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A Structurally and Functionally Biomimetic Biphasic Scaffold for Intervertebral Disc Tissue Engineering.

Choy AT, Chan BP - PLoS ONE (2015)

Bottom Line: On mechanical testing, the height of our engineered disc recovered by ~82-89% in an annulus-independent manner, when compared with the 99% recovery exhibited by native disc.Biphasic scaffolds comprised of 10 annulus fibrosus-like lamellae had the best overall mechanical performance among the various designs owing to their similarity to native disc in most aspects, including elastic compliance during creep and recovery, and viscous compliance during recovery.However, the dynamic mechanical performance (including dynamic stiffness and damping factor) of all the biphasic scaffolds was similar to that of the native discs.

View Article: PubMed Central - PubMed

Affiliation: Tissue Engineering Laboratory, Department of Mechanical Engineering, The University of Hong Kong, Hong Kong Special Administrative Region, China.

ABSTRACT
Tissue engineering offers high hopes for the treatment of intervertebral disc (IVD) degeneration. Whereas scaffolds of the disc nucleus and annulus have been extensively studied, a truly biomimetic and mechanically functional biphasic scaffold using naturally occurring extracellular matrix is yet to be developed. Here, a biphasic scaffold was fabricated with collagen and glycosaminoglycans (GAGs), two of the most abundant extracellular matrix components in the IVD. Following fabrication, the scaffold was characterized and benchmarked against native disc. The biphasic scaffold was composed of a collagen-GAG co-precipitate making up the nucleus pulposus-like core, and this was encapsulated in multiple lamellae of photochemically crosslinked collagen membranes comprising the annulus fibrosus-like lamellae. On mechanical testing, the height of our engineered disc recovered by ~82-89% in an annulus-independent manner, when compared with the 99% recovery exhibited by native disc. The annulus-independent nature of disc height recovery suggests that the fluid replacement function of the engineered nucleus pulposus core might mimic this hitherto unique feature of native disc. Biphasic scaffolds comprised of 10 annulus fibrosus-like lamellae had the best overall mechanical performance among the various designs owing to their similarity to native disc in most aspects, including elastic compliance during creep and recovery, and viscous compliance during recovery. However, the dynamic mechanical performance (including dynamic stiffness and damping factor) of all the biphasic scaffolds was similar to that of the native discs. This study contributes to the rationalized design and development of a biomimetic and mechanically viable biphasic scaffold for IVD tissue engineering.

No MeSH data available.


Related in: MedlinePlus

Series of images to show the fabrication of the biphasic scaffold an intervertebral disc.(A) A CG core was encapsulated in the first collagen layer before crosslinking. (B) Some of the photochemically-crosslinked CG cores were then encapsulated in a further 1 to 9 layers of collagen. (C) Immersion of the CG core with collagen layers in the photosensitizer, rose Bengal. (D) Irradiation of the construct with an argon laser at 514 nm for photochemical crosslinking. (E) Dehydration of the CG containing the 1st collagen layer by rolling it on absorbent filter paper. (F) Dehydration of the CG core now encapsulated in the 2nd to 10th collagen layers. (G) The rehydrated CG core in one of the AF-like collagen lamella (E1). (H) Rehydrated CG core in two to ten collagen lamellae (E2 to E10). (I) Condensed biphasic scaffold in top view. (J) Condensed biphasic scaffold in side view. (K) Rabbit disc harvest. (L) Native rabbit disc in top view. (M) Native rabbit disc in side view.
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pone.0131827.g001: Series of images to show the fabrication of the biphasic scaffold an intervertebral disc.(A) A CG core was encapsulated in the first collagen layer before crosslinking. (B) Some of the photochemically-crosslinked CG cores were then encapsulated in a further 1 to 9 layers of collagen. (C) Immersion of the CG core with collagen layers in the photosensitizer, rose Bengal. (D) Irradiation of the construct with an argon laser at 514 nm for photochemical crosslinking. (E) Dehydration of the CG containing the 1st collagen layer by rolling it on absorbent filter paper. (F) Dehydration of the CG core now encapsulated in the 2nd to 10th collagen layers. (G) The rehydrated CG core in one of the AF-like collagen lamella (E1). (H) Rehydrated CG core in two to ten collagen lamellae (E2 to E10). (I) Condensed biphasic scaffold in top view. (J) Condensed biphasic scaffold in side view. (K) Rabbit disc harvest. (L) Native rabbit disc in top view. (M) Native rabbit disc in side view.

