Preparation of Decellularized Tissue as Dual Cell Carrier Systems: A Step Towards Facilitating Re-epithelization and Cell Encapsulation for Tracheal Reconstruction.

Surgical treatment of tracheal diseases, trauma, and congenital stenosis has shown success through tracheal reconstruction coupled with palliative care. However, challenges in surgical-based tracheal repairs have prompted the exploration of alternative approaches for tracheal replacement. Tissue-based treatments, involving the cultivation of patient cells on a network of extracellular matrix (ECM) from donor tissue, hold promise for restoring tracheal structure and function without eliciting an immune reaction. In this study, we utilized decellularized canine tracheas as tissue models to develop two types of cell carriers: a decellularized scaffold and a hydrogel. Our hypothesis posits that both carriers, containing essential biochemical niches provided by ECM components, facilitate cell attachment without inducing cytotoxicity. Canine tracheas underwent vacuum-assisted decellularization (VAD), and the ECM-rich hydrogel was prepared through peptic digestion of the decellularized trachea. The decellularized canine trachea exhibited a significant reduction in DNA content and major histocompatibility complex class II, while preserving crucial ECM components such as collagen, glycosaminoglycan, laminin, and fibronectin. Scanning electron microscope and fluorescent microscope images revealed a fibrous ECM network on the luminal side of the cell-free trachea, supporting epithelial cell attachment. Moreover, the ECM-rich hydrogel exhibited excellent viability for human mesenchymal stem cells encapsulated for 3 days, indicating the potential of cell-laden hydrogel in promoting the development of cartilage rings of the trachea. This study underscores the versatility of the trachea in producing two distinct cell carriers-decellularized scaffold and hydrogel-both containing the native biochemical niche essential for tracheal tissue engineering applications.

Entropically Toughened Robust Biodegradable Polymer Blends and Composites for Bone Tissue Engineering.

Biodegradable polymers and composites are promising candidates for biomedical implants in tissue engineering. However, state-of-the-art composite scaffolds suffer from a strength-toughness dilemma due to poor interfacial adhesion and filler dispersion. In this work, we propose a facile and scalable strategy to fabricate strong and tough biocomposite scaffolds through interfacial toughening. The immiscible biopolymer matrix is compatible by the direct incorporation of a third polymer. Densely entangled polymer chains lead to massive crazes and global shear yields under tension. Weak chemical interaction and high-shear melt processing create nanoscale dispersion of nanofillers within the matrix. The resultant ternary blends and composites exhibit an 11-fold increase in toughness without compromising stiffness and strength. At 70% porosity, three-dimensional (3D)-printed composite scaffolds demonstrate high compressive properties comparable to those of cancellous bones. In vitro cell culture on the scaffolds demonstrates not only good cell viability but also effective osteogenic differentiation of human mesenchymal stem cells. Our findings present a widely applicable strategy to develop high-performance biocomposite materials for tissue regeneration.

Salivary gland regeneration: from salivary gland stem cells to three-dimensional bioprinting.

Hyposalivation and severe dry mouth syndrome are the most common complications in patients with head and neck cancer (HNC) after receiving radiation therapy. Conventional treatment for hyposalivation relies on the use of sialogogues such as pilocarpine; however, their efficacy is constrained by the limited number of remnant acinar cells after radiation. After radiotherapy, the salivary gland (SG) secretory parenchyma is largely destroyed, and due to the reduced stem cell niche, this gland has poor regenerative potential. To tackle this, researchers must be able to generate highly complex cellularized 3D constructs for clinical transplantation via technologies, including those that involve bioprinting of cells and biomaterials. A potential stem cell source with promising clinical outcomes to reserve dry mouth is adipose mesenchymal stem cells (AdMSC). MSC-like cells like human dental pulp stem cells (hDPSC) have been tested in novel magnetic bioprinting platforms using nanoparticles that can bind cell membranes by electrostatic interaction, as well as their paracrine signals arising from extracellular vesicles. Both magnetized cells and their secretome cues were found to increase epithelial and neuronal growth of in vitro and ex vivo irradiated SG models. Interestingly, these magnetic bioprinting platforms can be applied as a high-throughput drug screening system due to the consistency in structure and functions of their organoids. Recently, exogenous decellularized porcine ECM was added to this magnetic platform to stimulate an ideal environment for cell tethering, proliferation, and/or differentiation. The combination of these SG tissue biofabrication strategies will promptly allow for in vitro organoid formation and establishment of cellular senescent organoids for aging models, but challenges remain in terms of epithelial polarization and lumen formation for unidirectional fluid flow. Current magnetic bioprinting nanotechnologies can provide promising functional and aging features to in vitro craniofacial exocrine gland organoids, which can be utilized for novel drug discovery and/or clinical transplantation.

