Eleftherios A. Makris

Repair and tissue engineering techniques for articular cartilage

Chondral and osteochondral lesions due to injury or other pathology commonly result in the development of osteoarthritis, eventually leading to progressive total joint destruction. Although current progress suggests that biologic agents can delay the advancement of deterioration, such drugs are incapable of promoting tissue restoration. The limited ability of articular cartilage to regenerate renders joint arthroplasty an unavoidable surgical intervention. This Review describes current, widely used clinical repair techniques for resurfacing articular cartilage defects; short-term and long-term clinical outcomes of these techniques are discussed. Also reviewed is a developmental pipeline of acellular and cellular regenerative products and techniques that could revolutionize joint care over the next decade by promoting the development of functional articular cartilage. Acellular products typically consist of collagen or hyaluronic-acid-based materials, whereas cellular techniques use either primary cells or stem cells, with or without scaffolds. Central to these efforts is the prominent role that tissue engineering has in translating biological technology into clinical products; therefore, concomitant regulatory processes are also discussed.

Developing functional musculoskeletal tissues through hypoxia and lysyl oxidase-induced collagen cross-linking

Significance The inadequate mechanical properties of engineered tissues have prevented related therapies from clinical translation. Collagen cross-links correlate with the mechanical integrity of tissues; however, addressing the weakness of neotissues through enhancing collagen cross-links has not received the attention it deserves. The present study demonstrates, both in vitro and in vivo, that improvements in the mechanical properties of native and engineered tissues can be attained using endogenous (hypoxia-mediated) lysyl oxidase and exogenous application of lysyl oxidase-like 2, which are enzymes responsible for collagen cross-linking. By promoting an ∼16-fold increase in collagen cross-linking and, concomitantly, an approximately fivefold enhancement in the neotissue’s mechanical properties, this work creates new prospects for regenerative medicine. The methods developed here work across a spectrum of collagen-rich tissues and are clinically applicable. The inability to recapitulate native tissue biomechanics, especially tensile properties, hinders progress in regenerative medicine. To address this problem, strategies have focused on enhancing collagen production. However, manipulating collagen cross-links, ubiquitous throughout all tissues and conferring mechanical integrity, has been underinvestigated. A series of studies examined the effects of lysyl oxidases (LOXs), enzymes responsible for the formation of collagen cross-links. Hypoxia-induced endogenous LOX was applied in multiple musculoskeletal tissues (i.e., cartilage, meniscus, tendons, ligaments). Results of these studies showed that both native and engineered tissues are enhanced by invoking a mechanism of hypoxia-induced pyridinoline (PYR) cross-links via intermediaries like LOX. Hypoxia was shown to enhance PYR cross-linking 1.4- to 6.4-fold and, concomitantly, to increase the tensile properties of collagen-rich tissues 1.3- to 2.2-fold. Direct administration of exogenous lysyl oxidase-like 2 (LOXL2) was applied in native cartilage and neocartilage generated using a scaffold-free, self-assembling process of primary chondrocytes. Exogenous LOXL2 was found to enhance native tissue tensile properties 1.9-fold. LOXL2 concentration- and time-dependent increases in PYR content (∼16-fold compared with controls) and tensile properties (approximately fivefold compared with controls) of neocartilage were also detected, resulting in properties on par with native tissue. Finally, in vivo subcutaneous implantation of LOXL2-treated neocartilage in nude mice promoted further maturation of the neotissue, enhancing tensile and PYR content approximately threefold and 14-fold, respectively, compared with in vitro controls. Collectively, these results provide the first report, to our knowledge, of endogenous (hypoxia-induced) LOX and exogenous LOXL2 applications for promoting collagen cross-linking and improving the tensile properties of a spectrum of native and engineered tissues both in vitro and in vivo.

Combined use of chondroitinase-ABC, TGF-β1, and collagen crosslinking agent lysyl oxidase to engineer functional neotissues for fibrocartilage repair.

Patients suffering from damaged or diseased fibrocartilages currently have no effective long-term treatment options. Despite their potential, engineered tissues suffer from inferior biomechanical integrity and an inability to integrate in vivo. The present study identifies a treatment regimen (including the biophysical agent chondroitinase-ABC, the biochemical agent TGF-β1, and the collagen crosslinking agent lysyl oxidase) to prime highly cellularized, scaffold-free neofibrocartilage implants, effecting continued improvement in vivo. We show these agents drive in vitro neofibrocartilage matrix maturation toward synergistically enhanced Youngs modulus and ultimate tensile strength values, which were increased 245% and 186%, respectively, over controls. Furthermore, an in vitro fibrocartilage defect model found this treatment regimen to significantly increase the integration tensile properties between treated neofibrocartilage and native tissue. Through translating this technology to an in vivo fibrocartilage defect model, our results indicate, for the first time, that a pre-treatment can prime neofibrocartilage for significantly enhanced integration potential in vivo, with interfacial tensile stiffness and strength increasing by 730% and 745%, respectively, compared to integration values achieved in vitro. Our results suggest that specifically targeting collagen assembly and organization is a powerful means to augment overall neotissue mechanics and integration potential toward improved clinical feasibility.

