osteogenic and chondrogenic differentiations, but bone/cartilage interface regeneration and physical stimulus have been showing great promise. Besides the matrix-associated autologous chondrocyte implantation (MACI) procedure, the matrix-associated stem cells implantation (MASI) and layered scaffolds in acellular or cellular strategy are also applied in clinic. This review outlines the progresses at preclinical and clinical levels, and identifies the new challenges in osteochondral tissue engineering. Future perspectives are provided, e.g., the applications of extracellular matrix-like biomaterials, computer-aided design/manufacture of osteochondral implant and reprogrammed cells for osteochondral regeneration.The authors thank the Portuguese Foundation for Science and Technology (FCT) through the projects TISSUE2TISSUE (PTDC/CTM/105703/2008) and OsteoCart (PTDC/CTM-BPC/115977/2009). We also acknowledge European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement REGPOT-CT2012-316331-POLARIS. L-P.Y. acknowledges the PhD scholarship from FCT (SFRH/BD/64717/2009). The FCT distinction attributed to J.M.O. and A.L.O. under the Investigator FCT program (IF/00423/2012) and (IF/00411/2013) are also greatly acknowledged

Development of a bilayered scaffold based on silk fibroin and silk fibroin/nano-calcium phosphate for osteochondral regeneration

Objectives: Osteochondral defect is a common condtion in clinic. Satisfactory outcomes are rarely achieved by traditional methods. Tissue engineering might be a promising strategy for this hinder. The aim of this study is to mimick the stratified structure of osteochondral tissue, by developing a bilayered scaffold for osteochondral regeneration. The developed bilayered scaffold is composed of a porous silk fibroin scaffold as the cartilage-like layer and a porous silk fibroin/nano-calcium phosphate (CaP) scaffold as the bone-like layer

Silk Fibroin/Nano-CaP Bilayered scaffolds for osteochondral tissue engineering

In this study, bilayered silk and silk/nano-CaP scaffolds were developed for osteochondral (OC) tissue engineering. Aqueous silk solution (16 wt.%) was used for preparation of the cartilage-like layer and, for generation of the silk/nano-CaP suspension and the bottom layer (CaP/Silk: 16 wt.%). The scaffolds were formed by using salt-leaching/lyophilization approach. The scanning electron microscopy revealed that the both layers presented porous structure and integrated well. Micro-computed tomography images confirmed that the CaP phase was only retained in the silk/nano-CaP layer. The hydration degree and mechanical properties of the bilayered scaffold were comparable to the ones of each single layer. The apatite crystal formation was limited to the silk/nano-CaP layer, when soaking the scaffold in a simulated body fluid solution, which is a must for the application of the developed scaffolds in OC tissue engineerin

De novo bone formation on macro/microporous silk and silk/nano-sized calcium phosphate scaffolds

Macro/micro porous silk/nano-sized calcium phosphate scaffolds (SC16) with bioactive and superior physicochemical properties have been recently developed. In this study, we aim at evaluating the new bone formation ability of the SC 16 scaffolds in vivo, using silk fibroin scaffolds (S16) as control. The CaP distribution profile in the scaffolds was characterized by Micro-Computed Tomography. The in vitro mineralization behavior was examined by immersion in Simulated Body Fluid solution from 1 to 14 days. The long-term hydration degree and weight loss ratio of the scaffolds were evaluated by immersion in an Isotonic Saline Solution from 1 month to 1 year. In vivo osteogenesis properties of the scaffolds were screened by implantation into the rat femur defects for 3 weeks. The results showed that the CaP phase distributed homogeneously in the SC16 scaffolds. Mineralization was only observed in SC16 scaffolds, and both scaffolds gradually degraded with time. The staining of the explants showed that new bone formation with higher density was observed in the SC16 scaffolds as compared to S16 scaffolds, guiding the growth of new bone directly onto its surface. These results demonstrated that the SC16 hybrid scaffolds are osteoconductive and can be good candidates for bone tissue engineering as promoted superior de novo bone formation.This study was supported by the Portuguese Foundation for Science and Technology (FCT) projects OsteoCart (PTDC/CTM-BPC/115977/2009) and Tissue2Tissue (PTDC/CTM/105703/2008). Research leading to these results has received funding from the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement no REGPOT-CT2012-316331-POLARIS. Le-Ping Yan is an FCT PhD scholarship holder (SFRH/BD/64717/2009)

Macro/microporous silk fibroin scaffolds with potential for articular cartilage and meniscus tissue engineering applications