Plant molecular farming-derived epidermal growth factor revolutionizes hydrogels for improving glandular epithelial organoid biofabrication.

Epidermal growth factor (EGF) is a known signaling cue essential towards the development and organoid biofabrication particularly for exocrine glands. This study developed an in vitro EGF delivery platform with Nicotiana benthamiana plant-produced EGF (P-EGF) encapsulated on hyaluronic acid/alginate (HA/Alg) hydrogel to improve the effectiveness of glandular organoid biofabrication in short-term culture systems. Primary submandibular gland epithelial cells were treated with 5 - 20 ng/mL of P-EGF and commercially available bacteria-derived EGF (B-EGF). Cell proliferation and metabolic activity were measured by MTT and luciferase-based ATP assays. P-EGF and B-EGF 5 - 20 ng/mL promoted glandular epithelial cell proliferation during 6 culture days on a comparable fashion. Organoid forming efficiency and cellular viability, ATP-dependent activity and expansion were evaluated using two EGF delivery systems, HA/Alg-based encapsulation and media supplementation. Phosphate buffered saline (PBS) was used as a control vehicle. Epithelial organoids fabricated from PBS-, B-EGF-, and P-EGF-encapsulated hydrogels were characterized genotypically, phenotypically and by functional assays. P-EGF-encapsulated hydrogel enhanced organoid formation efficiency and cellular viability and metabolism relative to P-EGF supplementation. At culture day 3, epithelial organoids developed from P-EGF-encapsulated HA/Alg platform contained functional cell clusters expressing specific glandular epithelial markers such as exocrine pro-acinar (AQP5, NKCC1, CHRM1, CHRM3, Mist1), ductal (K18, Krt19), and myoepithelial (α-SMA, Acta2), and possessed a high mitotic activity (38-62% Ki67 cells) with a large epithelial progenitor population (∼70% K14 cells). The P-EGF encapsulation strikingly upregulated the expression of pro-acinar AQP5 cells through culture time when compared to others (B-EGF, PBS). Thus, the utilization of Nicotiana benthamiana in molecular farming can produce EGF biologicals amenable to encapsulation in HA/Alg-based in vitro platforms, which can effectively and promptly induce the biofabrication of exocrine gland organoids.

Biofabrication, biochemical profiling, and in vitro applications of salivary gland decellularized matrices via magnetic bioassembly platforms.

Trending three-dimensional tissue engineering platforms developed via biofabrication and bioprinting of exocrine glands are on the rise due to a commitment to organogenesis principles. Nevertheless, a proper extracellular matrix (ECM) microarchitecture to harbor primary cells is yet to be established towards human salivary gland (SG) organogenesis. By using porcine submandibular gland (SMG) biopsies as a proof-of-concept to mimic the human SG, a new decellularized ECM bioassembly platform was developed herein with varying perfusions of sodium dodecyl sulfate (SDS) to limit denaturing events and ensure proper preservation of the native ECM biochemical niche. Porcine SMG biopsies were perfused with 0.01%, 0.1%, and 1% SDS and bio-assembled magnetically in porous polycarbonate track-etched (PCTE) membrane. Double-stranded DNA (dsDNA), cell removal efficiency, and ECM biochemical contents were analyzed. SDS at 0.1% and 1% efficiently removed dsDNA (< 50 ng/mg) and preserved key matrix components (sulfated glycosaminoglycans, collagens, elastin) and the microarchitecture of native SMG ECM. Bio-assembled SMG decellularized ECM (dECM) perfused with 0.1-1% SDS enhanced cell viability, proliferation, expansion confluency rates, and tethering of primary SMG cells during 7 culture days. Perfusion with 1% SDS promoted greater cell proliferation rates while 0.1% SDS supported higher acinar epithelial expression when compared to basement membrane extract and other substrates. Thus, this dECM magnetic bioassembly strategy was effective for decellularization while retaining the original ECM biochemical niche and promoting SMG cell proliferation, expansion, differentiation, and tethering. Altogether, these outcomes pave the way towards the recellularization of this novel SMG dECM in future in vitro and in vivo applications.

