Engineering large, anatomically shaped osteochondral constructs with robust interfacial shear properties.

Despite the prevalence of large (>5 cm2) articular cartilage defects involving underlying bone, current tissue-engineered therapies only address small defects. Tissue-engineered, anatomically shaped, native-like implants may address the need for off-the-shelf, tissue-repairing therapies for large cartilage lesions. This study fabricated an osteochondral construct of translationally relevant geometry with robust functional properties. Scaffold-free, self-assembled neocartilage served as the chondral phase, and porous hydroxyapatite served as the osseous phase of the osteochondral constructs. Constructs in the shape and size of an ovine femoral condyle (31 × 14 mm) were assembled at day 4 (early) or day 10 (late) of neocartilage maturation. Early osteochondral assembly increased the interfacial interdigitation depth by 244%, interdigitation frequency by 438%, interfacial shear modulus by 243-fold, and ultimate interfacial shear strength by 4.9-fold, compared to late assembly. Toward the development of a bioprosthesis for the repair of cartilage lesions encompassing up to an entire condylar surface, this study generated a large, anatomically shaped osteochondral construct with robust interfacial mechanical properties and native-like neocartilage interdigitation.

Feasibility of in situ chondrogenesis for the entire umbilical cord in preliminary preparation for tracheal graft.

BACKGROUND: There remains a scarcity of both autografts and allografts for tracheal transplantation after long-segmental resection. Subsequently, tissue engineering has become a promising alternative for tracheal transplantation, which requires successful in vitro chondrogenesis. METHODS: To optimize the protocol for in situ chondrogenesis using the pig-derived whole Umbilical Cord (UC) as the starting material, it must be performed without using the UC-multipotent stromal cell (MSCs) isolation procedure. Nevertheless, chondrogenic induction is performed under a variety of conditions; with or without TGF-ß1 at different concentrations, and also in combination with either a rotatory or hollow organ bioreactor. The engineered explant sections were analyzed using various histochemical and immunohistochemical stains to assess the expression of chondrocyte markers. Cell viability was determined through use of the APO-BrdU TUNEL assay kit. RESULTS: The results showed that culture conditions induced heterogeneous chondrogenesis in various compartments of the UC. Moreover, explants cultured with 10 ng/ml TGF-ß1 under hypoxic (1% O2) in combination with a bioreactor, significantly enhanced the expression of aggrecan and type II collagen, but were lacking in the production of Glycosaminoglycans (GAGs), as evidenced by alcian blue staining. We speculated that whole segment UCs allowed for the differentiation into premature chondrocytes in our tissue-engineered environments. CONCLUSION: This study has provided exciting preliminary evidence showing that a stem cell-rich UC wrapped around an anatomical tracheal scaffold and implanted in vivo can induce nodes of new cartilage growth into a structurally functional tissue for the repairing of long-segmental tracheal stenosis.

De novo cartilage growth after implantation of a 3-D-printed tracheal graft in a porcine model.

BACKGROUND: Experiments were conducted on the assumption that vivid chondrogenesis would be boosted in vivo following previously preliminary chondrogenesis in a mesenchymal stem cell (MSC)-rich entire umbilical cord (UC) in vitro. METHODS: Virtual 3-D tracheal grafts were generated by using a profile obtained by scanning the native trachea of the listed porcine. Although the ultimate goal was the acquisition of a living specimen beyond a 3-week survival period, the empirical results did not meet our criteria until the 10th experiment, ending with the sacrifice of the animal. The categories retrospectively evolved from post-transplant modification due to porcine death using 4 different methods of implantation in chronological order. For each group, we collected details on graft construction, clinical outcomes, and results from both gross and histology examinations. RESULTS: Three animals died due to tracheal complications: one died from graft crush, and two died secondary to erosion of the larger graft into the great vessels. It appeared that the remaining 7 died of tracheal stenosis from granulation tissue. Ectopic de novo growth of neocartilage was found in three porcine subjects. In the nearby tissues, we detected neocartilage near the anastomosis containing interim vesicles of the vascular canals (VCs), perichondrial papillae (PPs) and preresorptive layers (PRLs), which were investigated during the infancy of cartilage development and were first unveiled in the tracheal cartilage. CONCLUSIONS: 3-D-printed anatomically precise grafts could not provide successful transplantation with stent-sparing anastomosis; nonetheless, de novo cartilage regeneration in situ appears to be promising for tracheal graft adaptability. Further graft refinement and strategies for managing granulated tissues are still needed to improve graft outcomes.

