3D-printed alginate/gelatin/PCL/hyaluronic acid scaffolds with metal–organic frameworks for quercetin delivery: Improving mechanical properties and cytocompatibility for cartilage tissue engineering applications

Growth Factor Delivery to a Cartilage-Cartilage Interface Using Platelet-Rich Concentrates on a Hyaluronic Acid Scaffold

Recent reports suggest the utility of extracellular matrix (ECM) molecules as raw components in scaffolding of engineered materials. However, rapid and tunable manufacturing of ECM molecules into fibrous structures remains poorly developed. Here we report on an immersion rotary jet-spinning (iRJS) method to show high-throughput manufacturing (up to ∼1 g/min) of hyaluronic acid (HA) and other ECM fiber scaffolds using different spinning conditions and postprocessing modifications. This system allowed control over a variety of scaffold material properties, which enabled the fabrication of highly porous (70-95%) and water-absorbent (swelling ratio ∼2000-6000%) HA scaffolds with soft-tissue mimetic mechanical properties (∼0.5-1.5 kPa). Tuning these scaffolds' properties enabled the identification of porosity (∼95%) as a key facilitator for rapid and in-depth cellular ingress in vitro. We then demonstrated that porous HA scaffolds accelerated granulation tissue formation, neovascularization, and reepithelialization in vivo, altogether potentiating faster wound closure and tissue repair. Collectively, this scalable and versatile manufacturing approach enabled the fabrication of tunable ECM-mimetic nanofiber scaffolds that may provide an ideal first building block for the design of all-in-one healing materials.

Controlled release of transforming growth factor-β3 from cartilage-extra-cellular-matrix-derived scaffolds to promote chondrogenesis of human-joint-tissue-derived stem cells

Treatment of focal lesions of the articular cartilage of the knee using chondrocytes in a hyaluronic acid (HA) scaffold is already being investigated in clinical trials. An alternative may be to use mesenchymal stem cells (MSC). We have compared articular chondrocytes with MSC from human bone marrow (BM) and adipose tissue (AT), all cultured in HA scaffolds, for their ability to express genes and synthesize proteins associated with chondrogenesis. The cells were expanded in monolayer cultures. After seeding into the scaffold, the chondrocytes were maintained in medium, while the two MSC populations were given a chondrogenic differentiation medium. Chondrogenesis was assessed by real-time RT-PCR for chondrocyte-associated genes, by immunohistochemistry and by ELISA for collagens in the supernatant. Redifferentiation of the dedifferentiated chondrocytes in the HA scaffold was shown by a modest increase in type II collagen mRNA (COL2A1) and reduction in COL1A1. BM-MSC expressed 600-fold higher levels of COL2A1 than chondrocytes after 3weeks in the scaffold. The levels of aggrecan (AGC1) and COL1A1 were similar for chondrocyte and BM-MSC scaffold cultures, while COL10A1 was higher in the BM-MSC. AT-MSC expressed levels of COL2A1 and COL1A1 similar to chondrocytes, but less AGC1 and COL10A1. Surprisingly, little collagen II protein was observed in the scaffold. Instead, collagen II was found in the culture medium. Chondrogenesis in HA scaffolds was more efficient using BM-MSC than AT-MSC or chondrocytes. Some of the secreted collagen II escaped entrapment in the extracellular space and was detected in the culture medium.

Mimicked cartilage scaffolds of silk fibroin/hyaluronic acid with stem cells for osteoarthritis surgery: Morphological, mechanical, and physical clues

Human adipose-derived mesenchymal stem cells (AD-MSCs) attracted much interest as a promising alternative to autologous chondrocytes and bone marrow-derived mesenchymal stem cells for cartilage regeneration. Developing a suitable culture technique to direct AD-MSCs into the chondrogenic lineage could be a crucial prerequisite for the cartilage defect repair application of AD-MSCs. Herein, we prepared the PEGDG-crosslinked porous three-dimensional (3D) hyaluronic acid (HA) scaffold and evaluated for its feasibility to induce proliferation and chondrogenic differentiation of the AD-MSCs. In addition, the effect of bone-morphogenetic protein-2 (BMP-2) and platelet-derived growth factor (PDGF) on chondrogenic differentiation was further investigated. Proliferation and chondrogenic differentiation were evaluated by cell morphology, DNA contents, s-GAG contents, and level of mRNA expression of relevant marker genes. When cultured with reference chondrogenic medium (RCM; serum-free DMEM-HG supplemented with 10 ng/mL of transforming growth factor-β1 (TGF-β1), 50 nM ascorbate, 100 nM dexamethasone, and 5 μg/mL of ITS), better proliferation and chondrogenic differentiation of AD-MSCs were obtained in the 3D HA scaffold culture as compared to the micromass culture, a standard 3D culture system. Moreover, the level of chondrogenic differentiation of AD-MSCs in the HA scaffold-RCM culture system was further increased by BMP-2, and decreased by PDGF. These results suggested that the HA scaffold with RCM was a promising chondrogenic culture system of AD-MSCs, and that BMP-2 could potentially serve as a chondrogenic supplement for AD-MSCs. However, PDGF was determined to be an inappropriate supplement based on its inhibition of the chondrogenic differentiation of AD-MSCs.