Using Vertebrate Stem and Progenitor Cells for Cellular Agriculture, State-of-the-Art, Challenges, and Future Perspectives.

Global food systems are under significant pressure to provide enough food, particularly protein-rich foods whose demand is on the rise in times of crisis and inflation, as presently existing due to post-COVID-19 pandemic effects and ongoing conflict in Ukraine and resulting in looming food insecurity, according to FAO. Cultivated meat (CM) and cultivated seafood (CS) are protein-rich alternatives for traditional meat and fish that are obtained via cellular agriculture (CA) i.e., tissue engineering for food applications. Stem and progenitor cells are the building blocks and starting point for any CA bioprocess. This review presents CA-relevant vertebrate cell types and procedures needed for their myogenic and adipogenic differentiation since muscle and fat tissue are the primary target tissues for CM/CS production. The review also describes existing challenges, such as a need for immortalized cell lines, or physical and biochemical parameters needed for enhanced meat/fat culture efficiency and ways to address them.

Magnetic bioassembly platforms towards the generation of extracellular vesicles from human salivary gland functional organoids for epithelial repair.

Salivary glands (SG) are exocrine organs with secretory units commonly injured by radiotherapy. Bio-engineered organoids and extracellular vesicles (EV) are currently under investigation as potential strategies for SG repair. Herein, three-dimensional (3D) cultures of SG functional organoids (SGo) and human dental pulp stem cells (hDPSC) were generated by magnetic 3D bioassembly (M3DB) platforms. Fibroblast growth factor 10 (FGF10) was used to enrich the SGo in secretory epithelial units. After 11 culture days via M3DB, SGo displayed SG-specific acinar epithelial units with functional properties upon neurostimulation. To consistently develop 3D hDPSC in vitro, 3 culture days were sufficient to maintain hDPSC undifferentiated genotype and phenotype for EV generation. EV isolation was performed via sequential centrifugation of the conditioned media of hDPSC and SGo cultures. EV were characterized by nanoparticle tracking analysis, electron microscopy and immunoblotting. EV were in the exosome range for hDPSC (diameter: 88.03 ± 15.60 nm) and for SGo (123.15 ± 63.06 nm). Upon ex vivo administration, exosomes derived from SGo significantly stimulated epithelial growth (up to 60%), mitosis, epithelial progenitors and neuronal growth in injured SG; however, such biological effects were less distinctive with the ones derived from hDPSC. Next, these exosome biological effects were investigated by proteomic arrays. Mass spectrometry profiling of SGo exosomes predicted that cellular growth, development and signaling was due to known and undocumented molecular targets downstream of FGF10. Semaphorins were identified as one of the novel targets requiring further investigations. Thus, M3DB platforms can generate exosomes with potential to ameliorate SG epithelial damage.

Development of high-throughput lacrimal gland organoid platforms for drug discovery in dry eye disease.

