Decellularized tissue engineered hyaline cartilage graft for articular cartilage repair.

Articular cartilage repair has been a long-standing challenge in orthopaedic medicine due to the limited self-regenerative capability of cartilage tissue. Currently, cartilage lesions are often treated by microfracture or autologous chondrocyte implantation (ACI). However, these treatments are frequently reported to result in a mixture of the desired hyaline cartilage and mechanically inferior fibrocartilage. In this study, by combining the advantages of cartilage tissue engineering and decellularization technology, we developed a decellularized allogeneic hyaline cartilage graft, named dLhCG, which achieved superior efficacy in articular cartilage repair and surpassed living autologous chondrocyte-based cartilaginous engraftment and ACI. By the 6-month time point after implantation in porcine knee joints, the fine morphology, composition, phenotype, microstructure and mechanical properties of the regenerated hyaline-like cartilaginous neo-tissue have been demonstrated via histology, biochemical assays, DNA microarrays and mechanical tests. The articular cartilaginous engraftment with allogeneic dLhCG was indicated to be well consistent, compatible and integrated with the native cartilage of the host. The successful repair of articular chondral defects in large animal models suggests the readiness of allogeneic dLhCG for clinical trials.

Full-Scale Osteochondral Regeneration by Sole Graft of Tissue-Engineered Hyaline Cartilage without Co-Engraftment of Subchondral Bone Substitute.

In this study, full-scale osteochondral defects are hypothesized, which penetrate the articular cartilage layer and invade into subchondral bones, and can be fixed by sole graft of tissue-engineered hyaline cartilage without co-engraftment of any subchondral bone substitute. It is hypothesized that given a finely regenerated articular cartilage shielding on top, the restoration of subchondral bones can be fulfilled via spontaneous self-remodeling in situ. Hence, the key challenge of osteochondral regeneration lies in restoration of the non-self-regenerative articular cartilage. Here, traumatic osteochondral lesions to be repaired in rabbit knee models are endeavored using novel tissue-engineered hyaline-like cartilage grafts that are produced by 3D cultured porcine chondrocytes in vitro. Comparative trials are conducted in animal models that are implanted with living hyaline cartilage grafts (LhCG) and decellularized LhCG (dLhCG). Sound osteochondral regeneration is gradually revealed from both LhCG and dLhCG-implanted samples 50-100 d after implantation. Quality regeneration in both zones of articular cartilage and subchondral bones are validated by the restored osteochondral composition, structure, phenotype, and mechanical property, which validate the hypothesis of this study.

Establishment of an in vitro three-dimensional model for cartilage damage in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease that leads to progressive joint destruction. To further understand the process of rheumatoid cartilage damage, an in vitro model consisting of an interactive tri-culture of synovial fibroblasts (SFs), LPS-stimulated macrophages and a primary chondrocyte-based tissue-engineered construct was established. The tissue-engineered construct has a composition similar to that of human cartilage, which is rich in collagen type II and proteoglycans. Data generated from this model revealed that healthy chondrocytes were activated in the presence of SFs and macrophages. The activated chondrocytes subsequently displayed aberrant behaviours as seen in a disease state such as increased apoptosis, decreased gene expression for matrix components such as type II collagen and aggrecan, increased gene expression for tissue-degrading enzymes (MMP-1, -3, -13 and ADAMTS-4, -5), and upregulation of inflammatory mediator gene expression (TNF-α, IL-1ß, IL-6 and IKBKB). Additionally, the inclusion of SFs and macrophages in the model enabled both cell types to more closely replicate an in vivo role in mediating cartilage destruction. This is evidenced by extensive matrix loss, detected in the model through immunostaining and biochemical analysis. Subsequent drug treatment with celecoxib has shown that the model was able to respond to the therapeutic effects of this drug by reversing cartilage damage. This study showed that the model was able to recapitulate certain pathological features of an RA cartilage. If properly validated, this model potentially can be used for screening new therapeutic drugs and strategies, thereby contributing to the improvement of anti-rheumatic treatment. Copyright © 2017 John Wiley & Sons, Ltd.

A novel DOPA-albumin based tissue adhesive for internal medical applications.

To date, existing tissue adhesives have various weak points in gluing kinetics and stability - particularly, in biocompatibility, which make most of them remain suboptimal for internal conditions. Herein, a novel mussel-inspired "BCD" tissue glue made of bovine serum albumin (BSA), citrate acid (CA) and dopamine was developed aiming at internal medical applications. BSA was employed as a natural and biocompatible macromolecular backbone; CA was introduced as a dual-functional intermediate to increase reactive carboxyl sites for engraftment of dopamine onto BSA backbone and also block the competing reactive amines from the proteinic backbone. Timely curing and stable adhesion were achieved between biological tissue substrates via instant chelation and gradual conjugation of DOPA-catechol groups in BCD glue. Within 30 min, this newly developed BCD tissue glue can provide over 10-fold greater adhesion stress than that of commercially available fibrin glue in wet environment. As a tissue adhesive for internal use, its superior properties also include ideal gelation kinetics and swelling behaviour, appropriate degradation rate, sound cytocompatibility in vitro, as well as fine biocompatibility in vivo. More importantly, successful animal experimentations in seroma prevention and instant hemostasis ultimately validated BCD tissue glue's preclinical efficacy as a tissue adhesive for various internal medical applications.