Anti-Adhesion Behavior from Ring-Strain Amine Cyclic Monolayers Grafted on Silicon (111) Surfaces.

In this manuscript, a series of amine tagged short cyclic molecules (cyclopropylamine, cyclobutylamine, cyclopentylamine and cyclohexylamine) were thermally grafted onto p-type silicon (111) hydride surfaces via nucleophilic addition. The chemistries of these grafting were verified via XPS, AFM and sessile droplet measurements. Confocal microscopy and cell viability assay was performed on these surfaces incubated for 24 hours with triple negative breast cancer cells (MDA-MB 231), gastric adenocarcinoma cells (AGS) endometrial adenocarcinoma (Hec1A). All cell types had shown a significant reduction when incubated on these ring-strain cyclic monolayer surfaces than compared to standard controls. The expression level of focal adhesion proteins (vinculin, paxilin, talin and zyxin) were subsequently quantified for all three cell types via qPCR analysis. Cells incubate on these surface grafting were observed to have reduced levels of adhesion protein expression than compared to positive controls (collagen coating and APTES). A potential application of these anti-adhesive surfaces is the maintenance of the chondrocyte phenotype during in-vitro cell expansion. Articular chondrocytes cultured for 6 days on ring strained cyclopropane-modified surfaces was able to proliferate but had maintained a spheroid/aggregated phenotype with higher COL2A1 and ACAN gene expression. Herein, these findings had help promote grafting of cyclic monolayers as an viable alternative for producing antifouling surfaces.

Overcoming Challenges in Engineering Large, Scaffold-Free Neocartilage with Functional Properties.

Although numerous cartilage engineering methods have been described, few report generation of constructs greater than 4 cm2, which is the typical lesion size considered for cell-based therapies. Furthermore, current cell-based therapies only target focal lesions, while treatment of large nonisolated lesions remains an area of great demand. The objective of this study was to scale up fabrication of self-assembled neocartilage from standard sizes of 0.2 cm2 to greater than 8 cm2. Passaged sheep articular chondrocytes were self-assembled into 5 or 25-mm-diameter scaffoldless neocartilage constructs. The 25-mm-diameter constructs grew up to 9.3 cm2 (areal scale-up of 23) and possessed properties similar to those of the 5-mm-diameter constructs; unfortunately, these large constructs were deformed and are unusable as a potential implant. A novel neocartilage fabrication strategy-employing mechanical confinement, a minute deadweight, and chemical stimulation (cytochalasin D, TGF-ß1, chondroitinase-ABC, and lysyl oxidase-like 2 protein)-was found to successfully generate large (25-mm diameter) constructs with flat, homogeneous morphologies. Chemical stimulation increased collagen content and tensile Young's modulus 140% and 240% in the 25-mm-diameter constructs and 30% and 70% in the 5-mm-diameter constructs, respectively. This study not only demonstrated that exceedingly large self-assembled neocartilage can be generated with the appropriate combination of mechanical and chemical stimuli but also that its properties were maintained or even enhanced.

Engineering biomechanically functional neocartilage derived from expanded articular chondrocytes through the manipulation of cell-seeding density and dexamethasone concentration.