Dysfunction and damage of the lacrimal gland (LG) results in ocular discomfort and dry eye disease (DED). Current therapies for DED do not fully replenish the necessary lubrication to rescue optimal vision. New drug discovery for DED has been limited perhaps because in vitro models cannot mimic the biology of the native LG. The existing platforms for LG organoid culture are scarce and still not ready for consistency and scale up production towards drug screening. The magnetic three-dimensional (3D) bioprinting (M3DB) is a novel system for 3D in vitro biofabrication of cellularized tissues using magnetic nanoparticles to bring cells together. M3DB provides a scalable platform for consistent handling of spheroid-like cell cultures facilitating consistent biofabrication of organoids. Previously, we successfully generated innervated secretory epithelial organoids from human dental pulp stem cells with M3DB and found that this platform is feasible for epithelial organoid bioprinting. Research targeting LG organogenesis, drug discovery for DED has extensively used mouse models. However, certain inter-species differences between mouse and human must be considered. Porcine LG appear to have more similarities to human LG than the mouse counterparts. We have conducted preliminary studies with the M3DB for fabricating LG organoids from primary cells isolated from murine and porcine LG, and found that this platform provides robust LG organoids for future potential high-throughput analysis and drug discovery. The LG organoid holds promise to be a functional model of tearing, a platform for drug screening, and may offer clinical applications for DED.

A novel gelatin/chitooligosaccharide/demineralized bone matrix composite scaffold and periosteum-derived mesenchymal stem cells for bone tissue engineering.

BACKGROUND: A novel biodegradable scaffold including gelatin (G), chitooligosaccharide (COS), and demineralized bone matrix (DBM) could play a significant part in bone tissue engineering. The present study aimed to investigate the biological characteristics of composite scaffolds in combination of G, COS, and DBM for in vitro cell culture and in vivo animal bioassays. METHODS: Three-dimensional scaffolds from the mixture of G, COS, and DBM were fabricated into 3 groups, namely, G, GC, and GCD using a lyophilization technique. The scaffolds were cultured with mesenchymal stem cells (MSCs) for 4 weeks to determine biological responses such as cell attachment and cell proliferation, alkaline phosphatase (ALP) activity, calcium deposition, cell morphology, and cell surface elemental composition. For the in vivo bioassay, G, GC, and GCD, acellular scaffolds were implanted subcutaneously in 8-week-old male Wistar rats for 4 weeks and 8 weeks. The explants were assessed for new bone formation using hematoxylin and eosin (H&E) staining and von Kossa staining. RESULTS: The MSCs could attach and proliferate on all three groups of scaffolds. Interestingly, the ALP activity of MSCs reached the greatest value on day 7 after cultured on the scaffolds, whereas the calcium assay displayed the highest level of calcium in MSCs on day 28. Furthermore, weight percentages of calcium and phosphorus on the surface of MSCs after cultivation on the GCD scaffolds increased when compared to those on other scaffolds. The scanning electron microscopy images showed that MSCs attached and proliferated on the scaffold surface thoroughly over the cultivation time. Mineral crystal aggregation was evident in GC and greatly in GCD scaffolds. H&E staining illustrated that G, GC, and GCD scaffolds displayed osteoid after 4 weeks of implantation and von Kossa staining confirmed the mineralization at 8 weeks in G, GC, and GCD scaffolds. CONCLUSION: The MSCs cultured in GCD scaffolds revealed greater osteogenic differentiation than those cultured in G and GC scaffolds. Additionally, the G, GC, and GCD scaffolds could promote in vivo ectopic bone formation in rat model. The GCD scaffolds exhibited maximum osteoinductive capability compared with others and may be potentially used for bone regeneration.

Carboxymethyl Cellulose Entrapped in a Poly(vinyl) Alcohol Network: Plant-Based Scaffolds for Cartilage Tissue Engineering.