A mussel-inspired double-crosslinked tissue adhesive on rat mastectomy model: seroma prevention and in vivo biocompatibility.

BACKGROUND: Seroma formation is a common postsurgical complication of breast cancer surgery. It delays wound healing and may lead to other more serious complications. Conventional methods of reducing seroma formation through suturing or placement of surgical drainage produce inconsistent clinical outcomes. Tissue adhesives are viable alternatives but most of them are unsuitable for internal use and for large-area applications because of weak tissue adhesion strength or biocompatibility issues. The aim of this study was to evaluate the efficacy and biocompatibility of a mussel-inspired double-crosslinked tissue adhesive (DCTA) in reducing seroma formation after mastectomy. MATERIALS AND METHODS: Thirty-six female Sprague-Dawley rats were randomly assigned to either the saline control group (n = 12), the TISSEEL sealant (Baxter) group (n = 12), or the DCTA group (n = 12). After performing a mastectomy and applying the corresponding treatment, the efficacy of DCTA was evaluated by measurement of seroma volume while its biocompatibility was assessed via micronuclei test and histopathologic examination. RESULTS: During the 1-wk postsurgical period, the average total seroma volume of DCTA was significantly lower than the saline control group. Importantly, the mean seroma volume in DCTA showed a decreasing trend, whereas those in TISSEEL and saline control groups showed otherwise. The application of DCTA showed no genotoxic effect on the host and no severe inflammation. CONCLUSIONS: This study demonstrates that the good tissue adhesion strength and stability of DCTA were successful in reducing seroma formation over a period of 1 wk. Furthermore, the results also showed that it is biocompatible, which makes it suitable for large-area, internal use.

Hydrogel based cartilaginous tissue regeneration: recent insights and technologies.

Hydrogels have been extensively employed as an attractive biomaterial to address numerous existing challenges in the fields of regenerative medicine and research because of their unique properties such as the capability to encapsulate cells, high water content, ease of modification, low toxicity, injectability, in situ spatial fit and biocompatibility. These inherent properties have created many opportunities for hydrogels as a scaffold or a cell/drug carrier in tissue regeneration, especially in the field of cartilaginous tissue such as articular cartilage and intervertebral discs. A concise overview of the anatomy/physiology of these cartilaginous tissues and their pathophysiology, epidemiology and existing clinical treatments will be briefly described. This review article will discuss the current state-of-the-art of various polymers and developing strategies that are explored in establishing different technologies for cartilaginous tissue regeneration. In particular, an innovative approach to generate scaffold-free cartilaginous tissue via a transient hydrogel scaffolding system for disease modeling to pre-clinical trials will be examined. Following that, the article reviews numerous hydrogel-based medical implants used in clinical treatment of osteoarthritis and degenerated discs. Last but not least, the challenges and future directions of hydrogel based medical implants in the regeneration of cartilaginous tissue are also discussed.

A preclinical evaluation of an autologous living hyaline-like cartilaginous graft for articular cartilage repair: a pilot study.

In this pilot study, an autologous synthetic scaffold-free construct with hyaline quality, termed living hyaline cartilaginous graft (LhCG), was applied for treating cartilage lesions. Implantation of autologous LhCG was done at load-bearing regions of the knees in skeletally mature mini-pigs for 6 months. Over the course of this study, significant radiographical improvement in LhCG treated sites was observed via magnetic resonance imaging. Furthermore, macroscopic repair was effected by LhCG at endpoint. Microscopic inspection revealed that LhCG engraftment restored cartilage thickness, promoted integration with surrounding native cartilage, produced abundant cartilage-specific matrix molecules, and re-established an intact superficial tangential zone. Importantly, the repair efficacy of LhCG was quantitatively shown to be comparable to native, unaffected cartilage in terms of biochemical composition and biomechanical properties. There were no complications related to the donor site of cartilage biopsy. Collectively, these results imply that LhCG engraftment may be a viable approach for articular cartilage repair.

Use of Interim Scaffolding and Neotissue Development to Produce a Scaffold-Free Living Hyaline Cartilage Graft.

The fabrication of three-dimensional (3D) constructs relies heavily on the use of biomaterial-based scaffolds. These are required as mechanical supports as well as to translate two-dimensional cultures to 3D cultures for clinical applications. Regardless of the choice of scaffold, timely degradation of scaffolds is difficult to achieve and undegraded scaffold material can lead to interference in further tissue development or morphogenesis. In cartilage tissue engineering, hydrogel is the highly preferred scaffold material as it shares many similar characteristics with native cartilaginous matrix. Hence, we employed gelatin microspheres as porogens to create a microcavitary alginate hydrogel as an interim scaffold to facilitate initial chondrocyte 3D culture and to establish a final scaffold-free living hyaline cartilaginous graft (LhCG) for cartilage tissue engineering.