Recent work has established methods to engineer self-assembled, scaffold-free neocartilage from an expanded articular chondrocyte (AC) cell source. In continuing such work, the objective of the present study was to investigate the effects of cell-seeding density and dexamethasone concentration on these neocartilage constructs. Neocartilage discs (5 mm diameter) were formed by self-assembling passaged leporine articular chondrocytes into non-adherent agarose moulds. The cell-seeding densities (2, 3, 4, 5 and 6 million cells/construct) and dexamethasone concentrations (10 and 100 nm) in the culture medium were varied in a full-factorial study. After 4 weeks, the neocartilage constructs were assessed for morphological, biochemical and biomechanical properties. The cell-seeding density profoundly affected neocartilage properties. The two dexamethasone concentrations explored did not induce overall significant differences. Constructs formed using lower cell-seeding densities possessed much higher biochemical and biomechanical properties than constructs seeded with higher cell densities. Notably, the 2 million cells/construct group formed hyaline-like neocartilage with a collagen wet weight (WW) content of ~7% and a Young's modulus of ~4 MPa, representing the high end of values achieved in self-assembled neocartilage. Excitingly, the mechanical properties of these constructs were on a par with that of native cartilage tissues tested under similar conditions. Through optimization of cell-seeding density, this study shows for the first time the use of expanded ACs to form homogeneous self-assembled neocartilage with exceptionally high tensile strength. With such functional properties, these engineered neocartilage constructs provide a promising alternative for treating articular lesions. Copyright © 2016 John Wiley & Sons, Ltd.

Effects of passage number and post-expansion aggregate culture on tissue engineered, self-assembled neocartilage.

UNLABELLED: Chondrocyte dedifferentiation presents a major barrier in engineering functional cartilage constructs. To mitigate the effects of dedifferentiation, this study employed a post-expansion aggregate culture step to enhance the chondrogenic phenotype of passaged articular chondrocytes (ACs) before their integration into self-assembled neocartilage constructs. The objective was twofold: (1) to explore how passage number (P2, P3, P4, P5, P6, and P7), with or without aggregate culture, affected construct properties; and (2) to determine the highest passage number that could form neocartilage with functional properties. Juvenile leporine ACs were passaged to P2-P7, with or without aggregate culture, and self-assembled into 5mm discs in non-adhesive agarose molds without using any exogenous scaffolds. Construct biochemical and biomechanical properties were assessed. With aggregate culture, neocartilage constructs had significantly higher collagen content, higher tensile properties, and flatter morphologies. These beneficial effects were most obvious at higher passage numbers. Specifically, collagen content, Young's modulus, and instantaneous compressive modulus in the P7, aggregate group were 53%, 116%, and 178% higher than those in the P7, non-aggregate group. Most interestingly, these extensively passaged P7 ACs (expansion factor of 85,000), which are typically highly dedifferentiated, were able to form constructs with properties similar to or higher than those formed by lower passage number cells. This study not only demonstrated that post-expansion aggregate culture could significantly improve the properties of self-assembled neocartilage, but also that chondrocytes of exceedingly high passage numbers, expanded using the methods in this study, could be used in cartilage engineering applications. STATEMENT OF SIGNIFICANCE: This work demonstrated that extensively passaged chondrocytes (up to passage 7 (P7); expansion factor of 85,000) could potentially be used for cartilage tissue engineering applications. Specifically, an aggregate culture step, employed after cell expansion and before cell integration into a neocartilage construct, was shown to enhance the ability of the chondrocytes to form neocartilage with better biochemical and biomechanical properties. The beneficial effects of this aggregate culture step was especially noticeable at the high passage numbers. Most interestingly, P7 chondrocytes, which are typically highly dedifferentiated, were able to form neocartilage with properties similar to or higher than those formed by lower passage number cells. The ability to obtain high chondrocyte yields with an enhanced chondrogenic potential could have a broad, beneficial impact in improving current therapies (e.g., using higher cell seeding densities for repair) or developing new strategies that require high cell numbers, such as a scaffold-free approach in forming engineered cartilage.

Cell-based tissue engineering strategies used in the clinical repair of articular cartilage.