Cartilage has a limited inherent healing capacity after injury, due to a lack of direct blood supply and low cell density. Tissue engineering in conjunction with biomaterials holds promise for generating cartilage substitutes that withstand stress in joints. A major challenge of tissue substitution is creating a functional framework to support cartilage tissue formation. Polyvinyl alcohol (PVA) was crosslinked with glutaraldehyde (GA), by varying the mole ratios of GA/PVA in the presence of different amounts of plant-derived carboxymethyl cellulose (CMC). Porous scaffolds were created by the freeze-drying technique. The goal of this study was to investigate how CMC incorporation and crosslinking density might affect scaffold pore formation, swelling behaviors, mechanical properties, and potential use for engineered cartilage. The peak at 1599 cm-1 of the C=O group in ATR-FTIR indicates the incorporation of CMC into the scaffold. The glass transition temperature (Tg) and Young's modulus were lower in the PVA/CMC scaffold, as compared to the PVA control scaffold. The addition of CMC modulates the pore architecture and increases the swelling ratio of scaffolds. The toxicity of the scaffolds and cell attachment were tested. The results suggest that PVA/CMC scaffolding material can be tailored in terms of its physical and swelling properties to potentially support cartilage formation.

Trends in Salivary Gland Tissue Engineering: From Stem Cells to Secretome and Organoid Bioprinting.

Xerostomia or dry mouth are commonly diagnosed in head and neck cancer patients due to salivary gland (SG) epithelial injury after radiotherapy. Regenerative medicine has fetched the opportunity to replace or regenerate the SG epithelia and restore its secretory function. Early adult stem cell transplantation strategies in rodents have recently shown to improve clinical outcomes in radiotherapy-induced xerostomia in Phase 1/2 human trials. Mesenchymal stem cells from adipose tissue are the most promising, although the ones from the labial mucosa, bone marrow, or dental pulp have an attractive therapeutic value after successful findings in ex vivo and in vivo mouse models of SG injury. Emerging approaches using cell-free therapy with cell "extracts", "soups" or secretome components also exhibit favorable outcomes in the same rodent models. When compared to cell-based approaches, extracellular vesicles (EV) from the secretome (i.e., exosomes) can be easily extracted, quantified, and are more stable for long-term storage and use in SG tissue engineering. Additive manufacturing and three-dimensional bioprinting or bioassembly have an important role on generating spheroids or organoids for cell transplantation to ameliorate SG injury. Moreover, organoids can secrete EV, which may have a therapeutic potential worth to explore in future studies. In this review, we will describe the technological advancements and challenges of these different cell-based and cell-free strategies in SG tissue engineering and regeneration. Impact statement Salivary gland (SG)-like innervated epithelial organoids and the secretome produced from stem cells may constitute feasible therapeutic alternatives to regenerate the SG due to their user-friendly, short-lived, consistent, and scalable additive manufacturing processes. Bioprinting such human SG organoids toward in vitro drug discovery may further reduce the incorporation of animal-derived components to the tissue constructs and minimize the use of animal experimentation in SG regeneration. Despite such advancements, transplantation with human adipose-derived mesenchymal stem cells is the only tissue engineering strategy that has reached Phase 1/2 clinical trials and shown to enlarge the serous SG epithelium and improve salivary flow.

Alginate/Pluronic F127-based encapsulation supports viability and functionality of human dental pulp stem cell-derived insulin-producing cells.

BACKGROUND: Current approach for diabetes treatment remained several adverse events varied from gastrointestinal to life-threatening symptoms. Regenerative therapy regarding Edmonton protocol has been facing serious limitations involving protocol efficiency and safety. This led to the study for alternative insulin-producing cell (IPC) resource and transplantation platform. In this study, evaluation of encapsulated human dental pulp-derived stem cell (hDPSC)-derived IPCs by alginate (ALG) and pluronic F127-coated alginate (ALGPA) was performed. RESULTS: The results showed that ALG and ALGPA preserved hDPSC viability and allowed glucose and insulin diffusion in and out. ALG and ALGPA-encapsulated hDPSC-derived IPCs maintained viability for at least 336 h and sustained pancreatic endoderm marker (NGN3), pancreatic islet markers (NKX6.1, MAF-A, ISL-1, GLUT-2 and INSULIN), and intracellular pro-insulin and insulin expressions for at least 14 days. Functional analysis revealed a glucose-responsive C-peptide secretion of ALG- and ALGPA-encapsulated hDPSC-derived IPCs at 14 days post-encapsulation. CONCLUSION: ALG and ALGPA encapsulations efficiently preserved the viability and functionality of hDPSC-derived IPCs in vitro and could be the potential transplantation platform for further clinical application.

