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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
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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
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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
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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
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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 (