Hypoxia-induced collagen crosslinking as a mechanism for enhancing mechanical properties of engineered articular cartilage

OBJECTIVE The focus of tissue engineering of neocartilage has traditionally been on enhancing extracellular matrix and thus biomechanical properties. Emphasis has been placed on the enhancement of collagen type and quantity, and, concomitantly, tensile properties. The objective of this study was to improve crosslinking of the collagen network by testing the hypothesis that hypoxia could promote pyridinoline (PYR) crosslinks and, thus, improve neocartilages tensile properties. METHODS Chondrocyte expression of lysyl oxidase (LOX), an enzyme responsible for the formation of collagen PYR crosslinks, was first assessed pre- and post- hypoxia application. Then, the mechanical properties of self-assembled neocartilage constructs were measured, after 4 weeks of culture, for groups exposed to 4% O2 at different initiation times and durations, i.e., during the 1st and 3rd weeks, 3rd and 4th weeks, 4th week only, continuously after cell seeding, or never. RESULTS Results showed that LOX gene expression was upregulated ∼20-fold in chondrocytes in response to hypoxia. Hypoxia applied during the 3rd and 4th weeks significantly increased PYR crosslinks without affecting collagen content. Excitingly, neocartilage tensile properties were increased ∼2-fold. It should be noted that these properties exhibited a distinct temporal dependence to hypoxia exposure, since upregulation of these properties was due to hypoxia applied only during the 3rd and 4th weeks. CONCLUSION These data elucidate the role of hypoxia-mediated upregulation of LOX and subsequent increases in PYR crosslinks in engineered cartilage. These results hold promise toward applying hypoxia at precise time points to promote tensile integrity and direct construct maturation.

A copper sulfate and hydroxylysine treatment regimen for enhancing collagen cross-linking and biomechanical properties in engineered neocartilage

The objective of this study was to improve the biomechanical properties of engineered neotissues through promoting the development of collagen cross‐links. It was hypothesized that supplementing medium with copper sulfate and the amino acid hydroxylysine would enhance the activity of lysyl oxidase enzyme to form collagen cross‐links, increasing the strength and integrity of the neotissue. Neocartilage constructs were generated using a scaffoldless, self‐assembling process and treated with copper sulfate and hydroxylysine, either alone or in combination, following a 2‐factor, full‐factorial study design. Following a 6‐wk culture period, the biomechanical and biochemical properties of the constructs were measured. Results found copper sulfate to significantly increase pyridinoline (PYR) cross‐links in all copper sulfate‐containing groups over controls. When copper sulfate and hydroxylysine were combined, the result was synergistic, with a 10‐fold increase in PYR content over controls. This increase in PYR cross‐links manifested in a 3.3‐fold significant increase in the tensile properties of the copper sulfate + hydroxylysine group. In addition, an 123% increase over control values was detected in the copper sulfate group in terms of the aggregate modulus. These data elucidate the role of copper sulfate and hydroxylysine toward improving the biomechanical properties of neotissues through collagen cross‐linking enhancement.—Makris, E. A., MacBarb, R. F., Responte, D. J., Hu, J. C., Athanasiou, K. A. A copper sulfate and hydroxylysine treatment regimen for enhancing collagen cross‐linking and biomechanical properties in engineered neocartilage. FASEB J. 27, 2421–2430 (2013). www.fasebj.org

Primary closure versus non-closure of dog bite wounds. A randomised controlled trial