Syndactyly release may require skin grafting to fill the skin defects, which might lead to complications or poor cosmetic outcomes. A simple graftless technique for syndactyly release with a hyaluronic acid (HA) scaffold used to cover the bare areas is described. Between 2008 and 2011, release of 26 webs in 23 patients was performed. All skin defects were covered with Hyalomatrix(®) PA. One patient was excluded due to early post-operative infection that required HA scaffold removal before its integration. Web creep, secondary deformities, scar quality, and patient and parental satisfaction were assessed. Mean follow-up of the group of 22 patients was 24 months. There were no secondary deformities and minimal degree of web creep. All patients had close to normal pigmentation and good pliability at the sites of scaffold application. The results confirm the use of a HA scaffold as a promising alternative to skin grafting in syndactyly release surgery.

ABSTRACTOptical techniques are increasingly employed for monitoring cell–matrix interactions in suitably prepared 3D scaffolds. The ability of designing and realizing synthetic extracellular matrix with well‐controlled optical properties is a crucial need in this field. For this purpose, a crosslinked hyaluronic acid (HA) scaffold is prepared. Fourier transform infrared and ultraviolet–visible spectroscopies enable to monitor the scaffold preparation process and to evidence scaffold high transparency and low fluorescence in the visible range. 3D optical characteristics of the HA scaffold are tested by two‐photon microscopy (TPM) imaging of embedded fluorescent microbeads and alive keratinocytes labeled with vital PKH67 dye at different depths from the scaffold surface. Some useful indications about the potentiality of TPM measurements for the determination of attenuation coefficient of turbid media are also reported. Moreover, the use of the presented HA scaffold for preparing tissue phantoms for fluorescence imaging or diffuse imaging is proposed. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017, 134, 45243.

Considering the absence of a definite cure for osteoarthritis and problems of existing approaches, designing scaffolds for cartilage regeneration by tissue engineering methods seems necessary. Optimization of scaffolds properties is one of the biggest challenges in this area. Due to contradicting reports regarding efficacy and safety of intra-articular injections of corticosteroids, further evaluation of these agents by their incorporation into a designed scaffold could be beneficial. On this basis, a hyaluronic acid (HA) scaffold was designed in two forms: shear-thinning injectable gel and lyophilized implantable disk. Polyethylene glycol diglycidyl ether (PEGDGE) was used as cross-linker. After optimization of scaffolds properties, in vivo efficacy of the most optimum formulation (HA 2%, PEGDGE 0.5%) in the presence and absence of prednisolone (0.1% W/V) was evaluated. In vivo studies in rats showed that after 10 weeks, HA scaffolds, in both forms, repaired damaged articular cartilage to some extent but incorporation of prednisolone into the scaffold showed no additional benefit. Overall it seems that implantable scaffolds of HA could be a potential therapeutic choice for cartilage regeneration and corticosteroids in long term may also reverse these beneficial effects. Hyaluronic acid (HA) is one of the major components of the extracellular matrix and cartilage tissue. There are numerous reports on the efficacy of different scaffolds of HA for cartilage regeneration. New engineering methods focus on retaining the scaffolds in the site for long time as well as loading therapeutic agents to accelerate the healing process. In situ injection of corticosteroids is one of the routine interventions for osteoarthritis. Here, the effect of loading prednisolone as an anti-inflammatory agent into the shear thinning gels and implantable disks of HA for treatment of an induced arthritis in rat knee was revisited. In future studies we will upgrade the scaffold by mixing HA with some other biopolymers to increase the strength and retention of the system and to improve drug loading capacity. It seems that loading specific growth factors would be an alternative to corticosteroids with higher efficacy and lower side effects.

A novel method to fabricate highly interconnected porous hyaluronic acid (HA) scaffolds with open surface pore structures was developed by using embossed ice particulates as a template. HA sponges were cross-linked by water-soluble carbodiimide (WSC) and the optimal cross-linking condition was analyzed by infrared spectroscopy. Cross-linking with 50 mM WSC in a 90% (v/v) ethanol/water solvent mixture assured the highest degree of cross-linking and most stable structure and, therefore, was used to cross-link the HA sponges. Observation with a scanning electron microscope showed that the HA scaffolds had funnel-like porous structures. There were large, open pores on the top surfaces and inner bulk pores under the top surface of the funnel-like HA sponges. The inner bulk pores were interconnected with the large, top surface pores and extended into the whole sponge. The pore morphology and density of the large, top surface pores were dependent on the dimension and density of the ice particulates. The size of the inner bulk pores was dependent on the freezing temperature. The funnel-like pore structures of the HA sponges facilitated cell penetration into the inner pores of the sponges and resulted in homogenous cell distribution in the sponges.