Mentions: The biphasic disc scaffold was fabricated by repeatedly laminating the NP-like CG core with photochemically crosslinked collagen lamellae to mimic the AF, as shown in Fig 1. The photochemical crosslinking procedure employed was a modified version of the one we previously reported for fabricating collagen membranes [15]. In brief, at 4°C acid soluble collagen solution was neutralized with sodium hydroxide and mixed with phosphate buffered saline (PBS) at pH 7.4 to prepare a 0.3% collagen gelling solution (w/v). One ml of the mixture was then transferred to a cylindrical mould of 16 mm diameter for gelation. At the start of gelation, the bottom of the mould was immersed in PBS pre-warmed to 37°C, and the top of the mould was in contact with a chilled plate (~4°C) for 5 min. This temperature difference between the bottom and top of the mould allowed just enough time for the dehydrated CG to be poured before it solidified. Further incubation of the whole mould at 37°C for 30 min resulted in the CG being encapsulated in a collagen hydrogel of 16 mm diameter and 5 mm tall (Fig 1A). This CG-gel composite was soaked in rose Bengal solution (R3877, Sigma) at a concentration of 0.001% (w/v) overnight (Fig 1C), and photochemically crosslinked by irradiating with an argon laser (Innova 300C, Coherent, California, USA; 514 nm) for 125 sec from the top to the bottom at a power of 200 mW (fluence = 25 J/cm2) (Fig 1D). The crosslinked CG-gel composite was dehydrated in a controllable manner by rolling on a piece of filter paper, until any loosely bound water in the collagen gel was removed (Fig 1E). The dehydrated gel of the composite thus made up one lamella (E1) of photochemically crosslinked collagen membrane surrounding the nuclear CG. (Fig 1G) This lamination process was repeated by encapsulating E1 in the 2nd collagen layer (Fig 1B), and the process was repeated until eventually, biphasic constructs containing 1, 2, 4 or 10 lamella(e) were obtained (Fig 1H). As a finishing step to further condense the scaffold structure into a dense fibrous meshwork with appropriate dimensions and to bring the construct to similar loaded physiologically conditions, biphasic constructs, right before mechanically tested, were submitted to a pre-load, comprising of 150 sec at -0.6 MPa and then 600 sec at -0.1 MPa. These stress values correspond to the pressure in a normal human disc, such that the lower stress value was measured during lying down, and the higher stress value was measured during standing [18].


A Structurally and Functionally Biomimetic Biphasic Scaffold for Intervertebral Disc Tissue Engineering.

Choy AT, Chan BP - PLoS ONE (2015)

Series of images to show the fabrication of the biphasic scaffold an intervertebral disc.(A) A CG core was encapsulated in the first collagen layer before crosslinking. (B) Some of the photochemically-crosslinked CG cores were then encapsulated in a further 1 to 9 layers of collagen. (C) Immersion of the CG core with collagen layers in the photosensitizer, rose Bengal. (D) Irradiation of the construct with an argon laser at 514 nm for photochemical crosslinking. (E) Dehydration of the CG containing the 1st collagen layer by rolling it on absorbent filter paper. (F) Dehydration of the CG core now encapsulated in the 2nd to 10th collagen layers. (G) The rehydrated CG core in one of the AF-like collagen lamella (E1). (H) Rehydrated CG core in two to ten collagen lamellae (E2 to E10). (I) Condensed biphasic scaffold in top view. (J) Condensed biphasic scaffold in side view. (K) Rabbit disc harvest. (L) Native rabbit disc in top view. (M) Native rabbit disc in side view.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482706&req=5