INTRODUCTION Dog bite wounds represent a major health problem. Despite their importance, their management and especially the role of primary closure remain controversial. In this randomised controlled trial, the outcome between primary suturing and non-closure was compared. METHODS 168 consecutive patients with dog bite injuries were included in this study. The wounds were allocated randomly in two treatment approaches: Group 1, consisting of eighty-two patients, had their wound sutured, whilst Group 2, consisting of eighty-six patients, did not have their wounds sutured. All wounds were cleansed using high-pressure irrigation and povidone iodine. All patients received the same type of antibiotic treatment. Our measured outcomes included presence of infection and cosmetic appearance. Cosmetic outcome was evaluated using the Vancouver Scar Scale (VSS). Wound and patient characteristics, such as time of management, wound location and size, and patient age, were recorded and analysed for their potential role in the resulting outcome. RESULTS The overall infection rate was 8.3%. No difference in the infection rate between primary suturing and non-suturing group was detected in the present study. The cosmetic appearance of the sutured wounds was significantly better (mean score 1.74) compared to the wounds that were left open (mean score 3.05) (p=0.0001). The infection rate was comparable among all age groups. Wounds treated within 8h of injury demonstrated an infection rate of 4.5%, which is lower compared to the 22.2% rate observed in wounds treated later than 8h. The wounds located at the head and neck exhibited better results in both infection rate and cosmetic outcome. Additionally, wounds >3 cm negatively affected the cosmetic appearance of the outcome. CONCLUSIONS Primary suturing of wounds caused by dog bites resulted in similar infection rate compared to non-suturing. However, primary suturing exhibited improved cosmetic appearance. Time of management appeared to be critical, as early treatment resulted in lower infection rate and improved cosmetic appearance regardless suturing or not. Furthermore, wounds located at the head and face demonstrated better results.

A chondroitinase-ABC and TGF-β1 treatment regimen for enhancing the mechanical properties of tissue engineered fibrocartilage

The development of functionally equivalent fibrocartilage remains elusive despite efforts to engineer tissues such as knee meniscus, intervertebral disc and temporomandibular joint disc. Attempts to engineer these structures often fail to create tissues with mechanical properties on a par with native tissue, resulting in constructs unsuitable for clinical applications. The objective of this study was to engineer a spectrum of biomimetic fibrocartilages representative of the distinct functional properties found in native tissues. Using the self-assembly process, different co-cultures of meniscus cells and articular chondrocytes were seeded into agarose wells and treated with the catabolic agent chondroitinase-ABC (C-ABC) and the anabolic agent transforming growth factor-β1 (TGF-β1) via a two-factor (cell ratio and bioactive treatment), full factorial study design. Application of both C-ABC and TGF-β1 resulted in a beneficial or positive increase in the collagen content of treated constructs compared to controls. Significant increases in both the collagen density and fiber diameter were also seen with this treatment, increasing these values by 32 and 15%, respectively, over control values. Mechanical testing found the combined bioactive treatment to synergistically increase the Youngs modulus and ultimate tensile strength of the engineered fibrocartilages compared to controls, with values reaching the lower spectrum of those found in native tissues. Together, these data demonstrate that C-ABC and TGF-β1 interact to develop a denser collagen matrix better able to withstand tensile loading. This study highlights a way to optimize the tensile properties of engineered fibrocartilage using a biochemical and a biophysical agent together to create distinct fibrocartilages with functional properties mimicking those of native tissue.

Induced Collagen Cross-Links Enhance Cartilage Integration

Articular cartilage does not integrate due primarily to a scarcity of cross-links and viable cells at the interface. The objective of this study was to test the hypothesis that lysyl-oxidase, a metalloenzyme that forms collagen cross-links, would be effective in improving integration between native-to-native, as well as tissue engineered-to-native cartilage surfaces. To examine these hypotheses, engineered cartilage constructs, synthesized via the self-assembling process, as well as native cartilage, were implanted into native cartilage rings and treated with lysyl-oxidase for varying amounts of time. For both groups, lysyl-oxidase application resulted in greater apparent stiffness across the cartilage interface 2–2.2 times greater than control. The construct-to-native lysyl-oxidase group also exhibited a statistically significant increase in the apparent strength, here defined as the highest observed peak stress during tensile testing. Histology indicated a narrowing gap at the cartilage interface in lysyl-oxidase treated groups, though this alone is not sufficient to indicate annealing. However, when the morphological and mechanical data are taken together, the longer the duration of lysyl-oxidase treatment, the more integrated the interface appeared. Though further data are needed to confirm the mechanism of action, the enhancement of integration may be due to lysyl-oxidase-induced pyridinoline cross-links. This study demonstrates that lysyl-oxidase is a potent agent for enhancing integration between both native-to-native and native-to-engineered cartilages. The fact that interfacial strength increased manifold suggests that cross-linking agents should play a significant role in solving the difficult problem of cartilage integration. Future studies must examine dose, dosing regimen, and cellular responses to lysyl-oxidase to optimize its application.

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.

Enhancing the mechanical properties of articular cartilage through enzyme-mediated collagen crosslinking as regulated by low oxygen tension

The poor ability of articular cartilage to repair following disease and injury makes the tissue a key target for reparative and regenerative medicine strategies. It has been shown that human knee joint resides under hypoxic conditions. Oxygen tension in healthy human synovium is between 7 and 11% and in articular cartilage may therefore be as low as 1–2.5% [1].© 2012 ASME