Exploring the Gelation Mechanisms and Cytocompatibility of Gold (III)-Mediated Regenerated and Thiolated Silk Fibroin Hydrogels.

Accelerating the gelation of silk fibroin (SF) solution from several days or weeks to minutes or few hours is critical for several applications (e.g., cell encapsulation, bio-ink for 3D printing, and injectable controlled release). In this study, the rapid gelation of SF induced by a gold salt (Au3+) as well as the cytocompatibility of Au3+-mediated SF hydrogels are reported. The gelation behaviors and mechanisms of regenerated SF and thiolated SF (tSF) were compared. Hydrogels can be obtained immediately after mixing or within three days depending on the types of silk proteins used and amount of Au3+. Au3+-mediated SF and tSF hydrogels showed different color appearances. The color of Au-SF hydrogels was purple-red, whereas the Au-tSF hydrogels maintained their initial solution color, indicating different gelation mechanisms. The reduction of Au3+ by amino groups and further reduction to Au by tyrosine present in SF, resulting in a dityrosine bonding and Au nanoparticles (NPs) production, are proposed as underlying mechanisms of Au-SF gel formation. Thiol groups of the tSF reduced Au3+ to Au+ and formed a disulfide bond, before a formation of Au+-S bonds. Protons generated during the reactions between Au3+ and SF or tSF led to a decrease of the local pH, which affected the chain aggregation of the SF, and induced the conformational transition of SF protein to beta sheet. The cytocompatibility of the Au-SF and tSF hydrogels was demonstrated by culturing with a L929 cell line, indicating that the developed hydrogels can be promising 3D matrices for different biomedical applications.

Effect of interface mechanical discontinuities on scaffold-cartilage integration.

A consistent lack of lateral integration between scaffolds and adjacent articular cartilage has been exhibited in vitro and in vivo. Given the mismatch in mechanical properties between scaffolds and articular cartilage, the mechanical discontinuity that occurs at the interface has been implicated as a key factor, but remains inadequately studied. Our objective was to investigate how the mechanical environment within a mechanically loaded scaffold-cartilage construct might affect integration. We hypothesized that the magnitude of the mechanical discontinuity at the scaffold-cartilage interface would be related to decreased integration. To test this hypothesis, chondrocyte seeded scaffolds were embedded into cartilage explants, pre-cultured for 14 days, and then mechanically loaded for 28 days at either 1N or 6N of applied load. Constructs were kept either peripherally confined or unconfined throughout the duration of the experiment. Stress, strain, fluid flow, and relative displacements at the cartilage-scaffold interface and within the scaffold were quantified using biphasic, inhomogeneous finite element models (bFEMs). The bFEMs indicated compressive and shear stress discontinuities occurred at the scaffold-cartilage interface for the confined and unconfined groups. The mechanical strength of the scaffold-cartilage interface and scaffold GAG content were higher in the radially confined 1N loaded groups. Multivariate regression analyses identified the strength of the interface prior to the commencement of loading and fluid flow within the scaffold as the main factors associated with scaffold-cartilage integration. Our study suggests a minimum level of scaffold-cartilage integration is needed prior to the commencement of loading, although the exact threshold has yet to be identified. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Synergistic effects of hypoxia and morphogenetic factors on early chondrogenic commitment of human embryonic stem cells in embryoid body culture.