pone.0131827.g001: Series of images to show the fabrication of the biphasic scaffold an intervertebral disc.(A) A CG core was encapsulated in the first collagen layer before crosslinking. (B) Some of the photochemically-crosslinked CG cores were then encapsulated in a further 1 to 9 layers of collagen. (C) Immersion of the CG core with collagen layers in the photosensitizer, rose Bengal. (D) Irradiation of the construct with an argon laser at 514 nm for photochemical crosslinking. (E) Dehydration of the CG containing the 1st collagen layer by rolling it on absorbent filter paper. (F) Dehydration of the CG core now encapsulated in the 2nd to 10th collagen layers. (G) The rehydrated CG core in one of the AF-like collagen lamella (E1). (H) Rehydrated CG core in two to ten collagen lamellae (E2 to E10). (I) Condensed biphasic scaffold in top view. (J) Condensed biphasic scaffold in side view. (K) Rabbit disc harvest. (L) Native rabbit disc in top view. (M) Native rabbit disc in side view.
Mentions: The biphasic disc scaffold was fabricated by repeatedly laminating the NP-like CG core with photochemically crosslinked collagen lamellae to mimic the AF, as shown in Fig 1. The photochemical crosslinking procedure employed was a modified version of the one we previously reported for fabricating collagen membranes [15]. In brief, at 4°C acid soluble collagen solution was neutralized with sodium hydroxide and mixed with phosphate buffered saline (PBS) at pH 7.4 to prepare a 0.3% collagen gelling solution (w/v). One ml of the mixture was then transferred to a cylindrical mould of 16 mm diameter for gelation. At the start of gelation, the bottom of the mould was immersed in PBS pre-warmed to 37°C, and the top of the mould was in contact with a chilled plate (~4°C) for 5 min. This temperature difference between the bottom and top of the mould allowed just enough time for the dehydrated CG to be poured before it solidified. Further incubation of the whole mould at 37°C for 30 min resulted in the CG being encapsulated in a collagen hydrogel of 16 mm diameter and 5 mm tall (Fig 1A). This CG-gel composite was soaked in rose Bengal solution (R3877, Sigma) at a concentration of 0.001% (w/v) overnight (Fig 1C), and photochemically crosslinked by irradiating with an argon laser (Innova 300C, Coherent, California, USA; 514 nm) for 125 sec from the top to the bottom at a power of 200 mW (fluence = 25 J/cm2) (Fig 1D). The crosslinked CG-gel composite was dehydrated in a controllable manner by rolling on a piece of filter paper, until any loosely bound water in the collagen gel was removed (Fig 1E). The dehydrated gel of the composite thus made up one lamella (E1) of photochemically crosslinked collagen membrane surrounding the nuclear CG. (Fig 1G) This lamination process was repeated by encapsulating E1 in the 2nd collagen layer (Fig 1B), and the process was repeated until eventually, biphasic constructs containing 1, 2, 4 or 10 lamella(e) were obtained (Fig 1H). As a finishing step to further condense the scaffold structure into a dense fibrous meshwork with appropriate dimensions and to bring the construct to similar loaded physiologically conditions, biphasic constructs, right before mechanically tested, were submitted to a pre-load, comprising of 150 sec at -0.6 MPa and then 600 sec at -0.1 MPa. These stress values correspond to the pressure in a normal human disc, such that the lower stress value was measured during lying down, and the higher stress value was measured during standing [18].

Bottom Line: On mechanical testing, the height of our engineered disc recovered by ~82-89% in an annulus-independent manner, when compared with the 99% recovery exhibited by native disc.Biphasic scaffolds comprised of 10 annulus fibrosus-like lamellae had the best overall mechanical performance among the various designs owing to their similarity to native disc in most aspects, including elastic compliance during creep and recovery, and viscous compliance during recovery.However, the dynamic mechanical performance (including dynamic stiffness and damping factor) of all the biphasic scaffolds was similar to that of the native discs.

View Article: PubMed Central - PubMed

Affiliation: Tissue Engineering Laboratory, Department of Mechanical Engineering, The University of Hong Kong, Hong Kong Special Administrative Region, China.

ABSTRACT
Tissue engineering offers high hopes for the treatment of intervertebral disc (IVD) degeneration. Whereas scaffolds of the disc nucleus and annulus have been extensively studied, a truly biomimetic and mechanically functional biphasic scaffold using naturally occurring extracellular matrix is yet to be developed. Here, a biphasic scaffold was fabricated with collagen and glycosaminoglycans (GAGs), two of the most abundant extracellular matrix components in the IVD. Following fabrication, the scaffold was characterized and benchmarked against native disc. The biphasic scaffold was composed of a collagen-GAG co-precipitate making up the nucleus pulposus-like core, and this was encapsulated in multiple lamellae of photochemically crosslinked collagen membranes comprising the annulus fibrosus-like lamellae. On mechanical testing, the height of our engineered disc recovered by ~82-89% in an annulus-independent manner, when compared with the 99% recovery exhibited by native disc. The annulus-independent nature of disc height recovery suggests that the fluid replacement function of the engineered nucleus pulposus core might mimic this hitherto unique feature of native disc. Biphasic scaffolds comprised of 10 annulus fibrosus-like lamellae had the best overall mechanical performance among the various designs owing to their similarity to native disc in most aspects, including elastic compliance during creep and recovery, and viscous compliance during recovery. However, the dynamic mechanical performance (including dynamic stiffness and damping factor) of all the biphasic scaffolds was similar to that of the native discs. This study contributes to the rationalized design and development of a biomimetic and mechanically viable biphasic scaffold for IVD tissue engineering.

No MeSH data available.


Related in: MedlinePlus