Content-Dependent Osteogenic Response of Nanohydroxyapatite: An

Aug 18, 2016 - MgAl layered double hydroxide/chitosan porous scaffolds loaded with PFTα to promote bone regeneration. Yi-Xuan Chen , Rong Zhu , Qin-F...
0 downloads 0 Views 1MB Size
Subscriber access provided by Northern Illinois University

Article

Content-dependent osteogenic response of nano-hydroxyapatite; an in vitro and in vivo assessment within collagen-based scaffolds Gráinne M Cunniffe, Caroline M Curtin, Emmet M Thompson, Glenn R Dickson, and Fergal J O'Brien ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b06596 • Publication Date (Web): 18 Aug 2016 Downloaded from http://pubs.acs.org on August 22, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Content-dependent osteogenic response of nano-hydroxyapatite; an in vitro and in vivo assessment within collagen-based scaffolds

Gráinne M. Cunniffe

a, b

†, Caroline M. Curtin

a, b, c

†, Emmet M. Thompson

a, b, c

, Glenn R.

Dickson c, Fergal J. O’Brien a, b, c *

a

Dr. G. M. Cunniffe†, Dr. C. M. Curtin†, Mr. E. M. Thompson, Prof. F. J. O’Brien*, Trinity

Centre for Bioengineering, Trinity College Dublin, Dublin 2, Ireland b

Dr. G. M. Cunniffe†, Dr. C. M. Curtin†, Mr. E. M. Thompson, Prof. F. J. O’Brien*, Advanced

Materials and BioEngineering Research (AMBER) Centre, Royal College of Surgeons in Ireland & Trinity College Dublin, Dublin 2, Ireland c

Dr. C. M. Curtin†, Mr. E. M. Thompson, Dr. G. R. Dickson, Prof. F. J. O’Brien*, Tissue

Engineering Research Group, Department of Anatomy, Royal College of Surgeons in Ireland, 123 St. Stephens Green, Dublin 2, Ireland † Dr. Gráinne M. Cunniffe and Dr. Caroline M. Curtin contributed equally to this work. * Corresponding Author Prof. Fergal J. O’Brien, Tissue Engineering Research Group (TERG), Department of Anatomy, Royal College of Surgeons in Ireland, 123 St. Stephen’s Green, Dublin 2, Ireland Email: [email protected]

Phone: 00 353 1 402 2149

Abstract 1 ACS Paragon Plus Environment

ACS Applied Materials & Interfaces

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 34

The use of collagen-based scaffolds in orthopedic applications has been limited due to poor mechanical properties, but this may be overcome by the introduction of a stiffer supporting phase. Thus, we have developed a synthesis technique to produce non-aggregating, stable nano-hydroxyapatite (nHA) particles, permitting the fabrication of biomimetic-inspired scaffolds through the combination of nano-sized HA with collagen, as found in native bone. This study evaluates the mechanical and biological impact of incorporating increasing concentrations of these nanoparticles into porous collagen scaffolds (1:1 and 5:1 weight ratios of nHA:collagen). Mechanical assessment demonstrated that increasing nHA incorporation correlated with increasing Young’s moduli, which could be further amplified using cross-linking treatments. Typically, the porosity of a scaffold is sacrificed to produce a stiffer material; however, through the use of nano-sized particles the inclusion of up to 5:1 nHA:collagen content still preserved the high 99% porosity of the composite scaffold, allowing for maximum cell infiltration. Moreover, increasing nHA presence induced significant bioactive responses, achieving superior cellular attachment and enhanced osteogenesis, promoting earlier expression of bone markers and cell-mediated mineralization vs. nHA-free collagen controls. Interestingly, these content-dependent results observed in vitro did not directly translate in vivo. Instead, similar levels of bone formation were achieved within critical-sized rat calvarial defects, independent of nHA content, following acellular implantation. The addition of nHA, both 1:1 and 5:1, induced significantly higher levels of mineralization and de novo bone ingrowth vs. collagen controls as demonstrated by microCT, histological and histomorphometric analyses. Ultimately, these results demonstrate the immense osteoinductivity of non-aggregated nanoparticles of HA incorporated into collagen-composite scaffolds, and emphasizes the importance of in vivobased evaluation of therapies intended for clinical use. 2 ACS Paragon Plus Environment

Page 3 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Keywords Collagen-based Scaffold; Nano-hydroxyapatite; Biomimetic; Osteogenesis; In vivo bone regeneration; nanoparticles; mineralization;

Introduction Scaffolds for use as bone graft substitutes must fulfill certain requirements to support and promote successful bone repair1. A highly porous 3D structure with an interconnected pore network is desired to facilitate cell migration and the transport of nutrients and metabolic waste products. In addition, the scaffold must be biocompatible and bioresorbable with controllable degradation and resorption rates to match cell/tissue growth in vitro and/or in vivo2. Also of importance is a suitable surface chemistry for cell attachment, proliferation, and differentiation, along with sufficient mechanical properties capable of supporting osteogenesis3. Ideally, to positively influence cellular activity, the scaffold should not only act as a physical template for tissue growth but also provide an instructive environment to direct cellular function, i.e. be osteoinductive. A scaffold with osteoinductive potential not only facilitates osteogenesis but also promotes it by inducing an osteogenic response from cells due to its inherent composition. However, this can be difficult to achieve based on composition alone, and typically requires the application of growth factors and cytokines such as bone morphogenetic proteins. Collagen is a protein which provides strength and structural stability to a number of tissues in the body including skin, tendon, blood vessels, cartilage and bone, and therefore has been widely investigated for use in tissue engineering. Collagen-based scaffolds are known 3 ACS Paragon Plus Environment

ACS Applied Materials & Interfaces

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 34

to be highly biocompatible, with porosity, pore size and permeability suitable for bone tissue engineering4-6. From a clinical perspective, however, collagen scaffolds are limited for use in orthopedic applications by their poor mechanical properties; therefore, this study sought to investigate the effect of incorporating a reinforcing nano-hydroxyapatite (nHA) phase into a highly porous collagen scaffold. This nHA-collagen pairing occurs naturally in bone, whereby nanocrystalline hydroxyapatite reinforces the organic matrix containing collagen and non-collagenous proteins. Scaffolds containing collagen, HA, and a combination of the two materials have displayed excellent biocompatibility in previous studies2,

7-11

. In addition, numerous investigations have shown HA scaffolds to be

osteoinductive, with studies attributing the upregulation of osteogenic gene expression to the calcium and phosphate ions12-15. However, the use of micron-sized HA is a source of concern, as it can lead to brittle scaffolds with poor resorbability. Therefore, attention has since turned to the use of nano-sized HA particles16-17. We have developed a technique to produce non-aggregating nano-sized particles (