Derivation of articular chondrocytes from human stem cells would advance our current understanding of chondrogenesis, and accelerate development of new stem cell therapies for cartilage repair. Chondrogenic differentiation of human embryonic stem cells (hESCs) has been studied using supplemental and cell-secreted morphogenetic factors. The use of bioreactors enabled insights into the effects of physical forces and controlled oxygen tension. In this study, we investigated the interactive effects of controlled variation of oxygen tension and chondrocyte-secreted morphogenetic factors on chondrogenic differentiation of hESCs in the embryoid body format (hESC-EB). Transient hypoxic culture (2 weeks at 5 % O2 followed by 1 week at 21 % O2) of hESC-EBs in medium conditioned with primary chondrocytes up-regulated the expression of SOX9 and suppressed pluripotent markers OCT4 and NANOG. Pellets derived from these cells showed significant up-regulation of chondrogenic genes (SOX9, COL2A1, ACAN) and enhanced production of cartilaginous matrix (collagen type II and proteoglycan) as compared to the pellets from hESC-EBs cultured under normoxic conditions. Gene expression profiles corresponded to those associated with native cartilage development, with early expression of N-cadherin (indicator of cell condensation) and late expression of aggrecan (ACAN, indicator of proteoglycan production). When implanted into highly vascularized subcutaneous area in immunocompromised mice for 4 weeks, pellets remained phenotypically stable and consisted of cartilaginous extracellular matrix (ECM), without evidence of dedifferentiation or teratoma formation. Based on these results, we propose that chondrogenesis in hESC can be synergistically enhanced by a control of oxygen tension and morphogenetic factors secreted by chondrocytes.

Silk microfiber-reinforced silk hydrogel composites for functional cartilage tissue repair.

Cartilage tissue lacks an intrinsic capacity for self-regeneration due to slow matrix turnover, a limited supply of mature chondrocytes and insufficient vasculature. Although cartilage tissue engineering has achieved some success using agarose as a scaffolding material, major challenges of agarose-based cartilage repair, including non-degradability, poor tissue-scaffold integration and limited processing capability, have prompted the search for an alternative biomaterial. In this study, silk fiber-hydrogel composites (SF-silk hydrogels) made from silk microfibers and silk hydrogels were investigated for their potential use as a support material for engineered cartilage. We demonstrated the use of 100% silk-based fiber-hydrogel composite scaffolds for the development of cartilage constructs with properties comparable to those made with agarose. Cartilage constructs with an equilibrium modulus in the native tissue range were fabricated by mimicking the collagen fiber and proteoglycan composite architecture of native cartilage using biocompatible, biodegradable silk fibroin from Bombyx mori. Excellent chondrocyte response was observed on SF-silk hydrogels, and fiber reinforcement resulted in the development of more mechanically robust constructs after 42 days in culture compared to silk hydrogels alone. Thus, we demonstrate the versatility of silk fibroin as a composite scaffolding material for use in cartilage tissue repair to create functional cartilage constructs that overcome the limitations of agarose biomaterials, and provide a much-needed alternative to the agarose standard.

Age-related carbonylation of fibrocartilage structural proteins drives tissue degenerative modification.

Aging-related oxidative stress has been linked to degenerative modifications in different organs and tissues. Using redox proteomic analysis and illustrative tandem mass spectrometry mapping, we demonstrate oxidative posttranslational modifications in structural proteins of intervertebral discs (IVDs) isolated from aging mice. Increased protein carbonylation was associated with protein fragmentation and aggregation. Complementing these findings, a significant loss of elasticity and increased stiffness was measured in fibrocartilage from aging mice. Studies using circular dichroism and intrinsic tryptophan fluorescence revealed a significant loss of secondary and tertiary structures of purified collagens following oxidation. Collagen unfolding and oxidation promoted both nonenzymatic and enzymatic degradation. Importantly, induction of oxidative modification in healthy fibrocartilage recapitulated the biochemical and biophysical modifications observed in the aging IVD. Together, these results suggest that protein carbonylation, glycation, and lipoxidation could be early events in promoting IVD degenerative changes.

Supplementation of exogenous adenosine 5'-triphosphate enhances mechanical properties of 3D cell-agarose constructs for cartilage tissue engineering.