Introduction: Gelatin and hyaluronic acid are two biopolymers with different applications in tissue engineering. They may be employed to construct diverse scaffolds that allow cells to differentiate and proliferate on them. In order to obtain the best functional and mechanical conditions in scaffolds, they must be crosslinked to form covalent links between gelatin and hyaluronic acid. The crosslinker 1-ethy-3-(3-dymethylaminopropyl) carbodiimide hydrochloride (EDC) is a compound widely used due to its low cytotoxicity. Besides, the concentration of the crosslinker may modify the physical properties and morphological characteristics of scaffolds when it forms covalent links between biopolymers, helping to construct different kinds of scaffolds used for developing soft tissues. However, the development of scaffolds made of gelatin and hyaluronic acid crosslinked with EDC has been poorly studied. In addition, the concentrations used for crosslinking gelatin and hyaluronic acid are contradictory. Therefore, the aim of this study was to analyze the structure of gelatin/hyaluronic acid scaffolds crosslinked with EDC. Methods: Gelatin-hyaluronic acid scaffolds were prepared by direct freeze-drying. Afterwards, They were crosslinked with different concentrations of EDC (6, 30, 50 and 60 mM) for 12 h. Results: This research has demonstrated that the gelatin/hyaluronic acid scaffolds crosslinked with the highest concentrations of the crosslinker had fewer water concentration absorbed, pore size diminished and pore number increased in comparison with control groups. Despite scaffolds composition has not changed in any of the concentrations, the bone marrow mesenchymal cells mortality percentage increased when cells were placed on the scaffolds of concentration 60 mM, perhaps for the residual 1-ethy-3-(3-dymethylaminopropyl) carbodiimide hydrochloride found in the scaffolds. Conclusion: Our results revealed that different EDC concentrations may modify the physical and biological characteristics of gelatin/hyaluronic acid scaffolds; as a result, the scaffolds obtained may be used for the manufacture of different tissues in regenerative medicine.

The current challenge in treating periodontitis is regenerating the periodontium. This motivates tissue-engineering researchers to develop scaffolds as artificial matrices that give mechanical support for osteoblasts, cementoblasts, gingival and periodontal ligament fibroblast cells. In this study, modified hyaluronic acid (HA) and chitosan (CS) were employed to create a hybrid CS-HA hydrogel scaffold for periodontal regeneration. CS, HA, and CS-HA scaffolds were obtained by freeze-drying technique, resulting in porous structures suitable for use in tissue engineering. Scaffolds were submitted to gamma and UV-sterilization without significant morphology changes. The ATR-FTIR spectra of CS-HA hydrogels showed peaks at 377 cm-1 , 1566 cm-1 , and 1614 cm-1 , representing secondary amide, primary amine, and carboxyl acid respectively, and it was also observed the emergence of peaks at 886 cm-1 , which probably represents the Schiff base formed in the case of hybrid CS-HA hydrogels. The scaffolds presented a high rate of PBS uptake, reaching values higher than 95%. Thermal degradation of HA scaffolds was around 225°C and CS was around 285°C. The ATR-FTIR spectra and swelling degree were slightly disturbed mainly after gamma sterilization, but degradation temperature did not change after sterilization. The performance of the CS-HA hydrogel scaffolds for in vitro cell culture was tested using NIH3T3 and MG63 cell lines. The Alamar Blue test showed a significant increase in cellular viability and high CD44 expression, suggesting that the cells migrated more when seeded onto the scaffolds. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1691-1702, 2016.

Human mesenchymal stem cells (hMSCs) are an alternative cell source in bioconstruct production for cartilage regeneration, and hyaluronic acid (HA) is a widely-used bioabsorbable scaffold material used for cartilage regeneration. In this work, the aims were to evaluate the mechanical competency of hMSC-seeded HA scaffolds compared with native intact human articular cartilage, and in relation to its cellular properties. Human MSCs were grown under static conditions in HA scaffolds and then tested, in stepwise, stress-relaxation indentation, 7, 14, and 21 days later. Scaffolds at days 14 and 21 showed a significant increase in mechanical measures when compared with day 7 and unseeded scaffold material, but did not achieve the same levels as human cartilage. There was consistent stiffness within the scaffold, with a decreased stiffness around the edge. In vitro culture of hMSC-seeded HA scaffolds over 3 weeks produces a white, solid tissue compared with unseeded constructs. Increased cell proliferation and collagen type II expression were also seen over this period of time. These results demonstrate the competency of the neo-formed cartilage-like tissue in relation to its mechanical and cellular properties, and further, the importance, for future clinical use, of implanting this construct after 14 days of culture.

Nanofibrous hyaluronic acid scaffolds delivering TGF-β3 and SDF-1α for articular cartilage repair in a large animal model

Three-dimensional printing of collagen and hyaluronic acid scaffolds with dehydrothermal treatment crosslinking