This study describes the developmental physicochemical properties of silk fibroin scaffolds derived from high concentration aqueous silk fibroin solutions. The silk fibroin scaffolds were prepared with different initial concentrations (8%, 10%, 12% and 16% (wt%)) and obtained by combining the salt-leaching and freeze-drying methodologies. The results indicated that the antiparallel β-pleated sheet (silk-II) conformation was present in the silk fibroin scaffolds. All the scaffolds possessed macro/micro porous structure. Homogeneous porosity distribution was achieved in all the groups of samples. As the silk fibroin concentration increased from 8% to 16%, the mean porosity decreased from 90.8±0.9% to 79.8±0.3%, and the mean interconnectivity decreased from 97.4±0.5% to 92.3±1.3%. The mechanical properties of the scaffolds exhibited a concentration dependence. The dry state compressive modulus increased from 0.81±0.29 MPa to 15.14±1.70 MPa, and the wet state dynamic storage modulus increased around 20-30 folds at each testing frequencies when the silk fibroin concentration increased from 8% to 16%. The water-uptake ratio decreased by means of increasing silk fibroin concentration. The scaffolds present favorable stability as their structure integrity, morphology and mechanical properties were maintained after in vitro degradation for 30 days. Based on these results, the scaffolds developed in this study are herein proposed to be used in meniscus and cartilage tissue engineering scaffolding.Tissue2Tissue project (PTDC/CTM/105703/2008

Combinatory approach for developing silk fibroin-based scaffolds with hierarchical porosity and enhanced performance for cartilage tissue engineering applications

Introduction: The combination of several processing technologies can open the possibility for producing scaffolds with superior performance for tissue engineering (TE) applications. Hydrogels are structurally similar to the natural extracellular matrix microenvironment presenting high elasticity and resistance to compression forces. They have been extensively used in biomedical devices fabrication and for TE applications, including for cartilage defects repair[1]. Recently, it was found that proteins like silk fibroin (SF), presenting tyrosine groups can be used to prepare fast formed hydrogels with controlled gelation properties, via an enzyme-mediated cross-linking reaction using horseradish peroxidase (HRP) and hydrogen peroxide (H2O2)[2],[3]. Moreover, the high versatility, processability and tailored mechanical properties of SF, make this natural polymer attractive for the development of innovative scaffolding strategies for cartilage TE applications[4],[5]. Materials and Methods: The present work proposes a novel route for developing SF-based scaffolds derived from high- concentrated SF (16wt%) enzymatically cross-linked by a HRP/H2O2 complex. The combination of salt-leaching and freeze-drying methodologies was used to prepare macro/microporous SF scaffolds with an interconnected structure and specific features regarding biodegradation and mechanical properties (Fig. 1a). The scaffolds morphology and porosity were analyzed by SEM and micro-CT. The mechanical properties (Instron) and protein conformation (FTIR, XRD) were also assessed. In order to evaluate the scaffolds structural integrity, swelling ratio and degradation profile studies were performed for a period of 30 day. This work also aims to evaluate the in vitro chondrogenic differentiation response by culturing human adipose derived stem cells (hASCs) over 21 days in basal and chondrogenic conditions. Cell behaviour in the presence of the macro/microporous structures will be evaluated through different quantitative (Live/Dead, DNA, GAGs, RT PCR) and qualitative (SEM, histology, immunocytochemistry) assays. Results and Discussion: The macro/microporous SF scaffolds showed high porosity and interconnectivity with the trabecular structures evenly distributed (Fig. 1b,c). A dramatic decrease of compressive modulus was observed for samples in hydrated state. Chemical analysis revealed that SF scaffolds displayed the characteristic peaks for β-sheet conformation. Swelling ratio data demonstrated a large swelling capacity, maintaining their structural integrity for 30 days. As expected, when immersed in protease XIV the degradation rate of SF scaffolds increased. Based on the promising morphology and physicochemical properties of the developed SF scaffolds, in vitro chondrogenic differentiation studies with hASCs are envisioned in order to validate their performance for cartilage regeneration applications. Conclusion: This study proposes an innovative approach to produce fast-formed porous SF scaffolds using enzymatically cross- linked SF hydrogels structured by the combination of salt-leaching and freeze-drying methodologies. The obtained results can provide a valuable reference of SF as a tunable and versatile biomaterial with great potential for applications in cartilage TE scaffolding. Portuguese Foundation for Science and Technology (FCT) project PEst-C/SAU/LA0026/201; ERDP funding through POCTEP Project 0687_NOVOMAR_1_P; Investigator FCT program IF/00423/2012 and IF/00411/2013 References: [1] Xia, L.-W., R. Xie, X.-J. Ju, W. Wang, Q. Chen, and L.-Y. Chu, Nano-structured smart hydrogels with rapid response and high elasticity. Nature communications, 2013. 4. [2] Sofia, S.J., A. Singh, and D.L. Kaplan, Peroxidase-catalyzed crosslinking of functionalized polyaspartic acid polymers. Journal of Macromolecular Science, Part A, 2002. 39(10): p. 1151-1181. [3] Reis, R.L., L.-P. Yan, A.L. Oliveira, J.M. Oliveira, D.R. Pereira, C. Correia, and R.A. Sousa, Hydrogels derived from silk fibroin: Methods and uses thereof. 2014. 107426. [4] Chen, C.-H., J.M.-J. Liu, C.-K. Chua, S.-M. Chou, V.B.-H. Shyu, and J.-P. Chen, Cartilage tissue engineering with silk fibroin scaffolds fabricated by indirect additive manufacturing technology. Materials, 2014. 7(3): p. 2104-2119. [5] Yan, L.-P., J.M. Oliveira, A.L. Oliveira, S.G. Caridade, J.F. Mano, and R.L. Reis, Macro/microporous silk fibroin scaffolds with potential for articular cartilage and meniscus tissue engineering applications. Acta biomaterialia, 2012. 8(1): p. 289-301.Â