Formation of tissue-engineered cartilage is greatly enhanced by mechanical stimulation. However, direct mechanical stimulation is not always a suitable method, and the utilization of mechanisms underlying mechanotransduction might allow for a highly effective and less aggressive alternate means of stimulation. In particular, the purinergic, adenosine 5'-triphosphate (ATP)-mediated signaling pathway is strongly implicated in mechanotransduction within the articular cartilage. We investigated the effects of transient and continuous exogenous ATP supplementation on mechanical properties of cartilaginous constructs engineered using bovine chondrocytes and human mesenchymal stem cells (hMSCs) encapsulated in an agarose hydrogel. For both cell types, we have observed significant increases in equilibrium and dynamic compressive moduli after transient ATP treatment applied in the fourth week of cultivation. Continuous ATP treatment over 4 weeks of culture only slightly improved the mechanical properties of the constructs, without major changes in the total glycosaminoglycan (GAG) and collagen content. Structure-function analyses showed that transiently ATP-treated constructs, and in particular those based on hMSCs, had the highest level of correlation between compositional and mechanical properties. Transiently treated groups showed intense staining of the territorial matrix for GAGs and collagen type II. These results indicate that transient ATP treatment can improve functional mechanical properties of cartilaginous constructs based on chondrogenic cells and agarose hydrogels, possibly by improving the structural organization of the bulk phase and territorial extracellular matrix (ECM), that is, by increasing correlation slopes between the content of the ECM components (GAG, collagen) and mechanical properties of the construct.

Transient hypoxia improves matrix properties in tissue engineered cartilage.

Adult articular cartilage is a hypoxic tissue, with oxygen tension ranging from <10% at the cartilage surface to <1% in the deepest layers. In addition to spatial gradients, cartilage development is also associated with temporal changes in oxygen tension. However, a vast majority of cartilage tissue engineering protocols involves cultivation of chondrocytes or their progenitors under ambient oxygen concentration (21% O(2)), that is, significantly above physiological levels in either developing or adult cartilage. Our study was designed to test the hypothesis that transient hypoxia followed by normoxic conditions results in improved quality of engineered cartilaginous ECM. To this end, we systematically compared the effects of normoxia (21% O(2) for 28 days), hypoxia (5% O(2) for 28 days) and transient hypoxia--reoxygenation (5% O(2) for 7 days and 21% O(2) for 21 days) on the matrix composition and expression of the chondrogenic genes in cartilage constructs engineered in vitro. We demonstrated that reoxygenation had the most effect on the expression of cartilaginous genes including COL2A1, ACAN, and SOX9 and increased tissue concentrations of amounts of glycosaminoglycans and type II collagen. The equilibrium Young's moduli of tissues grown under transient hypoxia (510.01 ± 28.15 kPa) and under normoxic conditions (417.60 ± 68.46 kPa) were significantly higher than those measured under hypoxic conditions (279.61 ± 20.52 kPa). These data suggest that the cultivation protocols utilizing transient hypoxia with reoxygenation have high potential for efficient cartilage tissue engineering, but need further optimization in order to achieve higher mechanical functionality of engineered constructs.

Silk hydrogel for cartilage tissue engineering.

Cartilage tissue engineering based on cultivation of immature chondrocytes in agarose hydrogel can yield tissue constructs with biomechanical properties comparable to native cartilage. However, agarose is immunogenic and nondegradable, and our capability to modify the structure, composition, and mechanical properties of this material is rather limited. In contrast, silk hydrogel is biocompatible and biodegradable, and it can be produced using a water-based method without organic solvents that enables precise control of structural and mechanical properties in a range of interest for cartilage tissue engineering. We observed that one particular preparation of silk hydrogel yielded cartilaginous constructs with biochemical content and mechanical properties matching constructs based on agarose. This finding and the possibility to vary the properties of silk hydrogel motivated this study of the factors underlying the suitability of hydrogels for cartilage tissue engineering. We present data resulting from a systematic variation of silk hydrogel properties, silk extraction method, gel concentration, and gel structure. Data suggest that silk hydrogel can be used as a tool for studies of the hydrogel-related factors and mechanisms involved in cartilage formation, as well as a tailorable and fully degradable scaffold for cartilage tissue engineering.