One of the most important issues facing cartilage tissue engineering is the inability to move technologies into the clinic. Despite the multitude of current research in the field, it is known that 90% of new drugs that advance past animal studies fail clinical trials. The objective of this review is to provide readers with an understanding of the scientific details of tissue engineered cartilage products that have demonstrated a certain level of efficacy in humans, so that newer technologies may be developed upon this foundation. Compared to existing treatments, such as microfracture or autologous chondrocyte implantation, a tissue engineered product can potentially provide more consistent clinical results in forming hyaline repair tissue and in filling the entirety of the defect. The various tissue engineering strategies (e.g., cell expansion, scaffold material, media formulations, biomimetic stimuli, etc.) used in forming these products, as collected from published literature, company websites, and relevant patents, are critically discussed. The authors note that many details about these products remain proprietary, not all information is made public, and that advancements to the products are continuously made. Nevertheless, by understanding the design and production processes of these emerging technologies, one can gain tremendous insight into how to best use them and also how to design the next generation of tissue engineered cartilage products.

Digoxin and adenosine triphosphate enhance the functional properties of tissue-engineered cartilage.

Toward developing engineered cartilage for the treatment of cartilage defects, achieving relevant functional properties before implantation remains a significant challenge. Various chemical and mechanical stimuli have been used to enhance the functional properties of engineered musculoskeletal tissues. Recently, Ca(2+)-modulating agents have been used to enhance matrix synthesis and biomechanical properties of engineered cartilage. The objective of this study was to determine whether other known Ca(2+) modulators, digoxin and adenosine triphosphate (ATP), can be employed as novel stimuli to increase collagen synthesis and functional properties of engineered cartilage. Neocartilage constructs were formed by scaffold-free self-assembling of primary bovine articular chondrocytes. Digoxin, ATP, or both agents were added to the culture medium for 1 h/day on days 10-14. After 4 weeks of culture, neocartilage properties were assessed for gross morphology, biochemical composition, and biomechanical properties. Digoxin and ATP were found to increase neocartilage collagen content by 52-110% over untreated controls, while maintaining proteoglycan content near native tissue values. Furthermore, digoxin and ATP increased the tensile modulus by 280% and 180%, respectively, while the application of both agents increased the modulus by 380%. The trends in tensile properties were found to correlate with the amount of collagen cross-linking. Live Ca(2+) imaging experiments revealed that both digoxin and ATP were able to increase Ca(2+) oscillations in monolayer-cultured chondrocytes. This study provides a novel approach toward directing neocartilage maturation and enhancing its functional properties using novel Ca(2+) modulators.

Bone regeneration with low dose BMP-2 amplified by biomimetic supramolecular nanofibers within collagen scaffolds.

Bone morphogenetic protein-2 (BMP-2) is a potent osteoinductive cytokine that plays a critical role during bone regeneration and repair. In the extracellular environment, sulfated polysaccharides anchored covalently to glycoproteins such as syndecan and also non-covalently to fibronectin fibers have been shown to bind BMP-2 through a heparin-binding domain and regulate its bioactivity. We report here on a synthetic biomimetic strategy that emulates biological BMP-2 signaling through the use of peptide amphiphile nanofibers designed to bind heparin. The supramolecular nanofibers, which integrate the biological role of syndecan and fibronectin, were allowed to form gel networks within the pores of an absorbable collagen scaffold by simply infiltrating dilute solutions of the peptide amphiphile, heparan sulfate, and BMP-2. The hybrid biomaterial enhanced significantly bone regeneration in a rat critical-size femoral defect model using BMP-2 amounts that are one order of magnitude lower than required for healing in this animal model. Using micro-computed tomography, we also showed that the hybrid scaffold was more effective at bridging within the gap relative to a conventional scaffold of the type used clinically based on collagen and BMP-2. Histological evaluation also revealed the presence of more mature bone in the new ossified tissue when the low dose of BMP-2 was delivered using the biomimetic supramolecular system. These results demonstrate how molecularly designed materials that mimic features of the extracellular environment can amplify the regenerative capacity of growth factors.