Silk bilayer scaffolds can induce fast integration with subchondral bone and support cartilage repair

Publicado em : J Tissue Eng Regen Med 2014; 8 (Suppl. 1)Introduction: Osteochondral defect (OCD) regeneration presents major challenges in orthopedics. Since healing of cartilage and bone should be simultaneously considered, ideal scaffolds should be those that can mimic both tissues properties. In this study, bilayered silk and silk-nano calcium phosphate (Silk/Silk-NanoCaP) scaffolds with tailored mechanical properties were developed for OCD tissue engineering application. Materials and methods: Aqueous silk solution (16%) was prepared.1 Nano calcium phosphate particles (16%) were synthesized in the silk solution (Silk-NanoCaP).2 The bony layer was prepared by addition of NaCl particles (500–1000 lm) into the Silk-NanoCaP suspension. After drying for 2 days and salt-leaching overnight, silk solution was added on top of the bony layer using the same procedure to produce the chondral layer. The !nal scaffolds were evaluated through in vitro culture of rabbit bone marrow stromal cells (RBMSCs) for 2 weeks, and in vivo implantation in a rabbit knee OCD for 4 weeks. Results: The RBMSCs cultured in the scaffolds presented increasing viability from day 1 to day 7 by MTS assay. Good adhesion and migration of the RBMSCs in the scaffolds were achieved, as observed under the scanning electron microscope. Cell proliferation was observed from day 7 to day 14 as determined by DNA quanti!cation. The bony layer induced higher alkaline phosphatase level as compared to the chondral layer, in osteogenic condition. Histological analysis (H&E) showed that the bilayered scaffolds integrated well with the host tissue, after 4 weeks of implantation in a critical size OC defect (Fig. 1). Abundant new bone formation was detected in the Silk-NanoCaP layer. Cartilage regeneration occurred in the silk layer. Discussion and conclusions: The bilayered scaffolds favored the attachment, proliferation, and differentiation of RBMSCs. The bony layer of the bilayered scaffolds possessed osteoconductive properties. The bilayered scaffolds were biocompatible in vitro and in vivo. These scaffolds also induced both subchondral bone regeneration and supported cartilage regeneration, thus showing great promise in OCD regeneration. Acknowledgments: The authors thank FCT projects Tissue2Tissue and OsteoCart, and the FP7 Programme POLARIS. Yan LP was awarded a FCT PhD scholarship. Investigador FCT program (IF/00423/2012) and (IF/00411/2013) are also greatly acknowledged. Disclosure: The authors declare that there is no con"ict of interest

Advanced mimetic materials for meniscus tissue engineering : targeting segmental vascularization

Meniscus lesions are among the most common orthopaedic injuries which can ultimately lead to degeneration of the knee articular cartilage. The human meniscus has a limited healing potential, partly due to a poor vasculature, and thus meniscus regeneration using tissue engineering strategies has recently been investigated as a promising alternative to total/partial meniscectomy [1]. Advanced scaffolds for tissue engineering of meniscus should be able to mimic and preserve the asymmetric vascular network of this complex tissue, i.e. enable controlling the segmental vascularization during the regeneration process. Novel scaffolds were produced combining a silk polymeric matrix (12 wt%) [2] and the methacrylated gellan gum hydrogel (iGG-MA), which has been shown to be able to prevent the ingrowth of endothelial cells and blood vessels into the hydrogels [3,4]. The angiogenic/ anti-angiogenic potential of acellular and cell-laden silk-12 scaffolds combined with iGG-MA hydrogel was investigated in vivo, using the chick embryo chorioallantoic membrane (CAM) assay. For producing the cell-laden scaffolds, human meniscus cells (HMC¢s) were isolated from morphologically intact human menisci using an enzymatic-based digestion and expanded using standard culture conditions. The HMC’sladen hydrogel/silk scaffolds were produced by encapsulating the HMC’s into a 2 wt% GG-MA hydrogel, followed by impregnation onto the 12 wt% silk scaffold and ionic-crosslinking in a saline solution. A CAM assay was used to investigate the control of segmental vascularization of the acellular and HMC¢s-laden hydrogel/silk scaffolds by the effect of GG-MA hydrogel, until day 14 of embryonic development. The in vivo study allowed investigating the number of macroscopic blood vessels converging to the implants. The evaluation of possible inflammation and endothelial cells ingrowths was performed by histological (haematoxylin and eosin - H&E - staining) and immunohistochemical methods (SNA-lectin staining). When the silk-12 scaffold was combined with the hydrogel, an inhibitory effect was observed as demonstrated by the low number of convergent blood vessels. Results have shown that iGG-MA hydrogel prevented the endothelial cells adhesion and blood vessels infiltration into the HMC’s hydrogel/silk scaffolds, after 4 days of implantation. This study showed that the hydrogel/silk scaffolds enabled controlling the segmental vascularization, thus it can possibly mimic the native vasculature architecture during meniscus regeneration