Optimization of chondrocyte isolation and phenotype characterization for cartilage tissue engineering.

Current protocols for chondrocyte isolation are inconsistent, resulting in suboptimal cell yield and compromised cell quality. Thus, there is a need for an improved isolation protocol that is able to give a maximum yield with optimal cell viability while preserving the chondrocyte phenotype. In light of this, we developed an improved isolation protocol based on enzymatic digestion using 0.1% (w/v) collagenase II. Different from existing methods of digesting minced cartilage for a prolonged period (usually 14-16 h), we performed two additional digestions, with a 5- and 3-h interval in between. The results showed that this multiple digestion method was able to yield a total number of cells that are more than a fivefold increase as compared to any of the common isolation protocols. More importantly, a high percentage of the isolated cells remained viable. Furthermore, an evaluation of the effect of additional digestions on chondrocyte phenotype indicated that cells harvested from the second and third digestion showed a comparable or higher proliferative capacity than the first digestion and all the cells expressed chondrocyte-specific markers tested, with cells from the third digestion showing exceptionally high gene expression levels for collagen type II (Col II), aggrecan, and COMP. Additionally, their ability to produce collagen type II as well as their morphology were not affected by the two additional digestions. Taken together, the results suggested that the use of this isolation protocol resulted in a higher cell yield and the quality of the isolated cells was maintained. Hence, we recommend this isolation protocol to be employed for more efficient cell harvesting especially from limited biopsied cartilage tissue samples.

A three-dimensionally engineered biomimetic cartilaginous tissue model for osteoarthritic drug evaluation.

Osteoarthritis (OA) is primarily characterized by focal cartilage destruction and synovitis. Presently, the pathogenesis of OA remains unclear, and an effective treatment methodology is an unmet need. To this end, a plethora of animal models and monolayer models have been developed, but they are faced with the limitation of high cost and inability to recapitulate a pure hyaline cartilaginous phenotype, which is important in studying the efficacy of therapeutic agents. We have previously developed a living hyaline cartilage graft (LhCG) that accurately presented a pure hyaline cartilage phenotype. Here, through the coculture of lipopolysaccharide (LPS)-activated macrophages with LhCG, we hypothesized that an accurate OA disease model may be developed. Subsequently, this model was evaluated for its accuracy for in vitro drug testing. Results indicated that chondrocyte proliferation and apoptosis were increased in the disease model. Additionally, extracellular matrix (ECM) synthesis increased as indicated by the increased anabolic gene expression levels, such as collagen type II and aggrecan. Up-regulation of matrix metalloproteinase-1 (MMP-1) and MMP-3 genes suggested increased degradative activity, while chondrocytic hypertrophic differentiation was observed. Furthermore, extensive degradation of collagen type II and glycosaminoglycans (GAGs) were also observed. The results of celecoxib treatment on our model showed inhibition of nitric oxide (NO) and prostaglandin E2 (PGE2) production, as well as down-regulation of MMP-1 and MMP-3 expression. Taken together, the results suggested that this coculture model was able to sufficiently mimic the native, diseased OA cartilage, while drug testing results confirmed its suitability as an in vitro model for predicting native cartilage response to drug treatment.

Three-dimensionally engineered biomimetic tissue models for in vitro drug evaluation: delivery, efficacy and toxicity.

INTRODUCTION: Three-dimensionally (3D) engineered biomimetic tissue models are sought after due to their high fidelity in mimicking various native tissues of the human body, this quality of which gives them an important role at the forefront of drug discovery and development. A multitude of studies have consistently indicated that gene expression profiles, cellular phenotypes, differentiation capabilities and functionalities are all affected by tissue architecture. Thus, the drug evaluation process will stand to gain immense benefits from the fairly accurate predictions of cellular responses displayed by 3D-engineered tissue models when exposed to the drugs of interest in vitro. Stemming from this fact, many studies have set out to capitalize on developing tissue models that are tailored to specific aspects of drug evaluation including the tests of novel drug delivery systems, drug efficacy and toxicity. AREAS COVERED: The areas covered include fabrication methods and usage of 3D in vitro tumor models in cancer research, focusing on the evaluation of delivery and efficacy of various anticancer drugs or other therapeutic agents. Also covered are the use of 3D in vitro inflammatory tissue models in anti-inflammation research, centering on osteoarthritis (OA) and rheumatoid arthritis (RA) and the use of 3D in vitro tissue models designed for drug toxicity evaluation specifically with liver-mimetic tissues. EXPERT OPINION: Currently available 3D tissue models in various fields of research have already displayed their capabilities in predicting cellular responses to various therapeutic agents and delivery methods with better accuracy than their 2D counterparts, albeit being in need of much refinement before they can be successfully applied for reliable drug evaluation. Given further development and improvement, it is highly probable that the 3D-engineered tissue models may perform as living platforms for dynamic drug evaluation in vitro.